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HTR8/SVneo Cells Display Trophoblast Progenitor Cell-Like Characteristics Indicative of Self-Renewal, Repopulation Activity, and Expression of “Stemness-” Associated Transcription Factors

Introduction. JEG3 is a choriocarcinoma—and HTR8/SVneo a transformed extravillous trophoblast—cell line often used to model the physiologically invasive extravillous trophoblast. Past studies suggest that these cell lines possess some stem or progenitor cell characteristics. Aim was to study whether these cells fulfill minimum criteria used to identify stem-like (progenitor) cells. In summary, we found that the expression profile of HTR8/SVneo (CDX2+, NOTCH1+, SOX2+, NANOG+, and OCT-) is distinct from JEG3 (CDX2+ and NOTCH1+) as seen only in human-serum blocked immunocytochemistry. This correlates with HTR8/SVneo's self-renewal capacities, as made visible via spheroid formation and multi-passagability in hanging drops protocols paralleling those used to maintain embryoid bodies. JEG3 displayed only low propensity to form and reform spheroids. HTR8/SVneo spheroids migrated to cover and seemingly repopulate human chorionic villi during confrontation cultures with placental explants in hanging drops. We conclude that HTR8/SVneo spheroid cells possess progenitor cell traits that are probably attained through corruption of “stemness-” associated transcription factor networks. Furthermore, trophoblastic cells are highly prone to unspecific binding, which is resistant to conventional blocking methods, but which can be alleviated through blockage with human serum.

Introduction
The master regulatory networks of human embryonic stem cell (hESC) transcription factors, OCT4, SOX2, and NANOG, as well as other cell fate determining transcription factors that are implicated in stem cell self-renewal capacities, such as NOTCH1 and STAT3, are expressed not only by embryonic stem cells, but also by a number of cancers [1]. Some of these factors are also expressed in choriocarcinoma (gestational trophoblastic disease) [2]. This has led to the thought that choriocarcinoma may also represent a group of tumors, in which hESC transcription factor deregulation has led to their transformation into cancer stem cells.
In mammalian development, the first cell differentiation step segregates trophoblast and embryonic cell lineages, thus resulting in the formation of the blastocyst's outer lining, the trophectoderm (TE), and its inner cell mass (ICM). The trophectoderm consists of trophoblast stem cells that express CDX2, a homeobox transcription factor, which is required for the emergence of these cells [3]. Physiological invasion is seen during blastocyst implantation, which is mediated through the trophectoderm. Interestingly, both CDX2 and SOX2 deficiency lead to implantation failure of the blastocyst secondary to trophoectoderm differentiation problems [4][5][6].
The trophectoderm also differentiates into several trophoblast subsets in order to create the placenta of the first trimester pregnancy. Of these subsets, the cytotrophoblast is considered a putative "progenitor cell, " which replenishes the outer layer of the villous (syncytiotrophoblast), but which is also able to invade the decidua in a cancer-like manner when necessary and desirable (extravillous trophoblast) [7]. This behaviour is often believed to be driven by hypoxia, and it is a well-orchestrated and closely controlled process, mostly through a network of interaction between the invading trophoblast, the decidua, the maternal endothelium, and the maternal immune system; the detailed description of which would tax the scope of this introduction [8]. The first trimester placenta is especially ample with invasive (cyto)trophoblast, while the term placenta trophoblast loses this capability [8].
The uniqueness of this situation, in which physiologic, spatially (limited to the decidua, first third of the myometrium, and the invasion into maternal spiral arteries), and temporally (limited to the first trimester of pregnancy) regulated invasion (by the trophoblast) and pathologic, deregulated, and malignant invasion (by choriocarcinoma) are set so close together, has drawn the attention of cancer researchers worldwide [8]. However, since isolation of primary trophoblast and choriocarcinoma cells is often cumbersome, in recent years, several trophoblastic cell lines have been utilized as imperfect models for the invasive trophoblast(ic) cell. Some of the most popular cell lines used constitute the immortalized first trimester trophoblast cell line, HTR8/SVneo, and the choriocarcinoma cell line JEG3. HTR8/SVneo cells are often considered a closer model of trophoblast cells, because the HTR8/SVneo cell lines were established by immortalizing a physiologic extravillous trophoblast cell via transfection with a plasmid containing the simian virus 40 large T antigen (SV40) [9], while the JEG3 cell line was cloned from a primary choriocarcinoma strain [10].
Our own recently published data, however, demonstrate that the miRNA profiles of these two cell lines are quite differing, surprisingly with JEG3 encompassing an miRNA profile that is closer to primary first trimester trophoblast cells than that of the HTR8/SVneo cell lines [11]. Villous cytotrophoblast and HTR8/SVneo cells have interestingly also been implicated in producing a "side population" that either demonstrates long-term repopulating properties or expresses classical hESC markers [12,13].
Following the idea that both JEG3 and HTR8/SVneo are transformed cells and have been proposed to produce cancer stem cell or progenitor (side population) cell populations, we aimed to characterize the putative cancer and trophoblast stem/progenitor cell traits of HTR8/SVneo and JEG3 cells on the basis of general minimum recommendations for identifying cancer stem cells or progenitor cells [14,15]. This is accomplished first by assessing the capacity of these cells to form spheroid bodies, second by determining the expression of various transcription factors related to progenitor or to cancer stem cell development, and finally by investigating the cells' ability to repopulate trophoblast tissue in a near in vivo model.

Spheroid Formation with Hanging Drops.
We chose the hanging drops protocol as reviewed by Kurosawa [21].
Briefly, 20 000 cells per 30 L drop supplemented RPMI (as described later in Section 2.2) were plated onto the lid of two Petri dishes in regular arrays (20 drops/Petri lid). The lid was inverted over the bottom of the PBS-filled Petri dish (see schematic representation Figure 1). The Petri dish with the hanging drops were cultured under standard conditions (37 ∘ C, 5% CO 2 , humidified atmosphere) for 48 hours. A schematic image of the hanging drop principle is seen in Figure 1. The experiment was carried out in the same manner for HTR8/SVneo cells and for JEG3 cells (40 drops per cell line).

Immunocytochemistry (ICH)
. Cells were trypsinized, centrifuged, and resuspended in 1 mL respective medium. Slides (SuperFrost/Plus slides; Menzel, Germany) were washed and sterilized with ethanol, coated with cells (200 L), and incubated over night at 37 ∘ C. The cells were fixed on the next day with ethanol/methanol and consequently used to perform immunocytochemistry. To inhibit endogenous peroxidise activity, the cells were incubated in methanol/H 2 O 2 for 5-10 min and washed for 5 min in phosphate-buffered saline (PBS, pH 7.4), followed by incubation firstly with and without 5% human AB serum (PAA), which corresponds to an approximate Fc-concentration of 0.6 mg/mL, in order to further eliminate the possibility of Fc-receptor crossreactions (as described in [22]), and secondly with goat serum at room temperature for 20 min (Vector Laboratories) to eliminate regular nonspecific background staining.
Samples were then incubated with the primary antibodies (please refer to Table 1) for 60 min at room temperature. Antibodies were diluted in DAKO Antibody Diluent with Background Reducing Components (DAKO, Denmark). A negative control was prepared by replacing the primary antibody with DAKO Antibody Diluent only. Isotype controls were prepared in the same manner as the primary antibody.
Analysis of staining intensity and gross estimation of stained cell numbers was accomplished by eye and by two blinded investigators (criteria similar to standard immunoreactive scoring).

