extfieldsofstudy: Medicine Biology

text:
RpoN and the Nps and Npa two‐component regulatory system control pilE transcription in commensal Neisseria

Abstract Over 20 genes are involved in the biogenesis and function of the Neisseria Type IV pilus (Tfp). In the pathogenic species, RpoD and the integration host factor (IHF) protein regulate expression of pilE, encoding the Tfp structural subunit. We previously reported that in commensal species, pilE transcription is regulated by RpoN, IHF, and activator Npa. Npa has many hallmarks of response regulators in two‐component regulatory systems, leading us to search for its response regulator partner. We report that Npa partners with sensor kinase Nps to control pilE transcription. Among the genes involved in Tfp biogenesis and function, only pilE is controlled by RpoN and Npa/Nps. We summarize our findings in a model, and discuss the implications of the differential regulation of pilE the context of Neisseria Tfp biogenesis.

and an output domain in the C terminus (Carrick, Fyfe, & Davies, 2000;Jung, Fried, Behr, & Heermann, 2012). A TCRS consists of a membranebound sensor kinase (SK) and a cytoplasmic response regulator (RR). The SK autophosphorylates upon detection of a signal, and transfers the phosphoryl group to the RR, which then activates or represses gene expression. In the RR, the phosphorylated amino acid is a highly conserved aspartate. We previously speculated that aspartate 58 (D58) in Npa is the phosphorylated residue (Rendón et al., 2013). Since Npa has hallmarks of a response regulator, we searched the genome of commensal Neisseria elongata (Nel) for its SK counterpart, and found Open Reading Frame (ORF) (NEIELOOT_00058) whose deduced amino acid sequence has domains typically found in SK. NEIELOOT_00058 is present in all other commensal Neisseria genomes published on the web.
In this work, we tested the hypothesis that NEIELOOT_00058 is the SK that activates Npa to regulate transcription of pilE, using Neisseria elongata (Nel) as the model commensal. We show that Nps is required for pilE transcription, and that Nps and Npa act in concert as a TCRS. Finally, we show that RpoN and Nps/Npa control transcription only of pilE and not the other Tfp biogenesis genes reported to date. The implication of these different control mechanisms for Type IV pilus biogenesis is discussed.

| Strains, plasmids, and growth conditions
All strains and plasmids used in this study are listed in Table S1.
Neisseria strains were routinely grown at 37°C with 5% CO 2 in GCB agar or liquid GCB containing Kellogg's supplements (Kellogg, Peacock, Deacon, Brown, & Pirkle, 1963), unless otherwise indicated. Escherichia coli strains were grown at 37°C in LB media (Bertani, 1951). Antibiotics were added when needed at specified concentrations. Chloramphenicol was added at a final concentration of 5 μg/ml, and kanamycin at 50 μg/ml for Neisseria. For E. coli, chloramphenicol was used at 30 μg/ml and kanamycin at 50 μg/ml.

| Construction of mutants
The nps and npa ORFs overlap by 11 bases, to ensure deletion of nps would not affect npa, we designed a construct in which 92% of nps was deleted. First, a kanamycin resistance gene from plasmid pHSS6 (Seifert, Chen, So, & Heffron, 1986) was amplified with primers MR327A and MR328A (Table S2). The purified PCR product was transformed into Nel 29315. Transformants were selected in GCB containing 50 mg/ml of kanamycin. Ten kanamycin-resistant colonies were sequenced to confirm nps replacement. One kanamycinresistant clone was selected and transformed with a 321 bp piece of DNA of which 117 bases were from nps. The transformation mix was serially diluted, and plated onto GCB agar. After 24 hr, individual colonies were selected and grown on GCB without antibiotic, and a replica in GCB-Km. Km-sensitive clones were selected and analyzed, first by PCR, and then by sequencing to confirm that the kanamycin resistance gene was removed ( Figure S7).

| Site directed mutagenesis
A kanamycin resistance gene was cloned between the HincII and HindIII sites in pUC19, to generate plasmid pUC19-Km. Primers MR313 and MR314 were used to amplify the nps-npa locus and the amplicon was inserted between the EcoRI and BamHI sites of pUC19-Km. The site directed mutagenesis protocol in (Heckman & Pease, 2007) was used to generate mutants Nel nps H325A, Nel npa D58A, Nel npaD58E, Nel Δnps-npa D58E, and Nel nps H325A-npa D58E. All the primers used are listed in Table S2.

| Complementation of mutant strains
Complemented strains were constructed by placing the wt gene of interest under control of the inducible lac promoter, and inserting the construct into the proB site in Nel. For this protocol, we constructed plasmid pML2 ( Figure S8) by modifying plasmid pKH37 (Kohler, Hamilton, Cloud-Hansen, & Dillard, 2007). Briefly, a 650 bp fragment of the 5′-end of proB gene, obtained by PCR using primers MR346 and MR347, was used to replace the aspC gene in the FspI and PciI sites generating plasmid pML1. The lctP gene was removed by digestion with SphI and KpnI. A 550 pb fragment of the 3′-end of proB, generated using primers MR348a and MR349, was cloned into the SphI and KpnI sites of pML1 creating plasmid pML2. The multiple cloning site, lacPO, lacIq, and cat remained intact.
Nps was amplified using primers MR350 and MR 351 and cloned into the PacI and SacI sites of pML2 generating plasmid pML2-inps.
Npa was amplified using primers MR216a and MR216b Nel and cloned into the PacI and XhoI sites of pML2 generating plasmid pML2-inpa. Nel mutants were transformed with the corresponding plasmid using the colony transformation protocol described in (Dillard, 2011). Transformants were selected in GCB with chloramphenicol (5 μg/ml). Complemented strains were verified by PCR, sequencing, and RT-PCR.

| RNA extraction and RT-PCR
Total Neisseria RNA was extracted using Trizol (Invitrogen) and treated with DNAse free (Ambion) to remove DNA, as recommended by the manufacturer. The integrity of the RNA was determined by agarose gel electrophoresis. cDNA was generated with 1,000 μg of RNA using M-MLV (Promega) per manufacturer's instructions.
Reactions without reverse transcriptase were used as negative controls. To ensure that equal amounts of RNA were used, a reaction to amplify 16S rRNA was performed. All primers used, and gene products are listed in Table S2.

| Polyclonal anti-PilE antibodies
Pili from Nel wt were purified from GCB agar plates as previously described (Rendón et al., 2013). Briefly, Nel was grown for 16 hr on GCB agar. Bacteria were collected in 2 ml of 0.3 M ethanolamine. Pili were sheared off by vortexing the bacteria for 1 min. Pili were precipitated by incubating the solution with 300 μl of saturated ammonium sulfate for 30 min at RT. Precipitated pili were collected by centrifugation (17,000 × g for 30 min). Polyclonal antibodies to this pilus preparation were raised by immunization of a rabbit with 4 weekly doses of purified pili (Alpha Diagnostic Intl. Inc). Nonspecific antibodies were removed from the immune sera by absorbing with intact Nel ΔpilE cells. Finally, specific anti-PilE antibodies were captured by affinity purification using purified Nel pilin. The antibodies were released by addition of 0.2 M glycine pH 2.5, following by incubation at RT for 5 min. The solution with antibodies was collected and neutralized with 1 M Tris-HCl pH 8. The specificity of the antibodies was tested by western blot ( Figure S9).

| Western blot
Equal amounts of bacteria (7 × 10 8 bacteria) were suspended in sample buffer. Ten microlitre of sample was added to each lane of a 15% acrylamide gel. To ensure a good separation, the gel was run at 90 volts for 2.5 hr. Proteins were transferred onto a nitrocellulose membrane using a Trans-Blot SD semidry transfer cell (Bio-Rad).
The membrane was dried and then blocked with 5% milk-TBST for 1 hr. Anti-PilE Nel antibodies were diluted 1:10,000 (in 5% milk-TBST) and incubated with the membrane for 1 hr. The membrane was washed 3× and incubated with secondary goat anti-rabbit antibodies (LICOR) for 1 hr. The western blot was visualized using an Odyssey Clx Infrared Imaging System (LICOR) instrument.

| Determination of transcription initiation site
Total RNA was purified as described above, followed by mRNA enrichment using the MICROBExpress bacterial mRNA enrichment kit (Ambion) per the manufacturer's instructions. A 5′ Rapid amplification of cDNA ends (RACE) reaction was used to determine the transcription initiation site (TIS) using the SMARTer ® RACE 5′/3′ kit (Takara-Clontech). Primers used to determine TIS and their targets are described in Table S2. The product obtained in each reaction was cloned into pCR2.1 (Life Technologies) and transformed into E. coli DH5. Colonies were selected for sequencing using universal primers M13 forward and M13 reverse.

| nps and npa form an operon
We reported that pilE transcription in commensal Neisseria requires RpoN and the activator Npa (Rendón et al., 2013). Several domains characteristic of TCRS response regulators are present in Npa.
Upstream of npa is ORF (NEIELOOT_00058, accession number: AJE19474.1) (Figure 1a), whose deduced amino acid sequence contains motifs typically found in SKs. We provisionally named this ORF Neisseria pilus sensor (Nps). Upstream of nps and npa is the DNA topoisomerase IV subunit A, parC (accession number: AJE19475.1); all three ORFs are arranged in the same orientation. This organization of the three genes is identical in the human commensals N. mucosa (Nmu) and N. sicca (Nsi), and in the animal commensals N. musculi (Nmus) and AP206, a Neisseria sp isolated from rhesus macaque (Weyand et al., 2013). In Neisseria weaverii (Nwe), Neisseria Neisseria flavescens (Nfl), Neisseria cinerea (Nci), Neisseria polysaccharea (Npo), and Neisseria lactamica (Nla), npa is followed by a fourth ORF, rsmJ, encoding a putative ribosomal RNA small subunit methyl transferase (locus tag NMA1806 in Nme). In pathogen Neisseria meningitidis (Nme), parC is followed by NMA1803, NMA1805, then rsmJ. NMA1803 is a homolog of Nps that lacks the last 76 bases of the ORF ( Figure S1). NMA1805 is a homolog of Npa that lacks the last 1260 bases of the ORF, the region that would have encoded the RpoN-and DNA-binding domains (Rendón et al., 2013). In pathogen Neisseria gonorrhoeae (Ngo), parC is followed by rsp, a fusion of npa and nps (Carrick et al., 2000;Rendón et al., 2013).
The organization of parC, nps, and npa, and the presence of promoter-like sequences upstream of parC suggest the three ORFs comprise an operon. We determined whether these genes are cotranscribed. Using primers specific for each gene (Table S2)

| Predicted domains of Nps
The closest Nps homologs are PilS of Kingella kingae (Kki) (47.5% identity and 75.5% similarity) and PilS of Pseudomonas aeruginosa (Pae) (28.8% identity and 64.3% similarity). In Kki and Pae, PilS is required for transcription of pilA, encoding the major subunit of their Tfp (Boyd, Koga, & Lory, 1994;Kehl-Fie, Porsch, Miller, & St Geme, 2009). The N-terminus of Nps is predicted to span the membrane, and the C terminus to be located in the cytoplasm (TMMOD server) (Kahsay et al., 2005). The N terminus is highly hydrophobic, and harbors the input domain that contains six transmembrane helices interspersed with short loops (residues 21-189) ( Figure S1). Residues 313-376 are predicted to function as a transmitter domain, as it contains two motifs, histidine (H) at position 325 and motif E/DxxN/T, that are commonly found in SKs (Figures 1c and S1) (Huynh, Noriega, & Stewart, 2010;Kilmury & Burrows, 2016). When the input domain of a SK detects a signal, the conserved H autophosphorylates, initiating a signaling cascade that activates transcription of the target gene. In Nps, H325 is predicted to participate in the phosphorylation cascade (Figures 1c and S1). These characteristics of Nps lead us to test the hypothesis that Nps is the SK that regulates pilE transcription by activating Npa.