Immunofluorescence Staining.
The cells were cultured on SuperFrost/Plus slides (Menzel, Germany) over night with serum-free media. Cells were fixed on the subsequent day with ethanol/methanol. To reduce nonspecific background staining, all samples were incubated either with goat serum or with 5% human AB serum (PAA, as recommended by [22]) at room temperature for 20 min (Vector Laboratories). Samples were incubated with the primary antibodies (please refer to Table 1 for company names and concentrations used) overnight at 4 ∘ C. Antibodies were diluted in DAKO Antibody Diluent with Background Reducing Components (DAKO, Denmark). On the next day, they were incubated with the secondary antibody labeled with Cy3 for 1 h at room temperature. Between each step, all samples were washed profusely with PBS. Sections were counterstained Vectashield Mounting Media with DAPI (VECTOR Laboratories) and then cover slipped.
A negative control was prepared by replacing the primary antibody with DAKO Antibody Diluent only. Isotype controls were prepared in the same manner as the primary antibody. All samples were analyzed with an AxioPlan2 microscope (Carl Zeiss, Jena, Germany). Assessment was accomplished by eye and by two investigators only in terms of positive or negative expression and pattern of expression.

Placental Explant Cultures and Confrontation Cultures.
Two biopsy-sized "explants" (2 mm diameter) each from villous tissue of five healthy human term placentae (after elective caesarian section) were collected. An approval by the local ethical committee exists. Prior to confrontation cultures, all spheroids were stained by application of 10 nM Mito Tracker dye (fluorescent green; Invitrogen) for 30 min at 37 ∘ C and then intensively washed in PBS (Biochrom, Germany). Commencing from the time that the placental "biopsies" or "explants" are placed in culture, these are termed villous explant cultures.
Each explant was then confronted in culture with one spheroid within the respective hanging drop for 48 h. Subsequently, the confronted tissues were incubated with a solution of 10% nonfat milk in PBS containing 1% Triton (AppliChem) to permeabilize cell membranes and to block nonspecific binding sites for 1 h. Following intensive washing step, tissues were incubated with a rat anti-human CD31 (anti-PECAM1; Millipore, Germany) for 2 h, followed by incubation with a goat anti-rat IgG-Cy5 conjugate (Milipore) for 90 min, all within the previously described solution (nonfat milk/PBS/Triton). After staining, descriptive analyses with the tissues and spheroids were accomplished on a confocal laser scanning microscope (Carl-Zeiss, Jena, Germany).

HTR8/SVneo Cells Have a High and JEG3 Cells a Low Propensity to Form Spheroid Bodies Within Hanging Drops.
The first step to confirm putative cancer stem/progenitor cell status is to confirm their capacity for self-renewal, which is often accomplished by propagation of these cells as spheroids in stem cell culturing conditions [14]. Cells with selfrenewing potential can be disaggregated from the spheroids and passaged multiple times with retention of spheroidforming ability [15].
Of the 40 hanging drops experiments per cell line, 100% of the incubated HTR8/SVneo cells and only 50% of the incubated JEG3 cells were able to form spheroids (data not demonstrated). The developed HTR8/SVneo spheroids regularly measured a diameter of approximately 700-750 m, which was also visible by the "naked" eye (Figures 2(a) and 2(b)). On the other hand, JEG3 spheroids were much smaller (as visible even by the "naked" eye) and irregularly shaped (Figures 2(c) and 2(d)). The JEG3 spheroids were also unstable and were disaggregated easily and thus could not easily be pipetted into the shortened tip of a pipette for transportation into, for example, a new hanging drop.
In order to verify that spheroid formation is not secondary to cell aggregation, we proceeded to disaggregate the spheroids, split these cells, and propagate them again in hanging drops to assess their continued ability to reform spheroids (as recommended in [15]). Under this experimental setting, we passaged the HTR8/SVneo spheroids multiple times (5 passages and continuing) without their loss of spheroid-forming abilities. As little as 5000 HTR8/SVneo cells are able to form a spheroid (splitting ongoing). Please note that we have not yet tried to form spheroids with less cells than the mentioned. Only 50% of the initial JEG3 spheroids were able to reform spheroids following disaggregation and splitting; further passages could not be maintained.

HTR8/SVneo and JEG3 Cells Express the Trophoblast
Stem Cell Marker CDX2. CDX2 is a transcription factor that is necessary for the first differentiation of hESC into the trophoblast stem cell (reviewed in [3]). Loss of CDX2 activity disables the blastocyst to implant correctly during murine pregnancy [23]. Following the hypothesis that HTR8/SVneo or JEG3 cells recapitulate features of a cancer stem cell or a trophoblast(ic) stem or progenitor cell, we sought to investigate whether the classic trophoblast stem cell "marker" is expressed in both cell lines.
Since it has been described that nonspecific binding in trophoblast(ic) cells is extreme and often cannot be alleviated through conventional blocking procedures [22], we initially blocked cell lines with normal goat serum and further blocked them with or without human serum. According to immunofluorescence staining results, both cell lines seem to stain positive for CDX2. However, since there was no alteration in the pattern of staining before and after application of human serum, and since it is rather unlikely that CDX2 is so prominently expressed in the cytoplasm [24,25], we conclude that unspecific binding is still too high in immunofluorescence stainings to be reliable.
In contrast, ICH results show that HTR8/SVneo cells express CDX2 in the nucleus, although the trophoblast marker expression appeared lower, especially in terms of number of nuclei, after blockage with human serum (Figure 3(b) without human serum versus Figure 3(a) with human serum). In JEG3 cells, a low-intensity CDX2 signal was observable even after blocking with human serum with numbers of positive-stained nuclei rather unchanged (Figures 4(a) and  4(b)). Via immunofluorescence staining procedures, CDX2 expression is made visible, and the signal patterns are again not affected by blocking procedure; thus, this procedure is again deemed unreliable (Figures 3(c), 3(d), 4(c), and 4(d)).

HTR8/SVneo Cells Express NOTCH1
, NANOG, and SOX2, but Not OCT4 according to ICH Analysis. In theory, cancer stem cells have probably reacquired properties similar to stem cells during transformation into a malignancy [1,2,26]. We analyzed the expression of the classic or core "stemness-" associated transcription factors OCT4, NANOG, and SOX2 (reviewed in [16]). We also chose to assess the expression of the cell fate determining transcription factor NOTCH1 due to its recent suggestion as a maintainer of "stemness, " as well its ever-emerging role in the invasion potential of tumors (reviewed in [17,20,27,28]).  . Since it has been described that nonspecific binding in trophoblast(ic) cells is extreme and often cannot be alleviated through conventional blocking procedures [22], we initially blocked cell lines with normal goat serum and further blocked them with or without human serum. Upon blocking of HTR8/SVneo cells with human serum, the positive signals for all investigated transcription factors were reduced, especially in the nuclei, or, as in the case of OCT4a, disappeared altogether ( Figure 3: (f), (j), (n), and (r) without human serum versus (e), (i), (m), and (q) with human serum). The expression intensity was highest for NOTCH1, and this expression was only slightly altered after blockage with human serum (Figures 3(i) and 3(j)). In contrast to peroxidase staining, we detected all stem cell markers via fluorescence staining, and there were no visible differences in staining pattern between the blocking procedures ( Figure 3, for NANOG: (g) and (h); for NOTCH1: (k) and (l); for OCT4: (o) and (p); for SOX2: (s) and (t)).
Taking the recommendations of Honig et al. [22] into consideration (meaning that we now deem the immunofluorescence procedure unreliable for staining of these transcription factors in these cells), we surmise that HTR8/SVneo cells express all of the investigated transcription factors except for OCT4.
JEG3 cells express NOTCH1, and, as in HTR8/SVneo cells, NOTCH1 was detectable with both blocking methods (Figures 4(i) and 4(j)). Interestingly, the NOTCH1 staining signal in JEG3 cells appears less intensive than that in HTR8/SVneo cells (Figure 3

HTR8/SVneo Spheroids Contain Chorionic Villi-Covering Cells Indicative of Repopulation.
To demonstrate repopulation capacity of cancer stem/progenitor cells, the progenitor cell candidates are usually injected in an immunocompromised mouse in the general vicinity of the target organ in which prior elimination of the target population has taken place (as the progenitor cells are supposed to replace them) [29]. If the progenitor cell candidates (e.g., mammary gland stem cell) possess repopulating potential, then they will be found in the stead of the original target cell population (e.g., mammary gland).
It has been observed before that altering in vitro culture standards can cause a side population of HTR8/SVneo cells to differentiate into trophoblast subpopulations, as made visible by ICH analysis of differentiation markers [12]. The HTR8/SVneo capacity to actually repopulate the villous in an in vivo setting has not yet been demonstrated. The syncytiotrophoblast layer within villous explant cultures is known to fully degenerate following 4 h of culture initiation [30].
We sought to determine whether the cells present in the HTR8/SVneo spheroids could repopulate the entire chorionic villous, which according to the previous citation is now replete of the syncytiotrophoblast layer, following confrontation with placental villous explants in an effort to remain in a near in vivo model of the placenta.
We excluded JEG3 from this experiment as the previous investigations suggest that JEG3 cells possess only low prominent cancer stem cell characteristics. Furthermore, as JEG3 spheroids were unstable and easily disaggregated, it was not possible to transfer them to new hanging drops in which coculture experiments were performed. In coculture of HTR8/SVneo with placental explants, spheroids are completely disaggregated after 48 h, and all retrievable HTR8/SVneo cells cover the chorionic villous ( Figure 5; HTR8/SVneo spheroid cells in green; endothelium of fetal capillaries within chorionic villi in red). Ten individual confrontation cultures have been performed with qualitatively similar results.
Assuming that the syncytiotrophoblast layer of placental explants truly do degenerate following 4 h cultivation, then the previous situation suggests that the HTR8/SVneo spheroid cells contain the progenitor cell characteristic of repopulating activity in a near in vivo model.