| pilE transcription requires Nps
We created an in-frame deletion of nps in Nel 29315 (see Methods).
NelΔnps, failed to produce detectable amounts of pilE mRNA (Table 1 and  Left three lanes: parC, nps and npa transcripts. All transcripts were produced by RT-PCR using primers shown in (a). Bottom panel: Right three lanes: parC to npa, parC-nps, and nps-npa amplicons. Left three lanes: parC, nps and npa amplicons. All amplicons were produced by PCR using primers shown in (a). MW, molecular weight markers. (c) Alignment of the H phosphotransfer domain in Nps from Neisseria species. The phosphorylatable histidine is in bold type and boxed in dark gray. The canonical phosphatase motif (ExxN) in Pseudomonas aeruginosa PilS that is present in the Nps homolog is boxed in light gray. The dashed line separates Nps in animal and human Neisseria. (*) identical residues; (:) highly conserved residues. SAM, S-adenosyl methyl transferase; Nmus, N. musculi; AP206, Neisseria sp isolated from rhesus macaque; Nwe, N. weaveri; Nsh, N. shayeganii; Nwa, N. wadsworthii; Nel, N. elongata; Nmu, N. mucosa; Nsi, N. sicca; Nsu, N. subflava; Nfl, N. flavescens; Nci, N. cinerea; Npo, N. polysaccharea; Nla, N. lactamica; Nme, N. meningitidis; Ngo, N. gonorrhoeae

| Nps and Npa form a two-component regulatory system
Upon activation, the SK transfers its phosphoryl group from the conserved H to a conserved aspartate (D) on the RR (Zschiedrich, Keidel, & Szurmant, 2016). We tested the hypothesis that the conserved H325 in Nps and D58 in Npa participate in the phosphorylation cascade, by replacing Nps H325 and Npa D58 with alanine (A) (Figure 3a).
npsH325A is predicted not to autophosphorylate, and consequently unable to activate Npa and pilE transcription. As expected, pilE mRNA and PilE levels were undetectable in npsH325A and in npaD58A (Figure 3b,c). These mutants formed colonies with a non-piliated morphology and did not form microcolonies (Table 1 and Figure S2).
Complementation of nps or npa with the cognate IPTG-inducible wt gene (Nel npsH325A+inps and npaD58A+inpa, respectively) restored pilE transcription, PilE production and microcolony formation (Table 1, Figures 3b,c and S2). Since these strains carry the mutated copy of nps or npa in addition to the inducible copy, the amount of IPTG needed to restore pilE levels was relatively high (5 μM for Nel npsH325 + inps and 10 μM for Nel npaD58A+inpa). We confirmed by RT-PCR that levels of nps and npa transcripts were not affected by the point mutations in Nel npsH325A and Nel npaD58A (Figure 3b). , and pilE or nps mRNA from the lysates was measured by RT-PCR in the presence (+RT) or absence (−RT) of reverse transcriptase, using nps-specific primers (see Table S2). The 16S rRNA reaction was used as a loading control and control for mRNA stability. Nel ΔpilE was included as negative control. Lane labeled "C" denotes control for Phosphorylation of the response regulator NtrC induces a conformational change (Hwang, Thorgeirsson, Lee, Kustu, & Shin, 1999).
To mimic such a change, the activatable aspartate (D) in NtrC was substituted with a glutamate (E) (Klose, Weiss, & Kustu, 1993;Lan & Igo, 1998;Moore, Shiau, & Reitzer, 1993). To confirm that D58 in Npa is the phosphorylated amino acid, we made an analogous mutation and created Nel npa D58E. Nel npaD58E produced pilE mRNA and PilE protein at levels similar to wt (Figure 3b,c) and produced piliated colonies on agar ( Figure S4). To further confirm Nel npaD58E behaves like a phosphorylated RR, we created double mutants Nel Δnps-npa D58E and Nel nps H325A-npa D58E. As expected, Nel Δnps-npa D58E and Nel nps H325A-npa D58E retained the ability to transcribe pilE (Table 1, Figure 3b,c). However, Nel nps H325A-npa D58E produced lower amounts of pilE mRNA and PilE than the wt parental strain (Figure 3c). The reduction in pilE expression in these double mutants was not caused by a defect in nps or npa expression, since these mutant genes were transcribed at the same level as their wt counterpart.
In sum, the data demonstrate that Nps and Npa work in concert as a TCRS to control pilE expression.

| Tfp genes other than pilE are not controlled by the RpoN/Nps-Npa regulatory system
In pathogenic Neisseria, assembly and function of the Tfp is a complex process that involves over 20 proteins (Carbonnelle et al., 2006).
Tfp biogenesis genes are present in all commensal Neisseria species (Marri et al., 2010), but how these commensal genes are regulated is unknown. We determined whether RpoN/Nps/Npa regulate expression of these genes in addition to pilE.
F I G U R E 3 pilE, nps, and npa transcript levels in Nel 29315 wt and nps and npa mutants. (a) Point mutations in the nps and npa genes are indicated by an asterisk. (b) Log phase cells were incubated with varying concentrations of IPTG for 4 hr, at 37°C. pilE, nps, or npa mRNA from the lysates was measured by RT-PCR in the presence (+RT) or absence (−RT) of reverse transcriptase, using gene-specific primers (see Table S2). The 16S rRNA reaction was used as a loading control and control for mRNA stability. Name of each mutant appears above each column. Primers targeting 16S rRNA were used as loading control and control of mRNA stability. IPTG concentration used to induce the genes for complementation appears above the columns. nps D58E

H325A D58E
The Tfp genes in Nel 29315 are clustered in ten regions (Figure 4a).
A fourth operon contains most of the minor pilin genes (pilH, pilI, pilJ, pilK, and pilX). Another minor pilin gene, pilV, is transcribed as a monocistronic unit (Figures 4a and S5d). Nel has only one copy of pilC, unlike Nme and Ngo (Marri et al., 2010), and its transcript is also monocistronic (Figure 4a). Like Ngo and Nme, Nel pilT and pilU are transcribed as one unit (Figures 4a and S5e). In Ngo and Nme, pilT2 is in a putative operon with pilZ (Brown, Helaine, Carbonnelle, & Pelicic, 2010), but in Nel, pilT2 is in a separate locus (Figure 4a). Finally, pilZ is flanked by ORFs 1 and 2 (NEIELOOT_01544 and NEIELOOT_01542) and the three genes form an operon (Figures 4a and S5f).
To determine whether RpoN and Nps-Npa control transcription of these nine gene clusters, we selected a gene from each operon and examined its expression in ΔrpoN, Δnps, and Δnpa mutants. RT-PCR was performed on mRNA from mid-log bacteria using specific primers (Table S2). 16s rRNA was use as the loading control and the control for mRNA integrity. A reaction on DNA was conducted as a control for primers specificity. In contrast to pilE, transcription of the tested genes was not altered in the ΔrpoN, Δnps, and Δnpa mutants when compared to the wt (Figure 4b). The amplicons were not a product of DNA contamination, as judged by lack of a product when RT was excluded from the reaction (data not shown). From these results, we conclude that RpoN and the TCRS Nps-Npa do not control transcription of these Tfp biogenesis genes other than pilE. Finally, we mapped the TIS of 4 Tfp loci by 5′ RACE (Rendón et al., 2013). In all cases, the TIS mapped to RpoD recognition sequences (Table S3). This confirms that a sigma factor of the RpoDfamily, and not RpoN, regulates expression of these loci.
In conclusion, of all the Tfp biogenesis and function genes described to date, only pilE is regulated by RpoN and the TCRS Nps-Npa.