Discussion
HTR8/SVneo and JEG3 cells are highly popular transformed cell lines often used as an imperfect model of the trophoblast, with HTR8/SVneo often deemed as closer to the physiologic setting. Both cell types have been proposed to possess "stemlike" or "progenitor-like" characteristics [2,12].
One aim of this study was to characterize stem/progenitor cell traits of these cell lines according to general minimum standards used to identify putative cancer stem cells 8 BioMed Research International [14,15]. Another aim of this study was to characterize the HTR8/SVneo and JEG3 cell lines for their expression pattern of various transcription factors associated with "stemness" (OCT4, NANOG, and SOX2), cell fate (NOTCH1), and trophoblast stem cells (CDX2) and to correlate this to their intrinsic progenitor/stem cell capacities, because their transformed character is likely to contribute to altered transcription factor expression, which in turn is likely to be responsible for stem/progenitor-like behavior. This is a first step in defining a cell population as a progenitor or cancer stem cell.
In our hands, HTR8/SVneo cells have a high propensity to form spheroid bodies, while expressing virtually all investigated transcription factors (specifically CDX2, NOTCH1, SOX2, and NANOG, and not OCT4). Furthermore, HTR8/SVneo spheroid cells demonstrate behavior reminiscent of self-renewal and replenishing properties. In contrast, JEG3 cells have only a limited ability to form spheroid bodies, and although they express the trophoblast stem cell marker CDX2 and a cell fate determinant NOTCH1, they did not express the investigated hESC cell markers or so-called "stemness-" associated transcription factors. Taken together, this is indicative of the fact that HTR8/SVneo cells are transformed in a manner that might make them closer in phenotype to a trophoblast progenitor cell than to a differentiated trophoblast cell, such as a syncytio-or extravillous trophoblast. Our experiments reveal traits that speak for JEG3 cells being a form of cancer stem cell as previously proposed.
As mentioned earlier, our own recent results indicate that HTR8/SVneo cells have less in common with the primary extravillous trophoblast cell than JEG3 cells [11]. GeneChip analyses of the expression signatures of primary trophoblast versus choriocarcinoma cell lines (including JEG3) and versus extravillous trophoblast derived cell lines (including HTR8/SVneo) have revealed that all three groups cluster distinctly [31]. Furthermore, the genes that are similarly expressed between EVT and choriocarcinoma cell lines are related to cell motility, signaling, vasculature and tissue development (all functional signs of differentiation), while HTR8/SVneo and EVT similarities are restricted to those genes regulating RNA transport and metabolism (housekeeping characteristics) [31]. Classic hallmark characteristics for primary, differentiated trophoblast cells are its expression of cytokeratin 7 and negative expression of vimentin [32,33]. HTR8/SVneo cells show an expression profile that is just the opposite [12,31]. In this aspect, it is interesting that HTR8/SVneo cells express N-cadherin, while their JEG3 counterparts do not [34], meaning that HTR8/SVneo express at least two known epithelial-mesenchymal transition (EMT) markers (N-cadherin and vimentin), which is also a sign of partial dedifferentiation (reviewed in [27]). Finally, HTR8/SVneo cells express HLA-G, a typical extravillous trophoblast differentiation marker [33], only weakly or not at all [12] and secrete -HCG, a hallmark characteristic of a syncytiotrophoblast cell (as reviewed in [35]), weakly, but more than primary cytotrophoblast, which do not secrete hCG at all [9].
In our study, we also demonstrate the self-renewal properties of HTR8/SVneo cells propagated as spheroids under the same culture conditions as human embryonic stem cell embryoid bodies. HTR8/SVneo cells not only survive multipassages after spheroid formation, but are continually able to reform spheroids after each passage even with fairly low cell numbers. This is also in line with a recent observation that HTR8/SVneo cells form a side population that displays selfrenewal characteristics [12]. We found it somewhat puzzling that all HTR8/SVneo cells incubated in hanging drops formed spheroids in our experiments, while Takao et al. describe only a very exclusive side-population that demonstrates selfrenewal properties [12]. However, since we excluded the possibility that HTR8/SVneo spheroids are mere cell aggregates, we propose that the hanging drop environment induces HTR8/SVneo cells to alter its phenotype towards that of a progenitor-like cell.
We have not proven in our own studies that the syncytiotrophoblast layer of placental explants has actually degenerated during the 48 h cultivation period we chose; however, other studies have described that this occurs after 4 h [30]. Following coculture of our placental explants with HTR8/SVneo cells for 48 h, we reveal for the first time that HTR8/SVneo cells have the propensity to "renew" or at least cover villous tissue in a model near to the in vivo situation. We speculate that the syncytiotrophoblast layer is lost, as described, and that HTR8/SVneo cells answer a distress call during the cultivation period, which allows the HTR8/SVneo cells to migrate to this area and cover or indeed replace the villous with syncytiotrophoblast-like cells. This is an indication for a certain degree of plasticity within the HTR8/SVneo cells, which we have not corroborated here with syncytiotrophoblast-specific expression of surface molecules. We believe that this is likely though, since Takao et al. have been able to demonstrate that a side-population of HTR8/SVneo cells is able to differentiate into syncytiotrophoblast and other trophoblast lineages [12]. Future studies are needed in order to unravel which transcription factor or other signal is responsible for regulating this migratory and replenishing characteristic.
Our investigations characterize for the first time the expression of "stemness-" associated cancer and trophoblast stem cell transcription factors in the HTR8/SVneo cell line. The fact that both cell lines produce CDX2, while only HTR8/SVneo shows progenitor cell capacities, could indicate that CDX2 is rather a sign of trophoblast lineage derivation instead of a trophoblast stem cell differentiation alone. However, CDX2 has been shown to be expressed only in first trimester trophoblast, while term placentae lose this capacity. Furthermore, the same study identified that CDX2+ELF5+ cells within the placentae characterize cytotrophoblast populations [36]. This in turn suggests that JEG3 cells have retained a certain cytotrophoblastic identity, which we have not been able to visualize as a progenitor-like function in this study.
The regulatory network between CDX2 and the "stemness-" associated transcription factors is tight and complicated; reiterating this in detail goes beyond the scope of this investigation. Briefly, CDX2 and the "stemness-" associated transcription factors are thought to reciprocate or mutually antagonize each other, also because this is the expression pattern found during blastocyst formation. At least in the mouse, 9 CDX2 is known to downregulate OCT4 and NANOG [37,38]. However, SOX2 and CDX2 are known to cooperate during trophectoderm formation [4]. In our analysis, while JEG3 cells did not express any of the so-called "stemness-" associated transcription markers, HTR8/SVneo cells expressed most of them (OCT4a neg , NANOG weak , and SOX2 weak ). Interestingly, a recent characterization of trophoblast progenitor cells derived from first trimester placentae reveals that undifferentiated trophoblast progenitor cells that form embryoid bodies and that are capable of multipassage also display OCT4 neg , NANOG weak , and SOX2 weak phenotypes [39]. Due to the association of this transcription marker expression profile with the presence of progenitor-like functions in HTR8/SVneo cells and in trophoblast progenitor cells, it is enticing to conclude that NANOG and SOX2 are responsible for the observed functions. Further studies with gain/loss-of-function analyses would, however, be necessary to finalize that conclusion. Furthermore, since CDX2 is coexpressed in HTR8/SVneo cells with certain hESC transcription factors, it is most plausible that the "stemness-" associated transcription factor regulatory network has been corrupted, probably through alterations secondary to transfection of HTR8 parent cells with a plasmid containing the simian virus 40 large T antigen (SV40).
We were surprised to see that NANOG was not expressed in JEG3 cells as described in a recent publication, in which NANOG was especially detected in the nuclear fraction of JEG3 cells [40]. Currently, we cannot explain this discrepancy other than in methodology differences, and further analyses on the mRNA and protein level are under way.
The NOTCH1 signal was remarkably visible in both cell lines. NOTCH1 has been associated with "stemness" properties, as well as with the differentiation of cancer stem cells (tumor stem-like cells) into endothelial progenitor cells [18,19,27]. Furthermore, NOTCH1 expression is linked with trophoblast, as well as with malignant types of invasion [17,20,28]. In the physiologic placenta, NOTCH1 expression is thought to be vital for placental angiogenesis, while defective NOTCH signaling is thought to contribute in the pathogenesis of preeclampsia (reviewed in [41]). With our current information, it is as yet impossible to conclude whether NOTCH1 expression is a sign of stem-like properties, of EMT or invasion potential. Further functional analyses would be helpful in unraveling this aspect.
In summary, though, all of the previously analyzed characteristics of HTR8/SVneo are in line with the idea that HTR8/SVneo cells share more similarities to a trophoblast progenitor cell, perhaps the cytotrophoblast, than a primary extravillous trophoblast. Our investigation, together with that of Takao et al., is first step in defining HTR8/SVneo progenitor cell characteristics; however, the final step of in vivo testing per xenotransplantation into immuncompromised mice is as yet pending (but seems likely to be successful, since xenografts of human placental explants into immuncompromised mouse is highly successful [42]).
In all finality, we hypothesize that transfection of the HTR8/SVneo cell line has altered the original (extravillous) trophoblast cell in a manner leading to development of the characteristics described in these investigations; thus, investigators should use caution when using the popular HTR8/SVneo cell line as a model for primary extravillous trophoblast cells.
We wish also to direct investigators to the expression alterations (or lack thereof) seen in our immunocytochemistry and immunofluorescence results before and after blockage with human serum. Our results suggest, as with [22], that trophoblastic cells are prone to unspecific binding, and caution should be exercised when interpreting immunostaining results.


extfieldsofstudy: Medicine Biology

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Detection of Mycobacterium tuberculosis by Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick in Clinical Samples

Tuberculosis (TB) is a communicable disease caused by the bacterium Mycobacterium tuberculosis (MTB) and is a persistent problem in the developing countries. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here, a LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect IS6110 gene of M. tuberculosis specifically and rapidly. The reaction was optimized at 63°C for 60 min, and the amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5 min was detected at the LFD test line 5 min after application. Excluding the step of DNA extraction, the test results could be generated approximately within 1 h. In addition to the advantage of short assay time, this technique could avoid the contact of carcinogenic ethidium bromide due to the exclusion of the electrophoresis analysis step. Furthermore, the data indicated that LAMP-LFD could detect M. tuberculosis genomic DNA as little as 5 pg. The technique showed a significant specificity since no cross-hybridization to M. intracellulare (MIC), M. fortuitum (MFT), M. avium (MAV), M. kansasii (MKS), and M. gordonae (MGD) genomic DNAs was observed. In the clinical unknown samples test, the sensitivity of LAMP-LFD was 98.92 % and the specificity was 100 % compared to those of the standard culture assay. Based on its sensitivity, specificity, rapidity, low cost, and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB.