| D ISCUSS I ON
The genes required for Tfp biogenesis and function are present in animal-and human-dwelling Neisseria species (Marri et al., 2010;Weyand et al., 2013Weyand et al., , 2016. Tfp gene expression has been studied mostly in the two pathogenic species. In the pathogens, RpoD regulates transcription of pilE, pilT/U, and pilC (Eriksson et al., 2015;Meyer et al., 1984;Taha, Giorgini, & Nassif, 1996). We previously reported that in commensal species of Neisseria, RpoN, IHF and the activator Npa regulate pilE transcription (Rendón et al., 2013).
Using Neisseria elongata as a model commensal, we showed that Nps and Npa act as the SK and response regulator, respectively, of a TCRS that regulates pilE transcription (Figures 2 and 3), and that Nps H325 and Npa D58 participate in the phosphorylation events.
nps H325A and npa D58A failed to transcribe pilE, while the complemented strains had this activity restored. A D58E substitution, which mimics an activated residue, resulted in constitutive expression of pilE. A similar mutation in NtrC altered the conformation of the response regulator, resulting in a constitutively active enzyme (Hwang et al., 1999;Klose et al., 1993;Lan & Igo, 1998;Moore et al., 1993). The npa D58E mutation overcomes the need of nps, as the double mutants Δnps+npa D58E and nps H325A+npaD58E are able to express pilE (Figure 4b). That the double mutant H325A+npaD58E produced significantly lower levels of pilE transcript than the wt is surprising. At the moment we cannot explain this observation. We know that this is not due to reduced expression of nps or npa since expression of these genes was not affected ( Figure 3b) and no mutation has occurred in the pilE locus. In the ΔpilE strain used as control, nps and npa transcripts appear to be slightly increased compared to the wt strain. It is unclear why this occurred. Further studies will determine whether this difference is due to a feedback mechanism in which the absence of pilE affects transcription of nps/npa.
Although the signal-sensing domains of some TCRS response regulators have been characterized (Hulko et al., 2006;Taylor & Zhulin, 1999;Xu & West, 1999), the signals detected by these regulators are unknown. In silico analysis indicates Nps lacks PAS (Per-Arnt-Sim), HAMP (domain present in histidine kinases, adenylate cyclases, methyl accepting proteins and phosphatases), and HPT (histidine containing phosphotransfer) motifs. Nps has six membrane-spanning regions interspersed with very short loops. Pae PilS, the Nps homolog, also has six such transmembrane domains.
Pae PilS is both a kinase and phosphatase; it senses the levels of membrane-bound pilin (PilA) and regulates pilA expression through its phosphatase activity (Boyd et al., 1994;Kilmury & Burrows, 2016). The PilS kinase and phosphatase motifs are highly conserved across all Neisseria Nps (Figures 1 and S1). The amino acids arginine 24 in PilS, and glutamate 5 and proline 22 in PilA are important for PilS/PilA interactions and PilS autoregulation (Kilmury & Burrows, 2016). These residues are also present in all Neisseria Nps and PilE ( Figures S1 and S6, respectively). This suggests Nps may, like PilS, sense and respond to membrane-bound PilE levels.
We attempted unsuccessfully to determine whether Nel Nps detects pH, oxygen, and growth phase (data not shown). Iron depletion affected pilE mRNA levels, but from the data we cannot definitely conclude that iron is the signal detected by Nps. Identifying the signal that Nps recognizes will allow us to understand how commensal Neisseria adapts to different environmental conditions.
Our model for pilE expression in commensal Neisseria is summarized in Figure 5. Upon detecting a signal (left), Nps autophosphorylates and transfers the phosphoryl group to Npa (this work).
Phosphorylated Npa binds the UAS, promoting RpoN activation of pilE transcription (Rendón et al., 2013). pilE transcription also requires IHF binding to its cognate sequence, which is situated between the RpoN and Npa recognition sites (Rendón et al., 2013). We hypothesize that this interaction of IHF with DNA bends the DNA, bringing Npa in close proximity to RpoN. In optimal conditions, when the Tfp components necessary for biogenesis are present, PilE would be assembled quickly into pili. If PilE accumulates in the membrane ( Figure 5, right panel), Nps would sense this, dephosphorylate Npa, and prevent it from activating RpoN. As a result, pilE transcription is abolished.
Our model implies that Tfp biogenesis genes other than pilE are constitutively expressed. Our findings indicate they are transcribed from a RpoD-dependent promoter (Figure 4), but they do not allow us to draw a conclusion about whether they are under more stringent regulation-for instance by an enhancer or a repressor. Future studies are needed to determine whether these genes are controlled by an intricate regulatory system(s).
Why have commensal and pathogenic Neisseria evolved different mechanisms to regulate pilE? This question might be explained by current information on Neisseria Tfp biogenesis. A large number of proteins situated in the inner membrane, periplasm and outer membrane are involved in the assembly and export of PilE and minor pilins (Brown et al., 2010;Carbonnelle et al., 2006;Winther-Larsen et al., 2005;Wolfgang, van Putten, Hayes, Dorward, & Koomey, 2000). The PilF ATPase incorporates PilE subunits into the Tfp fiber  and the PilT ATPase causes the Tfp fiber to retract, presumably by disassembling these subunits from the fiber base (Wolfgang, Lauer, et al., 1998). If the Tfp assembly/disassembly machinery malfunctions, PilE accumulates in the inner membrane and becomes toxic to the bacterium (Carbonnelle et al., 2006;Wolfgang et al., 2000). It is not unreasonable to speculate that the RpoN/Nps/Npa system serves not only to aid the bacterium to adapt to new niches but also to relieve the toxicity of membranebound PilE.

ACK N OWLED G M ENTS
This work was supported by National Institutes of Health grant R01AI107966 awarded to M.S. We thank A. Hockenberry, W. Kim, and K. Rhodes for helpful discussions and revision of this manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare not conflict of interest.

DATA ACCE SS I B I LIT Y
The authors adhere to sharing data and materials policies described in the guidelines for authors.


extfieldsofstudy: Chemistry Medicine

text:
Inhibitors of lysosomal function or serum starvation in control or LAMP2 deficient cells do not modify the cellular levels of Parkinson disease-associated DJ-1/PARK 7 protein

Mutations in PARK7/DJ-1 gene are associated with familial autosomal recessive Parkinson disease. Recently, lysosomes and chaperone mediated autophagy (CMA) has been reported to participate in the degradation of DJ-1/PARK7 protein. Lamp-2A isoform is considered as the lysosomal receptor for the uptake of proteins being degraded by the CMA pathway. We have used several cell lines with disrupted LAMP2 gene expression and their respective control cells to test the possible role of lysosomal degradation and in particular CMA in DJ-1 /PARK7 degradation. Interruption of LAMP-2 expression did not result in an increase of the steady-state protein levels of DJ-1 /PARK7, as it would have been expected. Furthermore, no change in DJ-1 /PARK7 protein levels were observed upon inhibition of lysosomal function with NH4Cl or NH4Cl plus leupeptin, or after activation of CMA by serum starvation for 24h. Accordingly, we have not found any evidence that DJ-1 /PARK7 protein levels are regulated via lysosomal degradation or the CMA pathway.

DJ-1 is a dimeric protein with a flavodoxin-like structure [3][4][5] [6,7] and ubiquitously expressed [8]. DJ-1 wild type protein is a rather stable protein since DJ-1 protein levels remain essentially unchanged after 24h of incubation of cells with cycloheximide [9] [10]. In addition, pulse-chase experiments did not reveal a clear decay in wild type DJ-1 protein levels within 24 hrs [11]. Surprisingly, Wang et al [12] recently reported that DJ-1 protein levels increase after treatment of SN4741 cells with NH 4 Cl or NH 4 Cl and leupeptin for 18h. Furthermore, they show that >80% of DJ-1 is degraded after prolonged (24 hrs) serum starvation, such treatment is known to activate the pathway of chaperone mediated autophagy (CMA). In CMA, proteins a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 with a conserved aminoacid motif selectively bind to the C-terminus of the integral lysosomal membrane protein Lamp-2A, one of the three isoforms generated from LAMP2 gene transcripts by alternative splicing. After binding to Lamp-2A, proteins are translocated to the lysosomal matrix for degradation [13]. The DJ-1 degradation observed under serum starvation conditions was prevented by addition of leupeptin and NH 4 Cl, and not by addition ofÁ3-methyl adenine  to the culture media of the cells. Those basic results, and other experiments, lead the authors [12] to conclude that DJ-1 protein is degraded by CMA pathway through binding to Lamp-2A [13]. Those results prompt us to replicate and validate the implication of the CMA pathway in degradation of wild type DJ-1. Using different cell lines which lack LAMP2 gene expression (and their respective controls) and after stimulation of CMA, we found no experimental evidence that CMA pathway is involved in the turnover of the DJ-1 wild type protein.

Ethical statement
Mice for isolation of mouse embryonic fibroblasts were handle following the ethical standards set by the National Animal Care Committee of Germany. Protocols were approved by the Ministerium für Energiewende, landwirtschaft und ländliche Räume, Schleswig-Holstein (V312-72241.121-3) to work by Dr. Paul Saftig group at the animal facilities of Institute of Biochemistry, Christian-Albrechts-Universität zu Kiel, Kiel, Germany. Human cell lines used in this study were described previously with identified source and approval for the B-cell line derived from a Danon patient [14].

Cell lines
Human B-lymphoblastoid cell lines (B-LCL) were grown in RPMI medium (Gibco BRL) supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin and 2 mM glutamine, as described [14]. HeLa, HEK, SN4741 cells (provided by Dr. José Luis Zugaza, Faculty of Science and Technology, University of the Basque Country, UPV/EHU, Bilbao, Spain) and the different control and Lamp-2 deficient cell lines were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL) supplemented with 10% foetal bovine serum (Sigma-Aldrich) and 100 μg/mL gentamycin. Mouse embryonic fibroblasts (MEF) were obtained from wild type (Wt) mice and MEF Lamp-2 -/y from Lamp-2 deficient mice [15] [16]. N2a cells transfected with shRNA scrambled sequence (shRNA scrmbl) and shRNA for Lamp-2 have been previously described [17] and were cultured in the presence of the selecting antibiotic G418 at 400 μg/mL. All cells were grown at 37˚C (except SN4741 that were cultured at 33˚C) and humidified 5% CO 2 . Both MEFs and N2a cell lines were provided by Drs. Judith Blanz and Paul Saftig from Institute of Biochemistry, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.

Studies of protein degradation
Exponentially growing cells were treated with 25 μg/ml of cycloheximide (CHX) for the times indicated. Cell viability by trypan blue exclusion was !95%. Cells were collected and processed for analysis by Western and immunoblot with anti-DJ-1 specific antibodies. To study the basal activity of the autophagic pathway, cells in complete medium (with serum) were untreated (controls) or treated with 20 mM NH 4 Cl or 20 mM NH 4 Cl in combination with 50 μM leupeptin (leup) for 24 h and processed for immunoblot analysis with anti-DJ-1, anti-LC3 or anti-IKappaBα antibodies. To study the effect of serum starvation on protein levels, exponentially growing cells were washed three times with HBSS with calcium and magnesium (Sigma, 55037) and incubated in serum free medium (serum starvation) for the times indicated. Cells were then cultured in the absence or in the presence of 20 mM NH 4 Cl, 20 mM NH 4 Cl and 50 μM Leup or 10 mM 3-MA. Afterwards cells were washed three times with cold PBS and lysed in SDS-Laemmli loading buffer without DTT and processed for Western and immunoblot analysis (see below).

Immunoblot analysis
After lysis, the samples were boiled for 5 min and loaded onto 10-14% SDS-PAGE (as required) and transfer to PVDF. Membranes were incubated with the corresponding primary antibodies (as indicated) and developed with anti-rabbit or anti-mouse peroxidaselabeled antibodies (1/5,000, Bio-Rad). Blots were imaged with a chemiluminiscent detector (MFChemiBIS 3.2, DNR Bio-Imaging Systems) and analyzed by quantitative densitometry using Totallab TL100 software. Protein levels were normalized respect to tubulin (protein loading control) and are expressed as mean ± s. e. m. from three different experiments.

RNA expression analysis
Total RNA from cells untreated or treated, as described above, was extracted by the using TRIzol reagent (Sigma). Isolated RNA was treated with DNase I (amplification grade from Invitrogen) to eliminate any remaining genomic DNA and inactivated. The RNA integrity was assessed by RNA chips using the Agilent 2100 Byoanalyzer. Afterwards, 1μg of total RNA was used for cDNA synthesis with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) using random primers. Quantitative real time PCR (qRT-PCR) was performed in a 7900 HT Fast Real-Time PCR System with Fast SYBR Green Master Mix (Applied Biosystems) using the suggested standard protocol. For the target mRNAs (mouse and human DJ-1 mRNA) the following oligonucleotides were used: forward human DJ-1, 5'-CCATAT-GATGTGGTGGTTCTAC-3'; reverse human DJ-1, 5'-ACTTCCACAACCTATTTCATGAG-3'; forward mouse DJ-1, 5'-ATCTGAGTCGCCTATGGTGAAG-3'; reverse mouse DJ-1, 5'-AC CTACTTCGTGAGCCAACAG -3'. The oligonucleotides used for normalization were: human β-actin: forward, 5'-AGCCTCGCCTTTGCCGA-3'; reverse, 5'-CTGGTGCCTGGGGCG-3' and mouse β-actin: forward, 5'-GACAGGATGCAGAAGGAGATTACTG-3'; reverse; 5'-GCTGATCCACATCTGCTGGAA-3'. Similar amplification efficiencies were obtained for target and reference mRNAs (100 ± 5%). Relative expression levels were analyzed by the ΔΔC T , as described [18]. Results are expressed as fold changes, taking the wild type cell (control) as a reference value of 1 (S2 Fig). The values presented are average from two independent experiments run in triplicates (technical replicas) with the corresponding range (minimum and maximum) indicated.

DJ-1 steady-state protein levels in control and LAMP2 interrupted cell lines
If Lamp-2A is implicated in the degradation of DJ-1 by the CMA pathway as reported [12], it would be expected that the interruption of LAMP2 gene expression would produce an increase in DJ-1 protein levels. To test the above prediction, DJ-1 protein levels were measured in mouse embryonic fibroblasts (MEF) derived from control and LAMP2 KO mice [16], as well as in control (scrambled shRNA) and LAMP2 -deficient (shRNA for LAMP2) N2a cells obtained by stable transfection of scrambled and LAMP-2 shRNA, respectively [17]. Lack of Lamp-2(A) expression in these cell lines has been validated by using Lamp-2 and Lamp-2A specific antibodies [17], Disruption of Lamp-2 expression does not affect Lamp-1 protein expression, another abundant integral membrane lysosomal protein (see S1 Fig). We also used B-lymphoblastoid cell lines (B-LCL) derived from a healthy control and Danon disease's patient [14], an X-linked human disease caused by mutations in LAMP2 gene. [19]. The data presented in Fig 1 show that no significant changes in DJ-1 protein levels were observed between controls and Lamp-2-deficient MEFs, N2a and B-LCL cells. Furthermore, we also found no significant changes in DJ-1 mRNA levels, as determined by qRT-PCR (see S2 Fig). These results reveal that DJ-1 steady state protein and mRNA levels are not significantly affected by lack of expression of Lamp-2.

Kinetic studies of DJ-1 stability after protein synthesis inhibition
The results shown above led us to test the stability of DJ-1 in the cell lines described above after protein synthesis inhibition with cycloheximide (CHX). In agreement with our previous results with N2a cells [10], treatment of these different cell lines with CHX up to 24h did not significantly change DJ-1 protein levels (Fig 2),

Effect of lysosomal inhibition on DJ-1 protein levels
Wang et al [12] reported that inhibition of lysosomal degradation by NH 4 Cl/leupeptin treatment (18hrs) leads to increased DJ-1 protein levels. This observation led the authors to conclude that DJ-1 is degraded in lysosomes. We aimed to reproduce those results in the same cell line (SN4741) used in their study, as well as, in other cell lines such as MEF obtained from wild type or Lamp-2-deficient mice, N2a cells either stably transfected with scrambled shRNA or LAMP-2-specific shRNA and in B-LCLs from a control or Danon disease's patient. As shown in Fig 3, our data showed that after 24h of treatment with NH 4 Cl or NH 4 Cl/leupeptin, there is no significant change in DJ-1 protein levels in any of the cell lines studied, including SN4741 cells. In contrast and as expected, NH 4 Cl and NH 4 Cl/leupeptin treatments increased the levels of LC3-II, as expected due to autophagic inhibition (Fig 3).

Effect of CMA activation on DJ-1 protein levels
Next we studied the effect on DJ-1 protein levels of activation of CMA pathway by serum starvation. Serum starvation for 24h did not produce any change in DJ-1 protein levels in MEF obtained from wild type or Lamp-2-deficient mice (Fig 4). Similar results were obtained in N2a cells either stably transfected with scrambled shRNA or LAMP-2-specific shRNA (Fig 5). Also no significant changes in DJ-1 protein levels were found in B-LCLs from a control or Danon disease's patient (Fig 6) [14], when starved for 8h. In the case of B-LCLs we could not apply 24h of serum starvation, since >60% cell died, as reported previously [14]. Finally, upon CMA activation we also did not observe any change of DJ-1 protein levels in SN4741 cells (Fig  7). The addition of either NH 4 Cl, NH 4 Cl/leupeptin or 3-MA during the starvation period did not modified DJ-1 protein levels in any of the cell lines studied (see . As expected, those co-treatments during starvation resulted in the accumulation of LC3-II (NH 4 Cl, NH 4 Cl/ leupeptin) and its inhibition in the presence of 3-MA (see also . In contrast, under the same experimental conditions IKappaBα, used as a control, was degraded (serum starvation) and its degradation significantly prevented by co-treatment with NH 4 Cl, NH 4 Cl/leupeptin (S5 Fig).

Discussion
The results presented here revealed that DJ-1 protein levels did not change in response to LAMP2 gene expression silencing (Fig 1) in different cell types (MEF, N2a and B-LCL). Also we showed that there was no difference in DJ-1 mRNA abundance between control and LAMP2 silenced cell lines (S2 Fig). As a consequence, the increased protein levels of DJ-1 reported by Wang et al [12] in SN4741 cells upon silencing LAMP2 gene expression can not be generalized to other cell types.
DJ-1 protein is a stable protein in the cell, here we have extended previous results to several cell lines, showing that treatment with CHX for 24h did not change the amount of DJ-1 protein in MEF from wild type or LAMP-2 knock-out mice, control N2a cells and interrupted with an shRNA for LAMP-2 or in B-LCL from control or from a patient with Danon disease.  [12].
Treatment of cells with NH 4 Cl or NH 4 Cl/leupeptin resulted in no change in DJ-1 protein levels either in control cells or in LAMP2 interrupted cells mentioned above (Fig 3) and also no change was observed in SN4741 ( Fig 3D). Furthermore, the results presented here show no change in DJ-1 protein levels by serum starvation for 24h, either in control or LAMP-2 interrupted cells (Figs 4-6) and in SN4741 (Fig 7). 4 Cl or 20 mM NH 4 Cl in combination with 50 μM leupeptin (leup) for 24 h. Total cell lysates were analysed by Western and immunoblot with the corresponding specific antibodies: DJ-1 and LC-3. Anti-tubulin antibodies were used as total protein loading control. Panels show the results for MEF (A), N2a (B), B-LCLs (C) and SN4741 (D). Graphs below each panel show the quantification of the levels of the proteins analysed respect to the levels in cells kept in complete growth medium, controls. Values are expressed as mean ± s.e.m. from three different experiments. Significant differences between groups ÃÃ at p<0.01 by Student t-test are indicated.

Fig 3. Protein expression levels of DJ-1 in control and Lamp-2-deficient cell lines in the presence of inhibitors of lysosomal function. Exponentially growing cells from control and Lamp-2-deficient cells were incubated in complete medium r suplemented with 20mM NH
https://doi.org/10.1371/journal.pone.0201152.g003 We have tried to find possible explanations for the discrepancy between our results and those of Wang et al [12]. We found differences in the buffer composition and procedure for cell lysis and protein extraction. In our hands, protein extraction using their buffer composition and procedure gave similar results to those presented here. Another difference is in the anti-DJ-1 antibody, they use Abcam, ab76008. We bought that antibody and in our hands gave similar results to Abcam ab18257 used in the present work, and we also obtained the same results using our in-house produced polyclonal anti-DJ-1 antibody [10]. As Wang al [12] only use SN4741, it could be that they are using a SN4741 cell line where DJ-1 protein for unknown reasons is more unstable.
Our results are in agreement with previous reported results on the stability of DJ-1 from typical biochemical studies [9] [11] [10]. Furthermore, quantitative proteomic studies using SILAC pulse-chase experiments and MS analysis also show that DJ-1 protein has a very long half-life (t1/2). For example; DJ-1 has a t1/2 = 187 h in mouse NIH3T3 cells [20]. The t1/2 values in HeLa and C2C12 cells are 59.2 h and 88h, respectively [21]. In vivo studies by deuterium labelling and MS also show that the t1/2 of DJ-1 in heart from mice varies from 199 to 299 h depending of the mouse strain under study [22]. Finally, recent proteomic studies by MS of the degradation of "young" (newly synthesized) and "old" (steady state) proteins in NIH3T3 report that DJ-1 degradation follows an exponential decay, exponential 1-state t1/2 = 150.26 h and steady-state 2-state model with a t1/2 = 217.1 h, similar exponential decay is observed in human RPE-1 cells with a t1/2 >300 h [23]. In all cases DJ-1 protein has a very long half-life, several days. These facts, together with the ubiquitous expression of DJ-1, are in agreement with a proposal to use DJ-1 peptides as reference for quantification of proteomic studies, being even better that commonly used peptides from house keeping proteins [24]. With a half-life of several days, even for the newly synthesized DJ-1 protein [23], the study of the pathway responsible for the degradation of DJ-1 is not easy to approach experimentally by applying treatments that can stimulate or inhibit its degradation. In particular, it could be that longer serum fasting times (> 24h) may be required to observe a significant change in DJ-1 protein levels. Unfortunately those experiments are not possible, because the cells (MEFs, N2a and In conclusion, DJ-1 protein has a long half-life in the cell and we have not found any change in DJ-1 protein levels by inactivation of LAMP2 gene expression, inhibition of lysosomal function or activation of the CMA pathway by serum starvation. Accordingly, we have been unable to reproduce in other cell lines, or even in SN4741 cells, the observations reported by Wang et al [12] on the role of CMA in DJ-1 protein turn-over in their SN4741 cells.  cells. Panel C shows the resutls obtained with SN4741 Total cell lysates were analysed by Western and immunoblot with the corresponding specific antibodies: as indicated. Anti-tubulin antibodies were used as total protein loading control. Graphs show the quantification of the levels of the different proteins analysed respect to the levels in cells kept in complete growth medium, controls. Values are expressed as mean ± s.e.m. from three different experiments. Significant differences between the indicated pairs analyzed by Student t-test are indicated by ÃÃ at p<0.01. and Ã p<0.05. (TIF)


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Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.