Introduction
TB is an airborne disease caused by the bacterium Mycobacterium tuberculosis (MTB). WHO reported that TB is a persistent problem in developing countries and ranks as the second leading cause of death from an infectious disease worldwide after the human immunodeficiency virus (HIV) [1]. This bacterium is a slow-growing bacterium that needs 1-2 months for growing in a culture; however, a rapid and timely diagnosis of tuberculosis is essential to combat this disease. The Ziehl-Neelsen (ZN) stain for direct specimen examination is a conventional diagnostic tool but lacks sensitivity. The tests based on PCR have shown promise for the detection of mycobacteria in clinical samples [2][3][4], but this amplification process requires additional processing time, reagents, and devices, which affect the cost of the assay. Moreover, PCR analysis needs well-trained personnel.
The LAMP assay allows DNA to be amplified at a constant temperature of 60-65 ∘ C [5]. After LAMP, the amplified DNA is normally detected by agarose gel electrophoresis, ethidium bromide staining, and UV transillumination. Due to the use of several primers, LAMP generates a complex mixture  of DNA products of different sizes; therefore, gel analysis cannot distinguish specific and nonspecific products. To avoid possible false positive results, the authenticity of LAMP DNA products can be confirmed by restriction endonuclease digestion [5] or by hybridization to specific probes [6]. Later, to further simplify and shorten the time needed to generate LAMP data, a biotin-labeled oligonucleotide probe and an FITC-labeled DNA probe captured by gold-labeled anti-FITC antibody following chromatography on a LFD (Milenia GenLine Hybri Detect) were developed. The techniques were successfully applied in the detection of viruses such as shrimp infectious hypodermal and hematopoietic necrosis virus [7] and shrimp Taura syndrome virus [8]. Using this strategy, the sensitivity is equivalent to that of PCR assay with similar time use of approximately 1 h. Described here was a LAMP-LFD method optimized for the detection of MTB. The sensitivity and specificity of this technique were investigated in comparison to those of a PCR assay.

Samples and DNA Extraction.
All clinical samples (101 unknown sputum samples) and standard strain (H37RVKK11-20) were provided from the National Tuberculosis Reference Laboratory (NTRL), Bureau of Tuberculosis, Department of Disease Control, Ministry of Public Health, Thailand. DNA was extracted from all clinical samples following the method of Rienthong et al. [9]. Briefly, Sputum unknown samples were decontaminated with N-acetyl-L-cysteine-sodium hydroxide. After centrifugation, the crude cell lysates were suspended in 300 L of distilled water, killed with heat at 95 ∘ C for 20 min in Thermoblock, and then underwent sonification for 15 min at the highest speed in an ultrasonic bath, followed by spinning the samples in a standard centrifuge with an aerosol-tight rotor at approximately 10,000 g for 5 min. The supernatant was used for the LAMP amplification. The total samples of unknown sputum were compared to those of the standard culture assay on Lowenstein-Jensen slants and incubated at 37 ∘ C for 8 weeks. The genomic DNA from the standard (H37RVKK11-20) was prepared following the method of Kaewphinit and coworkers [10]. The concentration and quality of genomic DNA in the LAMP reaction were determined by spectrophotometric analysis at 260 and 280 nm.

LAMP Primers and PCR Primers.
LAMP primers for MTB were designed according to the published sequences of the IS6110 gene specific for MTB genome (Gen-Bank accession no. X17348) using Primer Explorer version 4 (http://primerexplorer.jp/elamp4.0.0/index.html). The directions and details of the primers are shown in Figure 1 and Table 1. The normal primers and biotin-labeled FIP primer were synthesized by Bio Basic Inc., Canada.

Optimization of Temperature for LAMP.
To determine the optimum temperature for amplification, the LAMP reactions were carried out at 60, 63, and 65 ∘ C for 1 h, followed by the analysis of the LAMP products by gel electrophoresis. The reaction mixture contained 2 M, each of inner primers FIP and BIP, 0.

Lateral Flow Dipstick (LFD) Assay.
A 5 -FITC-labeled oligonucleotide probe designed according to the IS6110 gene of MTB between the F1c and B1c primer targets was synthesized by Bio Basic Inc., Canada. As recommended in similar tests [7,8], 20 picomoles of FITC-labeled probe (FITC-5 -ATCCGGCCACAGCCC-3 ) was added to the LAMP reaction and after hybridization at 65 ∘ C for 5 min, 8 L hybridized product was added to 120 L assay buffer in a new tube, and an LFD was dipped into the mixture for 5 min.

Sensitivity of LAMP by Gel Electrophoresis and LFD.
To determine detection sensitivity limits, 10-fold serial dilutions (10 −1 to 10 −6 ) of 50 ng of total DNA of MTB (H37RVKK11-20) were used as templates for biotin-labeling LAMP tests performed under optimized conditions. Amplified DNA was detected either by 2% agarose gel electrophoresis and ethidium bromide staining followed by DNA visualization using a UV transilluminator or by the use of LFD as described above.  . PCR was performed by using a DNA thermal cycler for 30 cycles. Each cycle consisted of denaturation at 94 ∘ C for 2 min, annealing at 53 ∘ C for 1 min, and extension at 72 ∘ C for 1 min and a final extension at 72 ∘ C for 10 min. PCR amplicon was analyzed by 1.5% agarose gel electrophoresis. The PCR assay was prepared following the method of Kaewphinit and colleagues [10]. compared to those of the standard culture assay and AFB smear.

Optimization of Reaction Temperature for MTB Detection.
When the LAMP was carried out at 60, 63, and 65 ∘ C for 60 min with 50 ng of MTB infected samples DNAs (Figure 2), all three temperatures tested generated similar LAMP product patterns with the clearest and strongest bands at 63 ∘ C. Based on this, the temperature of 63 ∘ C was used for all tests in this study.

Comparison of LAMP and PCR Sensitivity with Gel Electrophoresis.
Using equivalent quantities of DNA extracted from MTB-infected samples as templates at various dilutions, the detection limits for LAMP and PCR were both at 10 −4 dilution (Figures 3(a) and 3(b)).

Sensitivity of the Combined LAMP-LFD.
The LAMP-LFD detection of MTB showed a limit at 10 −4 DNA dilution (Figure 3(c)). This corresponded to the detection limit for LAMP or PCR methods followed by electrophoresis, as described above. The detection limit was also similar to those previously reported for hepatopancreatic parvovirus (HPV) detection in black tiger shrimp Penaeus monodon [11].

Specificity of LAMP-Gel Electrophoresis and LAMP-LFD.
A specificity test was manipulated by using 50 ng each of MTB genomic DNAs and other mycobacteria (i.e., MIC, MFT, MAV, MKS, and MGD). The data revealed that no cross-reactions were obtained from LAMP-gel electrophoresis (Figure 4(a)) and LAMP-LFD or LFD (Figure 4(b)). a FITC-Probe, and the results were detected by LFD and compared with those obtained from the traditional culture and AFB smear test. In brief, the sensitivity and specificity of this assay for clinical samples' diagnosis of MTB genomic DNAs were 98.92% and 100%, respectively, but the sensitivity of AFB smears test was 80.65%, both methods compared to those of the culture assay (Table 2).