Introduction
Cellulose, the most abundant organic and renewable biomass, has been considered a good candidate for the production of alternative biofuels and chemicals. 1 Cellulose consists of i.e., glucose, requires the synergistic action of many cellulolytic enzymes known as cellulases. Cellulases refer to a multienzyme complex composed of endoglucanase (endo-1,4-␤-glucanase, EC 3.2.1.4), exoglucanase (exo-1,4-␤-glucanase, EC 3.2.1.91), and cellobiase (␤-glucosidase, EC 3.2.1.21). 6,7 The enzymes are widely found in many organisms, such as actinomycetes, bacteria, fungi and plants. Among the three major groups of cellulases, endoglucanase is considered to be the most important enzyme because its action results in a nonreducing end that is necessary for the action of exoglucanase and cellobiase. 8 There are several sources of endoglucanase such as bacteria, fungi and plant, 7-10 but fungal endoglucanase is the major source of enzyme for industrial applications. Although several endoglucanases have been cloned, identified and characterized, most efforts have focused on the enzymes from the filamentous fungi, such as Aspergillus usamii, 7 A. kawachii, 12 Trichoderma reesei, 13 A. niger, 14 Volvariella volvacea, 15,16 Penicillium canescens, 11 P. funiculosum 17 and Verticillium dahlia. 18 Nierman et al. 19 reported that there are approximately eighteen different genes that encode endoglucanases in the entire sequence of the A. fumigatus Af293 genome. Most recently, the endoglucanase gene belonging to the family AA9 (formerly known as GH61) from the alkali-tolerant A. fumigatus MKU1, which is isolated from paper and pulp industry wastes, has also been cloned and sequenced. 3 Das et al. 20 reported the kinetic properties of the purified halostable endoglucanase from a culture extract of A. fumigatus ABK9, and the authors demonstrated that the calculated molecular weight of the enzyme was approximately 56.3 kDa. The enzyme displayed its maximum activity at a temperature of 50 • C and a pH value of 5.0. This enzyme is highly stable in a pH range from 4.0 to 7.0 and at NaCl concentrations up to 3.0 M. Xu et al. 21 reported the expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi and the authors demonstrated that the engineered L. starkeyi was capable of expressing and secreting both enzymes with high activity toward cellulose.
Currently, Kluyveromyces lactis, an ascomycetous budding yeast belonging to the endoascomycetales, is considered as one of the most favored systems for the expression of several heterologous proteins because it has several advantages including protein folding, protein processing and posttranslational modification. It has been reported that K. lactis produces and secretes heterologous proteins at a higher level than Saccharomyces cerevisiae. It also secretes few native proteins and a secreted recombinant product is therefore easily recovered and purified from the culture medium. 22,23 Most importantly, K. lactis can be grown in inexpensive media. Furthermore, it has been asserted as a "Generally Recognized As Safe" microorganism by the US FDA and can thus be applied in several industries such as food, beverage, flavor, vitamin and feed production. 24 In the current study, the cloning and molecular characterization of the gene encoding endoglucanase from the thermotolerant fungus A. fumigatus DBiNU-1 were investigated. In addition, the heterologous expression of the A. fumigatus endoglucanase gene in yeast K. lactis GG799 and the enzymatic properties of the gene product were also described.
We demonstrated in the current study that the endoglucanase from A. fumigatus DBiNU-1 is a thermostable enzyme.

Strains, plasmid vectors and culture conditions
The thermotolerant fungus A. fumigatus DBiNU-1 used in this work was obtained from Department of Biology, Faculty of Science, Naresuan University. It was isolated from a soil sample collected in the northern part of Thailand, and was identified by morphological analysis and DNA sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) of rDNA region. It was used as a source for genomic DNA isolation. The fungus was maintained on potato dextrose agar (PDA) and stored at 4 • C. K. lactis GG799 (New England Biolabs, US) was used as a host system for protein expression, while Escherichia coli DH5␣ was used as a host system for gene cloning. The pGEM-T Easy (Promega, Madison, WI, USA) and pKLAC2 vectors were used for gene cloning in E. coli and for gene expression in K. lactis, respectively.

Genomic DNA extraction
A. fumigatus DBiNU-1 was cultured in YPD medium on a rotary incubator shaker (220 rpm) at 30 • C for 24 h. The fungal mycelia were harvested by filtration and washed twice with sterile distilled water. Genomic DNA was isolated from the fungal mycelia using the GF-1 nucleic acid extraction kit (Vivantis, USA), and the purified genomic DNA was used as a DNA template for gene amplification using the polymerase chain reaction (PCR) technique.

PCR amplification, cloning and nucleotide sequencing of the endoglucanase gene from A. fumigatus DBiNU-1
Based on the alignment of the multiple amino acid sequences of the endoglucanases from A. fumigatus MKU1 (AFJ54162), A. niger ATCC10574 (ADC84000) and A. oryzae KBN616 (BAA22588), the conserved regions at the N-terminus (GALYLSEM) and Cterminus (IRWGDIG) were found. The design of the degenerate primers Cel7asper 1F (5 -GGNGCNYTNWSNGARATG-3 ) and Cel7asper 1R (5 -NCCDATRTCNCCCCANCKDAT-3 ) were based on these two regions, and the primers were used to amplify a partial sequence of the endoglucanase gene from A. fumigatus DBiNU-1. Since the partial sequence of the endoglucanase gene from A. fumigatus DBiNU-1 was highly homologous to that from A. fumigatus MKU1 (JQ796069), the amplification of the full length open reading frame (ORF) of the endoglucanase gene from A. fumigatus DBiNU-1 was performed using specific primers Cel7full-F (5 -ATGGACTCCAAAAGAGGCGTCGT-3 ) and Cel7full-R (5 -CTACAGACACTGAGAGTACCACGGA-3 ), which were synthesized based on the nucleotide sequences of the endoglucanase gene from A. fumigatus MKU1. The PCR reaction mixture (25 l) consisted of 2× KOD Xtreme TM PCR buffer, 2 mM dNTPs, 10 pmol/l each forward and reverse primer, 1.0 U/l KOD Xtreme TM Hot Start DNA polymerase, and 100 ng/l template DNA, as per the manufacturer's instructions (Merck, Germany). The template DNA was first denatured at 95 • C for 3 min. Thirty cycles of the amplification reaction were then performed using of the following conditions: denaturation for 1 min at 94 • C, annealing for 1 min at 50 • C and extension for 2 min at 72 • C. A final extension was performed at 72 • C for 7 min. The PCR product was visualized on 0.7% agarose gel, extracted and purified from the gel using the NucleoSpin Extract II Kit (Macherey-Nagel, Germany). The Rapid DNA Ligation and Transformation Kit (Fermentas, USA) was used to clone the purified PCR product into the pGEM T-easy vector and to transform the resulting plasmid into E. coli DH5␣. Transformants were screened at 37 • C on LB medium containing 100 g/ml ampicillin. The recombinant plasmid DNA was isolated from the selected transformants and subjected to DNA sequencing by the Sanger method using the MegaBACE 1000 automated DNA sequencer (Pharmacia Biotech, Sweden). The nucleotide and the deduced amino acid sequences of the endoglucanase gene product from A. fumigatus DBiNU-1 were analyzed using GENETYX (Software Development, Tokyo, Japan), while a homology analysis was carried out using the FASTA and BLAST program in the Gen-Bank database.

Construction of the expression vector and transformation of K. lactis
The ORF of the endoglucanase gene from A. fumigatus DBiNU-1 was amplified by PCR using the following primer pairs: CEL7PKLAC F: 5 CGCTCGAGAAAAGAATGG ACTCCAAAAGAGGC-3 (the underline bases represent the XhoI restriction site) and CEL7PKLAC R: 5 -CCGGAATTCCTACAGACACTGAGAGTACCA-3 (the underline bases represent the EcoRI restriction site). All PCR primers were synthesized by Integrated DNA technology (IDT, Singapore). The PCR reaction mixture (25 l) consisted of 2× KOD Xtreme TM PCR buffer, 2 mM dNTPs, 10 pmol/l each forward and reverse primer, 1.0 U/l KOD Xtreme TM Hot Start DNA polymerase and 20 ng/l template DNA, as per the manufacturer's instruction (Merck, Germany). The thermocycling conditions consisted of a 94 • C step for 2 min, followed by 35 cycles of successive incubation at 94 • C for 30 s, 60 • C for 1 min and 72 • C for 2 min. After the thermocycling, a final extension was performed at 72 • C for 7 min. To construct the expression cassette pKLAC2-Cel7, the purified amplified fragment was digested with XhoI and EcoRI and then ligated into the XhoI/EcoRI site of the K. lactis expression vector pKLAC2. The accuracy of the nucleotide sequence of the cloned endoglucanase gene was confirmed by DNA sequencing as described above.
The transformation of the expression cassette into K. lactis GG799 was performed using lithium acetate with the heat shock method. 25 To integrate the cloned endoglucanase gene into the LAC4 locus of the K. lactis chromosome, the expression cassette pKLAC2-Cel7 was linearized with BstXI before transformation. The transformants harboring the pKLAC2-Cel7 plasmid were screened and selected at 30 • C using yeast carbon base (YCB) agar medium supplemented with 5 mM acetamide. The selected transformants were further analyzed on YPD agar medium containing 1% carboxymethyl cellulose (CMC) (Fluka, Germany).

Detection of the recombinant endoglucanase using 1% Congo red
The selected transformants were grown in YPGal medium at 30 • C in an incubator shaker (200 rpm) for 24 h. Thereafter, 50 l of the yeast culture was transferred onto the YPD agar medium that contained 1% CMC and incubated at 30 • C. Sterile distilled water was used as a control in this study. After 7 days of incubation, the agar medium was flooded with 1% Congo red and subsequently incubated at room temperature (RT) for 15 min. The agar medium was rinsed twice with sterile distilled water and then washed with a 1 M NaCl solution for 15 min. The clear zone that appeared on the agar medium was monitored and photographed.

SDS-polyacrylamide gel electrophoresis and zymogram analysis of the recombinant endoglucanase
The selected transformant was grown in YPD liquid medium supplemented with 1% CMC at 30 • C in an incubator shaker (200 rpm) for 7 days. The culture supernatant was collected by centrifugation at 13,000 rpm and 4 • C for 15 min, and the endoglucanase activity in the clear supernatant (crude enzyme) was assayed. The protein concentration in the culture supernatant was measured by the Bradford method 26 using bovine serum albumin (BSA) as the standard. For SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the crude enzyme was prepared based on the method described by Eriksson and Pettersson 27 and Béguin, 28 using a sample buffer containing 2% SDS, 0.5 M Tris-HCl (pH 6.8), 10% glycerol and 0.01% bromophenol blue. The SDS-PAGE was carried out based on the standard method described by Li, 29 using 30 g of protein sample and a 12% polyacrylamide gel, and the separation of the protein bands in the gel was visualized by Coomassie brilliant blue R-250 (Sigma) staining.
The zymogram analysis was based on the method described by Grigorevski-Lima et al., 30 and was carried out to determine the enzyme activity. The crude enzyme was subjected to SDS-PAGE using a 12% polyacrylamide gel that contained 1% CMC as substrate. After electrophoresis, the gel was soaked in 2.5% (v/v) Triton X-100 for 2 h and then incubated in 50 mM sodium acetate buffer (pH 5.0) for 1 h at RT in order to remove the SDS and renature the enzyme. The resulting gel was washed with 50 mM sodium acetate buffer (pH 5.0) at 45 • C for 30 min, stained with 1% (w/v) Congo red dye solution at RT for 30 min, and subsequently destained with 1 N NaCl until the excess dye was removed. The clear zone, which corresponded to enzyme activity, was monitored and documented with a gel documentation system (Bio-Rad, Singapore).

Purification of protein using an aqueous two-phase system
The recombinant endoglucanase was purified using an aqueous two-phase system. 31 Various concentrations of polyethylene glycol 6000 (PEG 6000) and dextran T70 were used in this system. To prepare the two-phase system, a stock solution composed of 20% (w/w) dextran and 40% (w/w) PEG and the enzyme solution were mixed and left to settle for at least 1.5 h. Protein samples in the top and bottom phases were taken, and the enzyme activity in both phases was further determined.
The activity of the purified recombinant endoglucanase was assayed using the method described by Li et al., 16 using CMC as substrate. One unit (U) of enzyme activity was defined as the amount of enzyme capable of releasing 1 mol of reducing sugar from CMC per minute under the assay conditions. d-Glucose was used as the standard.

Effect of pH on enzyme activity and stability
The effect of pH on the activity of the purified recombinant endoglucanase was evaluated using the standard method described by Li et al., 16 with different buffers including sodium acetate buffer (pH 3.0-6.0) and phosphate buffer (pH 7.0-11.0). The stability of the purified endoglucanase at different pH values was evaluated by incubating the purified enzyme at 50 • C for 1 h in the same buffers in the pH range from 3.0 to 11.0 and measuring the remaining enzyme activity under standard conditions.

Effect of temperature on enzyme activity and stability
The effect of temperature on the activity of the purified recombinant endoglucanase was evaluated at the optimum pH at temperatures ranging from 30 to 80 • C. The thermal stability of the purified enzyme was evaluated by preincubating the enzyme solution in 50 mM sodium acetate buffer (pH 5.0) at various temperatures (30-80 • C) for 1 h. The remaining enzyme activity was assayed under standard conditions.

Cloning and nucleotide sequencing analysis of the endoglucanase gene from A. fumigatus DBiNU-1
The ORF of the gene encoding the endoglucanase in A. fumigatus DBiNU-1 was amplified by PCR using a genomic DNA template that was isolated from A. fumigatus DBiNU-1 and specific primers whose design was based on the nucleotide sequences of the endoglucanase gene from A. fumigatus MKU1, as described in the Materials and methods section. A PCR product of approximately 1.3 kb was detected after agarose gel electrophoresis analysis, and it was purified and cloned, and the resulting vector was transformed into E. coli DH5␣. The recombinant plasmid DNA that was isolated from the selected transformants was then subjected to DNA sequencing. As found in the present study, the complete nucleotide sequence of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1,383 nucleotides in length and encoded a polypeptide of 460 amino acid residues (Fig. 1). The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were found to be 48.19 kDa and 5.03, respectively. Based on the sequence similarities in the carbohydrateactive enzyme (CAZy) database (http://www.cazy.org/), the deduced amino acid sequence of the endoglucanase gene product from A. fumigatus DBiNU-1 was classified into glycoside hydrolase family 7 (GH7). [32][33][34] These comprise two domains, i.e., a catalytic domain at the N-terminus, which contains the glycosyl hydrolase family 7 active-site residues (amino acids at positions 22-398), and a fungal type cellulosebinding domain/module (CBD/CBM) at the C-terminus (amino acids at positions 428-456), which is involved in substrate binding, as observed in most fungal cellulases. 35 The prediction of the amino acid sequences by the SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/) revealed the signal peptide (MDSKRGVVAAVLALLPLVSA) at the N-terminal region and a signal peptide cleavage site between Ala 20 and Gln 21 .
The homology analysis of the deduced amino acid sequence showed that the endoglucanase from A. fumigatus DBiNU-1 shared 98%, 74%, 59%, 51%, 46% and 34% identity with the GH7 endoglucanases from A. fumigatus KMU1 (AFJ54162), A. terreus MS-31 (ADR78837), T. reesei M5 (ADM08177), A. oryzae KBN616 (BAA22589), A. nidulans FGSCA4 (EAA63386), and Fusarium oxysporum (AAA65586), respectively (Fig. 2). The putative catalytic amino acid residues Asp 191 and Glu 369 were found to be highly conserved among the GH7 endoglucanases and are similar to those reported in the GH12 and AA9 family. 3,7 A three-dimensional structure of the predicted protein sequence of the endoglucanase from A. fumigatus DBiNU-1 was predicted using the Swiss-PdbViewer (http://www/expasy.org/spdbv/), and the result is shown in Additional information section, Fig. 1. The protein structure was observed at the tertiary level, and two completely distinct structures corresponding to the catalytic domain and CBM1 domain were detected.

Expression of the endoglucanase gene from A. fumigatus DBiNU-1 in K. lactis GG799
The expression vector pKLAC2-Cel7 was constructed and transformed into K. lactis GG799, and the transformants were selected using YCB agar medium, as described in the Materials and methods section. The selected transformants were further analyzed on YPD agar medium containing 1% CMC. Transformants harboring the pKLAC2-Cel7 exhibited a clear zone on agar plates after Congo red staining, suggesting that the cloned endoglucanase gene from A. fumigatus DBiNU-1 was expressed in the K. lactis (Fig. 3).
The crude enzyme in the cell-free supernatant was analyzed by SDS-PAGE using a 12% polyacrylamide gel, and the resolved proteins were visualized using Coomassie brilliant blue R-250 staining. As shown in Fig. 4A, a protein band of approximately 48 kDa that corresponded to the predicted molecular weight of the endoglucanase from A. fumigatus DBiNU-1 was detected. The endoglucanase activity was also determined based on the degradation of CMC in the polyacrylamide gel using a zymogram analysis. A protein band of approximately 48 kDa that coincided with the band that was observed in the SDS-PAGE analysis was detected on a polyacrylamide gel after Congo red staining (Fig. 4A), suggesting that this protein band was the recombinant endoglucanase from A. fumigatus DBiNU-1. The endoglucanase activity in the cell-free supernatant from K. lactis harboring the pKLAC2-Cel7 plasmid was measured, and the highest enzyme activity was found to be 0.80 U/ml with a specific activity of 3.08 U/mg protein. There was no endoglucanase activity in the cell-free supernatant from the negative control (K. lactis transformed with pKLAC2) under the same cultural conditions.

Purification and characterization of the purified recombinant endoglucanase
The recombinant endoglucanase from the K. lactis strain that harbored pKLAC2-Cel7 was purified using the aqueous twophase system, as described in the Materials and methods section. A maximum enzyme activity and specific activity of 1.57 U/ml and 15.39 U/mg protein, respectively, were achieved using the two-phase system that contained 7% PEG and 5% dextran. Fig. 4B shows the SDS-PAGE analysis of the purified recombinant endoglucanase that was obtained from aqueous two-phase system. Most of the protein band at approximately 48 kDa, which corresponded to the monomer of the Cel7 gene product from A. fumigatus DBiNU-1, was observed, while those low-molecular weight proteins that were contained in the cellfree supernatant were mostly removed.
The effect of pH on the activity and stability of the purified recombinant endoglucanase was evaluated, and the results are summarized in Fig. 5A. The purified recombinant endoglucanase displayed its highest activity at pH 5.0, and more than 80% of its original activity remained in the pH range from 4.0 to 8.0.
The effect of temperature on the recombinant endoglucanase activity and stability is illustrated in Fig. 5B. The maximal activity of the purified recombinant endoglucanase at pH 5.0 was attained at a temperature of 60 • C. The activity of the purified enzyme remarkably decreased at temperatures above 60 • C. More than 80% of the original activity of the purified enzyme remained in a temperature range from 30 to 60 • C, whereas only half of the original activity was detected at 70 • C.

Discussion
Based on the whole genome sequence of A. fumigatus Af293, 18 different genes encoding endoglucanases have been reported. 19 They belong to various families, such as GH5, GH7, GH12, GH45, AA9 (formerly known as GH61), and GH81, based on their amino acid sequence similarities. According to a nucleotide sequence analysis, three endoglucanase genes from A. fumigatus are intronless (one from GH7 and two from AA9), and two out of these three known intronless endoglucanase genes are predicted to have a cellulose-binding domain/module in the C-terminal region. In the current study, a gene that encodes an intronless endoglucanase in A. fumigatus DBiNU-1 was cloned, and its nucleotide and putative amino acid sequences were analyzed using GENETYX (Software Development, Tokyo, Japan). The results showed that the complete sequence of the gene that encodes the endoglucanase in A. fumigatus DBiNU-1, designated Cel7, was 1383 bp in length and encoded a polypeptide of 460 amino acid residues (Fig. 1) 36 the CBD/CBM is important for the binding of cellulose. Cellulases that lack the CBD/CBM exhibited a reduction in cellulose degradation. It should be noted from the current study that a signal peptide containing 20 amino acid residues was detected in the N-terminal region, suggesting that the endoglucanase from A. fumigatus DBiNU-1 is an extracellular enzyme because the same feature has been found in an intronless endoglucanase from A. fumigatus MKU1. 3 In addition to a signal peptide, two regions, GALYLSEM and IRWGDIG, that are highly conserved in all fungal endoglucanases in the GH7 family were detected in the endoglucanase from A. fumigatus DBiNU-1. This finding also verified that the A. fumigatus Cel7 gene product was a member of the fungal GH7 family. No putative N-glycosylation site (N-W-T) was found in the predicted protein sequences, suggesting that the endoglucanase from A. fumigatus DBiNU-1 is not an N-glycosylated protein, which is distinct from the reported case of the endoglucanase from A. usamii E001. 7 The predicted molecular weight of the endoglucanase from A. fumigatus DBiNU-1, as confirmed by SDS-PAGE analysis, was approximately 48 kDa, which was distinct from other endoglucanases from other Aspergillus strains, e.g., A. terreus M11 (25 kDa), 37 A. awamori VTCC-F099 (32 kDa), 38 A. fumigatus KMU1 (35 kDa), 3 A. oryzae AG1 (45 kDa), 39 A. usamii E001 (24 kDa), 7   glycoside hydrolases such as GH7, GH12, GH16 and AA9 have a modular structure that consists of a catalytic domain and a substrate-binding domain. 40 The Swiss-PdbViewer (http://www/expasy.org/spdbv) was used to predict the threedimensional structure of the putative amino acid sequence of the endoglucanase from A. fumigatus DBiNU-1 (Additional information section, Fig. 1). The GH7 endoglucanase from this thermotolerant fungus exhibited an arrangement of catalytic residues and a fold that are similar to those of the ␤-1,3glucanases and ␤-1,3-1,4-glucanases of family 16. 41 The aqueous two-phase system has been widely used to purify several enzymes. Compared with conventional purification methods, this technique provides a high yield of recovery with high level of enzyme activity and stability. 31 In the current study, the recombinant endoglucanase from A. fumigatus DBiNU-1 that was expressed in K. lactis with the pKLAC2-Cel7 vector was purified using the aqueous two-phase system. A maximum enzyme activity and specific activity of 1.57 U/ml and 15.39 U/mg protein, respectively, were achieved; these values were approximately 2-and 5-fold greater than the corresponding original activities. The optimum pH of the activity of the purified recombinant endoglucanase from A. fumigatus DBiNU-1 was 5.0, and the enzyme was highly stable in a pH range from 4.0 to 8.0 (Fig. 5A). The purified recombinant endoglucanase also exhibited its highest activity at 60 • C, and it over 80% of its original activity in a temperature range from 30 to 60 • C (Fig. 5B). The results of the current study were in good agreement with those reported for most of the endoglucanase from Aspergillus strains, such as A. terreus DSM 826, 42 A. awamori VTCC-F099, 38 A. usamii E001 7 and A. fumigatus ABK9. 20 However, a high thermal stability (70 • C) was also reported for endoglucanases from T. aurantiacus 43 and A. fumigatus MKU1. 3 The differences in thermal stability indicated the different molecular properties of the enzymes, including the bonding that stabilizes in the structures and the conformations among the various species. It has been reported that endoglucanases with optimal activities between 55 and 80 • C are considered thermostable enzymes. 44 The high thermal stability of the endoglucanase from A. fumigatus DBiNU-1 (60 • C) make this enzyme a good candidate for application in the paper industry and in saccharification processes for the production of biofuel and other chemicals using cellulosic materials.