Discussion
The LAMP-LFD technique was used for the detection of MTB in clinical double blind samples. In this study, the LAMP method was carried out at 63 ∘ C for 60 min, which was faster than typical PCR methods that require 2-3 h for PCR cycling.
Referring to the test, no expensive equipment was required and the results could be determined within approximately 1 h (not including DNA preparation time). LAMP-LFD was faster than LAMP-gel electrophoresis (2 h 30 min) and much faster than PCR-gel electrophoresis (3 h 30 min) [12].
The LAMP detection method that targeted a 178 bp sequence of the IS6110 gene was successfully developed for the detection of MTB, with the sensitivity of the LAMP being comparable to that of common PCR amplification for MTB, which was at the concentration level 5 pg of genomic DNA, a sensitivity higher than that reported previously [13].
In the clinical unknown sample test (Table 2), the sensitivity of the LAMP-LFD was 98.92% (92/93), but AFB smear test was 80.65% (75/93), as compared to those of the culture assay, and the specificity of the LAMP-LFD was 100%. For the one sample containing LFD assay was false negative MTB as compared with those of the culture assay. It was reasoned that possibly only a small amount of bacterial DNA was extracted from sputum samples. When compared to positive cultures, the sensitivity, specificity, positive predictive value, and negative predictive value of the method for MTB diagnosis were higher than that method, and the overall time for the procedure was faster than that reported previously [13,14]. In addition, the LAMP-LFD assay for detection step confirms the identity of the specific amplicon by hybridization and avoids the use of carcinogens such as ethidium bromide. The test platform can be adapted easily for rapid detection of other mycobacteria agents simply by designing appropriate sets of LAMP primers and specific probes. Since the cost for the LAMP-LFD detection is comparable with that for standard PCR followed by electrophoresis, it constitutes a highly sensitive, safe and rapid alternative for the detection of MTB. Hence, the technique could be applicable for detecting minute amounts of MTB in clinical specimens. According to the specificity test, no cross-reactions to other mycobacteria were observed. This indicated that the LAMP method was specific for MTB. The introduction of LFD for analysis of LAMP amplification products could reduce the time and complications associated with usual detection by electrophoresis. This resulted in a total analysis time (excluding the DNA extraction step) of less than 75 min. The high sensitivity and specificity, the relatively short analysis time, and the use of relatively inexpensive equipment were key advantages of the LAMP-LFD assay. In addition, the technique did not involve the use of carcinogens such as ethidium bromide. The test platform can be adapted easily for rapid detection of other mycobacteria agents simply by designing appropriate sets of LAMP primers and specific FITC probes to be used with the generic LFD employed. According to the sensitivity, specificity, less time consumption, low cost and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB.


extfieldsofstudy: Biology Medicine

text:
Characterization of Mycobacterium orygis

1. Datta K, Bartlett KH, Baer R, Byrnes E, Galanis E, Heitman J, et al. Spread of Cryptococcus gattii into Pacific Northwest region of the United States. Emerg Infect Dis. 2009;15:1185–91. http://dx.doi. org/10.3201/eid1508.081384 2. Brandt ME, Hutwagner LC, Klug LA, Baughman WS, Rimland D, Graviss EA, et al. Molecular subtype distribution of Cryptococcus neoformans in four areas of the United States. Cryptococcal Disease Active Surveillance Group. J Clin Microbiol. 1996;34:912–7. 3. Meyer W, Aanensen DM, Boekhout T, Cogliati M, Diaz MR, Esposto MC, et al. Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii. Med Mycol. 2009;47:561–70. 4. Harris J, Lockhart S, Chiller T. Cryptococcus gattii: where do we go from here? Med Mycol. 2012;50:113–29. http://dx.doi.org/ 10.3109/13693786.2011.607854 5. Bovers M, Hagen F, Kuramae EE, Boekhout T. Six monophyletic lineages identified within Cryptococcus neoformans and Cryptococcus gattii by multi-locus sequence typing. Fungal Genet Biol. 2008;45:400–21. http:// dx.doi.org/10.1016/j.fgb.2007.12.004 6. Kidd SE, Hagen F, Tscharke RL, Huynh M, Bartlett KH, Fyfe M, et al. A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). Proc Natl Acad Sci U S A. 2004;101:17258–63. http://dx.doi.org/10.1073/pnas.0402981101 7. Byrnes EJ III, Bildfell R, Frank SA, Mitchell TG, Marr KA, Heitman J. Molecular evidence that the range of the Vancouver Island outbreak of Cryptococcus gattii infection expanded into the Pacific Northwest in the United States. J Infect Dis. 2009;199:1081–6. http://dx.doi.org/10.1086/597306 8. Duncan C, Stephen C, Campbell J. Clinical characteristics and predictors of mortality for Cryptococcus gattii infection in dogs and cats of southwestern British Columbia. Can Vet J. 2006;47:993–8. 9. Chan ED, Kong PM, Fennelly K, Dwyer AP, Iseman MD. Vertebral osteomyelitis due to infection with nontuberculous Mycobacterium species after blunt trauma to the back: 3 examples of the principle of locus minoris resistentiae. Clin Infect Dis. 2001;32:1506–10. http://dx.doi. org/10.1086/320155 10. Centers for Disease Control and Prevention. Emergence of Cryptococcus gattii—Pacific Northwest, 2004–2010. MMWR Morb Mortal Wkly Rep. 2010;59:865–8.

Characterization of Mycobacterium orygis
To the Editor: We thank Gey van Pittius and colleagues for their addition to the markers that identify Mycobacterium orygis as a distinct subspecies in the M. tuberculosis complex (1). Its isolation from a wild buffalo broadens the host range of M. orygis. Gey van Pittius and colleagues raise 3 issues: the utility of the gyrB oryx single-nucleotide polymorphism (SNP) being equally specific as the reported SNP in Rv2042 38 , the presence of genomic regions RD701 and RD702 in M. orygis, and the addition of the sequence type (ST) 701 spoligotype to M. orygis-specific spoligotypes.
We agree that use of the gyrB oryx mutation is more practical for routine daily use because this gene helps identify several subspecies of the M. tuberculosis complex. However, use of the partial Rv2042 sequencing is similarly practical because it can be combined with sequencing of the adjacent pncA gene, which enables identification of several M. tuberculosis complex species and some subspecies (i.e., M. orygis, M. bovis, M. canettii) (2), to identify the CAS genotype of M. tuberculosis (J. van Ingen, unpub. data) and, to some degree, assess susceptibility to pyrazinamide (3).
With the added data, we can conclude that M. orygis is an M. tuberculosis complex subspecies defined by the presence of genomic regions RD1, RD2, RD4, RD5a, RD6, RD13-RD16, RD701, and RD702, by the C-to-G SNP in mmpL6 551 , and by the deletion of regions RD3, RD5b, RD7-RD12, RDoryx_1, RDoryx_4, and RDoryx_wag22. Subspeciesspecific SNPs are present in gyrB and Rv2042. Spoligotypes ST587, ST701, and closely related types are characteristic of M. orygis, and this subspecies yields 17-20 copies of insertion sequence 6110 and a distinct 24-locus variable number tandem repeats pattern (4,5). Given the rapid progress in genome sequencing, additional markers specific for the different subspecies will further enrich this panel of differences.   (1) is the high frequency of mutation to rifampin resistance by 2 Mycobacterium tuberculosis Beijing strains, which might play a role in the association between the Beijing strains and multidrug-resistant tuberculosis. Earlier reported frequency of mutation to rifampin resistance by M. tuberculosis has been 10 −8 CFU (2,3), including the Beijing genotype (3,4). Of note, the Beijing 2002-1585 strain, for which frequency of mutation to rifampin resistance is 10 -3 CFU (1 mutant/1,000 CFU), showed a moderate frequency of 10 −8 CFU in another study (4). We think that a mutation frequency increase of 100,000× is remarkably high. In contrast, rifampinresistant mutants of the Beijing 1585 strain did not emerge in low-density cultures (5 × 10 5 CFU/mL) used for time-kill kinetics experiments, al-though frequency of mutation to rifampin resistance was determined to be 10 -3 CFU.

Jakko van Ingen, Roland Brosch, and Dick van Soolingen
Mutation frequency is determined by fluctuation assays. To exclude preexisting mutants, which would bias the mutation frequency by so-called jackpots, a series of low-inoculum cultures is typically used (5). However, for unknown reasons, de Steenwinkel et al. used only 1 highdensity culture of 10 10 CFU of each strain to determine mutation frequency. This strategy is not recommended because mutations can occur early or late, resulting in substantial mutation frequency fluctuation between test episodes. A strain with known mutation rates should preferably be included to rule out possible technical errors.
We propose the following explanations for the remarkable results: 1) the rifampin concentration for selecting mutants might have been too low, enabling growth of some colonies of drug-susceptible bacteria; 2) rifampin mutants arose early or preexisted in the cultivation of Beijing strains 1585 and 1607, producing jackpots; or 3) the 2 Beijing isolates might contain rifampin-resistant subpopulations (heteroresistance). The capacity of the Beijing strain to develop and, especially, transmit multidrug-resistant tuberculosis remains to be further analyzed.  (3) found a mutation frequency of 4-24 × 10 −8 at a subculture concentration of 8 mg/L, whereas we found a mutation frequency of 3-4 × 10 −3 at a subculture concentration of 1 mg/L and a lower mutation frequency at 2 mg/L. Thus, the concentration of drugs in subculture plates is crucial to mutation frequency assays. Absent a subculture concentration standard, we applied rifampin at 1 mg/L (4) because bacteria growing at this concentration are considered resistant to rifampin. Our mutation frequency and time-kill kinetics assay results are not contradictory


s2fieldsofstudy: Biology Chemistry

text:
Structure and ubiquitination-dependent activation of TANK-binding kinase 1.

Upon stimulation by pathogen-associated inflammatory signals, TANK-binding kinase 1 (TBK1) induces type I interferon expression and modulates nuclear factor κB (NF-κB) signaling. Here, we describe the 2.4 Å-resolution crystal structure of nearly full-length TBK1 in complex with specific inhibitors. The structure reveals a dimeric assembly created by an extensive network of interactions among the kinase, ubiquitin-like, and scaffold/dimerization domains. An intact TBK1 dimer undergoes K63-linked polyubiquitination on lysines 30 and 401, and these modifications are required for TBK1 activity. The ubiquitination sites and dimer contacts are conserved in the close homolog inhibitor of κB kinase ε (IKKε) but not in IKKβ, a canonical IKK that assembles in an unrelated manner. The multidomain architecture of TBK1 provides a structural platform for integrating ubiquitination with kinase activation and IRF3 phosphorylation. The structure of TBK1 will facilitate studies of the atypical IKKs in normal and disease physiology and further the development of more specific inhibitors that may be useful as anticancer or anti-inflammatory agents.

Introduction
The NF-κB transcription factors are central regulators of innate immunity, inflammation, cell proliferation and apoptosis (Dolcet et al., 2005;Karin, 2006). Their activity is tightly regulated through the control of the IκB kinase (IKK) family of proteins. The canonical IKK complex consists of the catalytically active IKKα and IKKβ subunits as well as the regulatory subunit, IKKγ/NEMO (Hayden and Ghosh, 2004). In response to stimuli such as cytokines, non-degradative Lys63(K63)-linked and linear (Met1)-linked polyubiquitination of NEMO results in the activation of the IKKα and IKKβ kinases (Bianchi and Meier, 2009;Tang et al., 2003;Zhou et al., 2004). These activated kinases phosphorylate the inhibitor of NF-κB (IκB) proteins, resulting in their degradative Lys48(K48)-linked polyubiquitination and subsequent proteasome-mediated degradation. Upon degradation of the IκB proteins, the NF-κB dimers translocate into the nucleus and activate the transcription of effector genes that mediate immune and inflammatory responses and regulate cell survival (Hacker and Karin, 2006).
In addition to the IKKα and IKKβ kinases, two closely related serine-threonine kinases, Tank-binding kinase (TBK1) and inhibitor of κB kinase ε (IKKε) play key distinct roles in innate immune responses to viral infection and other pathogen-associated inflammatory stimuli by inducing type-I interferon expression and modulating NF-κB signaling (Bonnard et al., 2000;Peters et al., 2000;Pomerantz and Baltimore, 1999;Shimada et al., 1999). TBK1 and IKKε are found together in a complex and share several binding partners including TANK (Chariot et al., 2002;Goncalves et al., 2011), which facilitates interregulation of the canonical IKKs (Clark et al., 2011b). TBK1 is constitutively expressed and TBK1-deficent mice exhibit embryonic lethality due to widespread hepatic apoptosis, a phenotype that closely resembles IKKβ-deficient mice (Bonnard et al., 2000;Li et al., 1999). By contrast, the expression of IKKε is inducible and largely immune cell-specific, reflected in the observation that IKKε-deficient mice are viable, but hypersensitive to viral infection (Tenoever et al., 2007). IKKε-deficient mice are also less prone to diet-induced obesity and inflammation (Chiang et al., 2009).
Upon activation by Toll-like receptors (TLRs) or cytoplasmic RIG-1 like receptors (RLRs), TBK1 and IKKε stimulate type I interferon production via direct phosphorylation of transcription factors IRF3 and IRF7 (Chau et al., 2008). TLR-mediated activation of TBK1 involves TRIF or MYD88-dependent pathways, while engagement of RLRs activates the mitochondrial adaptor MAVS, which facilitates TBK1/IKKε mediated activation of IRF3/7 and NF-κB. Recently, the adaptor protein STING was found to play an essential role in the signaling response to cytoplasmic dsDNA, promoting TBK1-specific activation of IRF3 as well as STAT6 Ishikawa and Barber, 2008). TBK1 also plays a role in mediating autophagy in response to intracellular bacterial pathogens (Radtke et al., 2007;Thurston et al., 2009;Wild et al., 2011). Thus, TBK1 and IKKε play essential roles in both antiviral and antibacterial innate immunity.
In addition to their role in innate immunity, TBK1 and IKKε contribute directly to cell transformation (Shen and Hahn, 2011). IKKε is a breast cancer oncogene amplified in 30% of breast cancers. In these cancers, IKKε-mediated activation of NF-κB signaling is required for transformation, at least in part through phosphorylation of the tumor suppressor CYLD (Boehm et al., 2007;Hutti et al., 2009) and TRAF2 (Shen et al., 2012). In cancers dependent on KRAS-signaling, RALB-mediated activation of TBK1 promotes cell survival (Barbie et al., 2009;Chien et al., 2006;Xie et al., 2011). These observations implicate these two serine-threonine kinases as potential therapeutic targets in cancer.
Although the IKK-related kinases exhibit partial homology to the IKKα and IKKβ kinases, these kinases play distinct roles in both normal and malignant physiology. Indeed, the kinase domain of TBK1 shares only ~35% sequence identity with that of the canonical IKKs and the SDD domain is quite divergent with only ~10% identity over 250 residues. To understand the biochemical differences among these major immune-signaling kinases, we have undertaken structural and biochemical studies and describe a high-resolution crystal structure of nearly full-length TBK1 in complex with small molecule inhibitors MRT67307 (Clark et al., 2011a) and BX795 (Clark et al., 2009). The structure reveals a novel dimeric assembly that is stabilized by an extensive network of interactions among the kinase, ubiquitin-like (ULD) and scaffold/dimerization (SDD) domains. The contacts that stabilize the dimer are largely conserved in the close homolog IKKε, but not in the canonical IκB kinase IKKβ, which dimerizes in an unrelated manner (Xu et al., 2011). In addition, the SDD domain in TBK1 adopts a very different conformation from that recently observed in the corresponding domain of IKKβ (Xu et al., 2011). We find that TBK1 undergoes K63linked ubiquitination at sites in the kinase domain (Lys30) and SDD domain (Lys401). These modifications require an intact dimer, and are in turn required for TBK1 activation. Our data provide a structural foundation for understanding TBK1 regulation and for further dissection of the functional differences observed between the canonical IκB kinases and the IKK-related kinases TBK1 and IKKε.