Conflicts of interest
The authors declare that they have no conflict of interest.


s2fieldsofstudy: Biology Medicine Chemistry

text:
MLN4924, a Protein Neddylation Inhibitor, Suppresses the Growth of Human Chondrosarcoma through Inhibiting Cell Proliferation and Inducing Endoplasmic Reticulum Stress-Related Apoptosis.

Chondrosarcoma, a heterogeneous malignant bone tumor, commonly produces cartilage matrix, which generally has no response to conventional therapies. Studies have reported that MLN4924, a NEDD8-activating enzyme inhibitor, achieves antitumor effects against numerous malignancies. In this study, the suppressive effects of MLN4924 on human chondrosarcoma cell lines were investigated using in vitro and in vivo assays, which involved measuring cell viability, cytotoxicity, apoptosis, proliferation, cell cycles, molecule-associated cell cycles, apoptosis, endoplasmic reticulum (ER) stress, and tumor growth in a xenograft mouse model. Our results demonstrated that MLN4924 significantly suppressed cell viability, exhibited cytotoxicity, and stimulated apoptosis through the activation of caspase-3 and caspase-7 in chondrosarcoma cell lines. Furthermore, MLN4924 significantly inhibited cell proliferation by diminishing the phosphorylation of histone H3 to cause G2/M cell cycle arrest. In addition, MLN4924 activated ER stress⁻related apoptosis by upregulating the phosphorylation of c-Jun N-terminal kinase (JNK), enhancing the expression of GRP78 and CCAAT-enhancer-binding protein homologous protein (CHOP, an inducer of endoplasmic ER stress⁻related apoptosis) and activating the cleavage of caspase-4. Moreover, MLN4924 considerably inhibited the growth of chondrosarcoma tumors in a xenograft mouse model. Finally, MLN4924-mediated antichondrosarcoma properties can be accompanied by the stimulation of ER stress⁻related apoptosis, implying that targeting neddylation by MLN4924 is a novel therapeutic strategy for treating chondrosarcoma.

autophagy, and abnormal cellular responses [26]. Nevertheless, the mechanism through which MLN4924 inhibits human chondrosarcoma remains unclear.
In this study, we conducted in vitro and in vivo experiments to explore the mechanism and therapeutic potential of MLN4924 for treating human chondrosarcoma.

MLN4924 Reduces Cell Viability And Causes Cytotoxicity in Human Chondrosarcoma Cells
Because an effective treatment for chondrosarcoma is necessary and MLN4924 has exhibited promise for treating various cancers [27,28], the expressing significance of neddylation pathway and effects of MLN4924 on the viability and cytotoxicity in human chondrosarcoma cell lines were first investigated. The chondrosarcoma cell lines jj012 and sw-1353 exhibited higher NAE-1 expression than the normal chondrocyte cell line, C28/I2 (Figure 1a). Furthermore, MLN4924 significantly reduced the cell viability of both jj012 and sw-1353 but not C28/I2 in a dose-dependent manner (Figure 1b), indicating that the upregulated neddylation pathway is a therapeutic target of human chondrosarcoma. Based on the results of Figure 1a,b, the suppressing effects of MLN4924 were only focused on chondrosarcoma cell lines jj012 and sw-1353. The result further showed that MLN4924 significantly reduced the cell viability of both jj012 and sw-1353 in a time-dependent manner ( Figure 1c). A lactate dehydrogenase (LDH) assay was used to examine the effect of MLN4924 on the cellular cytotoxicity and cytolysis of chondrosarcoma cell lines. The results also demonstrated that MLN4924 caused significant cytotoxicity in both jj012 and sw-1353 cells (Figure 1d). Generally, jj012 cells were more sensitive to neddylation inhibition than sw-1353 cells in terms of repressed cell viability and induced cytotoxicity. These results indicated that the inhibition of neddylation by the NAE inhibitor MLN4924 holds therapeutic potential for treating human chondrosarcoma.    The jj012, sw-1353, and C28/I2 cells were treated with various concentrations of  MLN4924 for 24 h. A WST-1 assay was performed to assess cell viability. (c) The jj012 and sw-1353 cells  were treated with various concentrations of MLN4924 for 24 and 48 h in a WST-1 assay. (d) The jj012  and sw-1353 cells were treated with dimethyl sulfoxide ([DMSO], as a control treatment) or MLN4924 (500 and 750 nM) for 48 h, and cytotoxicity was determined through an LDH assay. * p < 0.05.

MLN4924 Suppresses Cell Proliferation by Hindering G2/M Cell Cycle Progression
We examined the effect of MLN4924 exposure on the proliferation and cell cycle progression of chondrosarcoma cell lines. Figure 2a illustrates that MLN4924 (750 nM) significantly reduced cell proliferation by decreasing BrdU incorporation in jj012 and sw-1353 cells. Moreover, Figure 2b indicates that MLN4924 caused G2/M phase arrest. Figure 2c further illustrates that MLN4924 reduced the phosphorylation of the mitosis marker histone H3 serine 10 in jj012 and sw-1353 cells. These results indicate that MLN4924 suppressed the proliferation of chondrosarcoma cells through inducement of G2/M phase arrest by diminishing histone H3 serine 10 phosphorylation.

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The results are representative of at least three independent experiments. * P < 0.05.

MLN4924 Induces Cellular Apoptosis through Intrinsic and Extrinsic Apoptotic Pathways in Human Chondrosarcoma Cells
After demonstrating that MLN4924 significantly inhibits cell proliferation in chondrosarcoma cells, we examined whether MLN4924 induces apoptosis in chondrosarcoma cells. Furthermore, because activated caspase-3 and -7 are indicators of early-stage apoptosis [29], the effect of MLN4924 on the activation of caspase-3 and -7 in cells was assessed. Flow cytometry analysis was used to examine caspase-3 and -7 activation, and it was determined that MLN4924 (750 nM) treatment considerably stimulated apoptotic caspase-3 and -7 activation in jj012 and sw-1353 cells after 48 h (Figure 3a). Moreover, the expression levels of antiapoptotic Bcl-2 and Bcl-XL, which are intrinsic apoptotic pathway regulators, were dose-dependently downregulated, and the cleavages of caspase-3 and -7 were dose-dependently enhanced by MLN4924 treatment in jj012 and sw-1353 cells (Figure 3b). The protein level of the pro-form of extrinsic caspase-8 was also dose-dependently reduced, indicating an increase in cleaved caspase-8 during apoptosis (Figure 3b). These results ( Figure 3) indicated that both intrinsic and extrinsic apoptotic pathways were involved in MLN4924-mediated apoptosis in chondrosarcoma cells.

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The ER is the cellular organelle responsible for the synthesis of proteins. Once unfolded or 168 misfolded proteins are accumulated in the lumen of the ER, cells activate the unfolded protein 169 response (UPR) to protect themselves against ER stress, which activates ER transmembrane proteins 170 to increase the expression of GRP78 to reestablish ER homeostasis. When ER stress is excessive, the

MLN4924 Promotes ER Stress-Related Signaling and Apoptosis in Human Chondrosarcoma Cells
The ER is the cellular organelle responsible for the synthesis of proteins. Once unfolded or misfolded proteins are accumulated in the lumen of the ER, cells activate the unfolded protein response (UPR) to protect themselves against ER stress, which activates ER transmembrane proteins to increase the expression of GRP78 to reestablish ER homeostasis. When ER stress is excessive, the UPR provokes the activation of c-Jun N-terminal kinase (JNK) and expression of the CCAAT/enhancer binding protein-homologous protein (CHOP), which can activate the cleavage of caspase-4 to elicit ER-stress-mediated apoptotic cell death to remove severely damaged cells. In this study, MLN4924-mediated neddylation inhibition interfered with the degradation and turnover of proteins for altering the proteomic profile of chondrosarcoma cells, which a study has determined may augment ER stress-related signaling and induce cell death [30]. Figure 4 illustrates how MLN4924 dose-dependently increased the expression of GRP78 and CHOP, the phosphorylation of JNK, and the cleavage of caspase-4 in jj012 and sw-1353 cells. This finding indicated that MLN4924 increased cellular stress to induce ER stress-associated apoptosis in human chondrosarcoma cells.

MLN4924 Significantly Inhibits Chondrosarcoma Xenograft Growth in Vivo
Because neddylation inhibition significantly affects the cellular functions of chondrosarcoma cells, the therapeutic efficacy of MLN4924 for suppressing chondrosarcoma xenograft growth in nude mice was examined. Images of jj012 and sw-1353 xenograft tumors are depicted in Figure 5a,c. MLN4924 markedly suppressed the volume of jj012-and sw-1353-xenograft tumors after 4 weeks of treatment compared with the DMSO control group (Figure 5b,d). By contrast, chondrosarcoma cells in the control group exhibited a trend of continual growth, reaching a peak of approximately 750 mm 3 (Figure 5b,d). Figure 5 indicates that the inhibition of neddylation could inhibit chondrosarcoma cell growth in vivo and suggests that MLN4924 has considerable therapeutic potential for treating human chondrosarcoma.

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Chondrosarcoma cells also actively excrete FGF2 and VEGF, which promotes angiogenesis by

Discussion
Chondrosarcoma exhibits a diverse histopathology and clinical behavior; its clinical management is exceptionally challenging because of its inherent resistance to conventional chemotherapy and radiation therapy; thus, new therapeutic approaches are urgently required [31]. Chemotherapy is generally ineffective for treating chondrosarcoma, especially the most frequently observed conventional type and the rare (low-grade) clear-cell variant [32]. Chondrosarcomas with a high percentage of small cells and limited cartilage content are thought to be most sensitive to chemotherapy and radiotherapy, as with other small-cell sarcomas [33]. Moreover, the efficacy of anticancer agents may be impeded by the considerable amount of extracellular matrices and poor vascularity associated with chondrosarcomas, which necessitate the anticancer agents to diffuse over a relatively long distance. This chemoresistance may also be attributed to the expression of the multidrug-resistance 1 gene, p-glycoprotein, resulting in resistance to doxorubicin in vitro. Some research has elucidated the molecular events underlying the pathogenesis of this bone malignancy with the goal of developing novel molecularly targeted therapies [34]. Chondrosarcoma signaling involves numerous growth factors and cytokine-regulated signaling pathways such as FGFR1, integrins, ADIPOR, CCR5, and the CXCR4-inducing MAPK-ERK and PI3K-AKT signaling pathways, leading to the activation of MMP, RANKL, VEGF, NF-κB, and p38 MAPK signaling. Chondrosarcoma cells also actively excrete FGF2 and VEGF, which promotes angiogenesis by attracting endothelial cells [27,34]. Furthermore, chondrosarcomas elicit autocrine and paracrine Hh signaling and may be downregulated by the inhibition of Hh pathway signaling by binding to Smoothened [35]. The dependent and independent pathways of EIF2α also regulate the invasion and motility of sw-1353 chondrosarcoma cells and the inactivation of Src, Rac1, and MMP13 by Sal [36]. The dual inhibition of the PI3K/Akt/mTOR and RAF/MEK/ERK signaling pathways has also been demonstrated to have synergistic antiproliferative effects in chondrosarcoma cells [37]. Mutations in IDH1/2, present in over half of primary conventional chondrosarcomas, render the development of IDH inhibitors a promising treatment option. However, these treatments are still under clinical investigation [38]. Recently, the ubiquitin-proteasome system (UPS) was determined to be associated with chondrosarcoma [39].
Cancer cells take advantage of the UPS by changing the turnover of specific proteins to increase growth and reduce apoptosis. Therefore, the components of the UPS are potential anticancer targets [40]. The UPS is dysregulated in various cancers [41,42]. A study demonstrated that UPS-regulated NF-κB signaling in bone morphogenetic protein-2-activated β1 integrin expression promotes the migration of human chondrosarcoma cells [43]. Moreover, p53, a major tumor-suppressing gene, affects numerous cancers. Low expression of P53 is associated with the metastasis of chondrosarcoma cells [44], indicating the importance of the UPS in chondrosarcoma because the expression level of P53 is controlled by the E3 ubiquitin ligase MDM2, which mediates protein degradation. However, how the UPS is involved in the carcinogenesis and progression of chondrosarcoma is not well characterized. In a recent study, the first-in-class proteasome inhibitor bortezomib inhibited cellular proliferation by stimulating caspase-dependent apoptosis and autophagy [39], suggesting that the UPS is crucial for maintaining the growth of chondrosarcoma cells. However, bortezomib inhibits the overall activity of the UPS, which could cause severe side effects [45]. This indicates that more specific targeting of the UPS is necessary to treat cancers.
The neddylation pathway of NAE-activated CRLs has been demonstrated to be overexpressed and overactivated in cancer cells [18], indicating that targeting neddylation can more specifically regulate the degradation of proteins and control cancer growth. However, the functional activity and expression pattern of the neddylation pathway in chondrosarcoma cells have not been sufficiently investigated. Researchers have determined that a specific NAE inhibitor, MLN4924, imitates the AMP structure and establishes an adduct with NEDD8 through NAE-1, a protein that participates in the first step of NEDD8 adenylation [46]. MLN4924 can restrict the neddylation of cullins, which leads to tumor-suppressive CRL substrate accumulation. As a result, cell death occurs, leading to the suppression of tumor growth, migration, and invasion. Additionally, other studies have suggested that NEDD8 abrogation may boost carcinogenesis and drug resistance, which has encouraging implications for the development of NEDD8 pathway-regulation strategies for cancer therapy [47,48]. This study first revealed that MLN4924, by specifically targeting NAE-activated neddylation, plays a vital role in suppressing the growth of chondrosarcoma cells, which indicates that the neddylation pathway is crucial for the growth of chondrosarcoma cells and is thus a potential therapeutic target for treating human chondrosarcoma.
Studies have demonstrated that MLN4924 is less toxic than bortezomib, an agent that blocks proteasome chymotrypsin-like activity [45,49,50]; the lower toxicity may be achieved by specifically targeting CRLs. MLN4924 is currently the focus of numerous preclinical and clinical trials for cancer treatment, possibly because of its low toxicity as well as its specificity [51]. Cullin-RING E3 ubiquitin ligases control protein ubiquitination and resulting degradation. Moreover, they are integral in stress responses, cell cycle progression, signal transduction, and DNA replication [52]. The existence of these aforementioned therapeutic activities accords with the results of our study, which demonstrated that MLN4924 induces caspase activation, cellular stress, and apoptosis in human chondrosarcoma cells.
We also determined that MLN4924 treatment is associated with the UPR and other proteins related to cellular stress. We determined that MLN4924 activates JNK, which is the corresponding downstream transcription factor belonging to the MAPK signaling pathway. This finding is supported by a related study that demonstrated the sensitivity of the JNK signaling pathway to stress stimuli and controlling cell death, differentiation, and proliferation [53]. Moreover, the current study determined that MLN4924 caused cell death related to the UPR. Protein aggregation in the cytosol or organelles can be induced by CRL substrate protein accumulation, subsequently prompting a UPR [30,54], which then activates ER-resident-chaperone-encoding gene transcription, thereby easing protein folding. The aforementioned UPR is involved in ameliorating cellular stress and restoring ER homeostasis. We further determined that MLN4924 leads to the expression of CHOP, the downstream effector of apoptosis related to the UPR, and GRP78, the key protein for ER integrity and gatekeeper for the UPR. This indicates that apoptosis related to the UPR can induce MLN4924 cytotoxicity in chondrosarcoma cells.

Measurement of Cell Viability
Cell viability was determined using WST-1 cell proliferation and a cytotoxicity assay kit (Biotools, New Taipei City, Taiwan). First, the cells were cultured in 96-well microplates (5000 cells/well) and incubated at 37 • C for 24 h; subsequently, they were exposed to the WST-1 reagent according to the manufacturer's instructions. The absorbance at 450 nm was detected using a Thermo Scientific Multiskan FC microplate photometer (Thermo Scientific, Rockford, IL, USA).

Lactate dehydrogenase Activity Assay
To determine cytotoxicity, the level of LDH activity in the supernatant of the cultured cell was detected according to the manufacturer's instructions (Roche, Indianapolis, IN, USA). The absorbance of each sample was measured using the aforementioned microplate photometer.

Apoptosis Assays in Vitro
Whether and to what extent MLN4924 induced apoptosis in chondrosarcoma cell lines was evaluated. The cultures of chondrosarcoma cell lines were treated with various concentrations of MLN4924 in culture medium for 48 h. After treatment, the cells were washed with ice-cold phosphate-buffered saline (PBS) and used for the assay activation of caspase-3/7 according to the manufacturer's instructions; this was done by quantifying the bioluminescent signal of Z-DEVD-aminoluciferin (Muse Caspase-3/7 assay kit, Millipore, Burlington, MA, USA) using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) [29].

Cell Proliferation Assay
To determine the inhibitory influence of MLN4924 on the proliferation of chondrosarcoma cells, 5-bromo-2 -deoxyuridine incorporation assays (Roche) were performed. Specifically, we seeded the chondrosarcoma cells in 96-well microplates (4000 cells/well) for 24 h, which were then exposed to MLN4924 for 48 h. After treatment, cells were cultured with 5-bromo-2 -deoxyuridine according to the manufacturer's instructions. We used a Thermo Scientific Multiskan FC microplate photometer operated at dual wavelengths (450-540 nm) to measure absorbance.

Cell Cycle Analysis
Propidium iodide (BD Biosciences) staining was used to evaluate the effect of MLN4924 on the cell cycle progression of chondrosarcoma cells. Chondrosarcoma cells were starved in serum-free medium to synchronize cell cycle progression. Following starvation, chondrosarcoma cells were treated with various concentrations of MLN4924 for 24 h. Following incubation, the cells were collected and fixed for staining with PI and then analyzed using flow cytometry. The ratios of cells in G0/1, S, and G2/M were calculated using the ModFit computer program (BD Biosciences).

Western Blot Analysis
For protein phosphorylation and expression analyses, the cells were washed with PBS and lysed using cell lysis buffer (Cell Signaling Technology) on ice for 15 min and were subjected to centrifugation at 14,000 rpm at 4 • C for 15 min. After supernatants were harvested, we conducted a bicinchoninic acid protein assay (Thermo Scientific) to determine protein concentrations. Equal quantities of each sample were resolved using SDS-PAGE and subsequently transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Thereafter, membranes were blocked using bovine serum albumin (5%) with Tris-buffered saline and Tween (TBST) for a minimum period of 1 h. Subsequently, membranes were incubated overnight with the primary antibodies at 4 • C. The resulting membranes were then washed twice using TBST for 10 min and incubated with specific horseradish peroxidase-conjugated secondary antibodies (Genetex, Irvine, CA, USA) for 1 h at room temperature. Subsequently, the antibody-bound membranes were washed twice with TBST and the TOOLS Ultra ECL-HRP substrate detection reagent (Biotools, New Taipei City, Taiwan) was used to visualize protein bands.

In Vivo Xenograft Experiments
This study was approved by the Institutional Animal Care and Use Committee of National Taiwan University (IACUC No. 20180156, 27 July 2018). The jj012 or sw-1353 cells (5 × 10 5 ) were suspended in 200 µL of serum-free medium, and then an equal volume of Matrigel (BD Biosciences) was added. Subsequently, the cells were subcutaneously injected into the dorsal flanks of eight-week-old nude mice (n = 7 in each group; Taiwan National Laboratory Animal Center, Taipei, Taiwan). Once their tumors grew to approximately 150 mm 3 , the mice were grouped and subjected to intraperitoneal injections of 10 mg/kg MLN4924 in normal saline twice a day for 5 weeks. The mice that received the treatment were designated as the MLN4924-treated group, and the mice that received only DMSO in normal saline were referred as the control group. Using calipers, tumor volume was measured twice a week using the following formula: V = LD × (SD) 2 /2, where V, LD, and SD represent tumor volume, longest tumor diameter, and shortest tumor diameter, respectively.

Statistical Analyses
The GraphPad Prism 5 software program (La Jolla, CA, USA) was used to assess data. Data are expressed as mean ± standard deviation. Statistical significance was analyzed using an unpaired Student's t test, and a p value of <0.05 was considered statistically significant.

Conclusions
This study is the first to demonstrate that specifically targeting NAE-activated neddylation could suppress the growth of chondrosarcoma cells, indicating that the activity of the neddylation pathway is critical for the growth of chondrosarcoma cells. MLN4924 caused the activation of ER-stress-associated proteins involved in cellular apoptosis and cell cycle inhibition in chondrosarcoma cells. This may have been achieved through inhibition of the neddylation of cullins, leading to tumor-suppressive CRL substrate accumulation. These findings provide support for using MLN4924 as a potential treatment for chondrosarcoma.