Structure of TBK1
We crystallized and determined the structure of TBK1 (residues 1-657) in complex with inhibitors MRT67303 and BX795 at resolutions of 2.4Å and 2.5Å, respectively (see Supplementary Table S1 for crystallographic details). In addition, we refined a third structure crystallized under different buffer conditions at a resolution of 3.3 Å. To obtain protein in a homogeneous, non-phosphorylated state amenable to crystallization, we expressed the kinase-dead D135N mutant and truncated the C-terminal domain (CTD, Figure 1A). The crystal structure reveals an elongated dimer with a central stalk formed by the two SDD domains, which run roughly parallel to each other along the length of the dimer. The kinase and ULD domains interact with each other and with the SDD domain to form a globular head at one end of the stalk ( Figure 1B). As discussed below, the kinase and ULD domains also contribute to dimerization via multiple contacts with the SDD domain of the opposite subunit in the dimer. The SDD domain resembles a triple-helical coiled-coil and organizes the structure as a whole ( Figure 1B, Supplementary Figure S1B). Two of the SDD helices are continuous along the entire length of the domain; the first spans residues 408-481(α1) and the third spans residues 577-651(α3). The intervening segment is mostly helical (α2a, α2b, and α2c), but is interrupted by two loop segments that appear to accommodate the overall superhelical twist of the domain. The ULD and kinase domains associate primarily with the first helix in the SDD ( Figure 1C, E). The interactions of the ULD domain are particularly extensive and largely hydrophobic. The saddle-shaped surface formed by strands β1-β5 in the ULD domain arch across the SDD at approximately its midpoint, centered on Gly442. Phe380 in the ULD domain packs against Gly442, which is conserved in both IKKs and IKK-related kinases (Supplementary Figure S1A). Additional ULD/SDD interactions are illustrated in Figure 1C. The linker connecting the ULD and SDD domain adopts an extended conformation and packs in a hydrophobic groove between the first and third helices in the SDD ( Figure 1D). The extensive hydrophobic contact among the ULD and SDD domains and the intervening linker suggest that these elements comprise a fixed structural unit that is unlikely to dissociate or rearrange in the context of TBK1 recruitment and activation. The kinase C-lobe is also well anchored but largely by polar interactions with the SDD ( Figure 1E) and ULD domains ( Figure 1F). Structures have recently been reported for the isolated ULD  and kinase domains of TBK1 (Ma et al., 2012), and for a fragment containing both the kinase and ULD domains (Ma et al., 2012). Comparison with these structures reveals essentially identical interdomain orientations, indicating that the kinase/ULD interaction is well-defined irrespective of scaffolding interactions with the SDD (Supplementary Figures S1C, D).
The TBK1 kinase domain exhibits the conserved protein kinase architecture consisting of Nand C-terminal lobes with the active site cleft at the interface. In the present structures, the kinase domain adopts an inactive conformation, with the C-helix and key residue Glu55 displaced from the active site (Figures 2A, S1C). The loop that connects strand β3 to the Chelix is partially disordered. Rotations of the C-helix are a regulatory feature of many kinases, and a conserved glutamic acid in this helix makes a key salt bridge interaction with an active site lysine residue when the C-helix pivots into the inward, active position. In some kinases, the non-phosphorylated activation loop plays a role in maintaining the outward, inactive position of the C-helix. This appears not to be the case in TBK1, as the activation loop is largely disordered in the present structure. The most N-terminal portion of the activation loop, the DFG motif, is well resolved, but the remainder of the loop (residues 160-175) is not visible in the electron density map. TBK1 is activated by phosphorylation of Ser172 in the activation loop, and comparison with the recently described structure of the Ser172-phosphorylated kinase domain shows that the activation loop is indeed organized by phosphorylation (Ma et al., 2012), and that Ser172 phosphorylation also promotes the active position of the C-helix (Supplementary Figure 1C).

Inhibitor binding
The compound BX795 was developed as an inhibitor of the kinase PDK1 (Feldman et al., 2005) and was later discovered to potently inhibit TBK1 (Clark et al., 2009). The related compound MRT67307 was developed as a more specific inhibitor of TBK1 (IC 50 = 19 nM) (Clark et al., 2011a). These compounds share a common anilinopyrimidine core and bind in a similar manner, with the pyrimidine and aniline nitrogen atoms hydrogen bonding with the backbone amide and carbonyl groups, respectively, of Cys89 in the "hinge" segment at the edge of the ATP-binding cleft ( Figure 2B, Supplemental Figure S2). The pyrimidine and aniline rings are roughly co-planar, and in contact with Met142 and Gly92 on the floor of the nucleotide binding cleft. In MRT67307, the methylmorpholino substituent contacts Phe88 in the hinge, while the cyclobutyl group located on the pyrimidine 4-substituent packs between P-loop residues Gly18 and Val23 and Asp157 in the DFG motif ( Figure 2B). The lack of activity of MRT67307 on the canonical IKKs appears to stem from several structural differences within the binding site, in particular Phe88 in TBK1 is replaced with a tyrosine in IKKβ (which would interfere with the observed orientation of the morpholino group), and Thr156 is replaced with isoleucine (which would eliminate a water-mediated hydrogen bond with the carbonyl in the inhibitor).

TBK1 dimer contacts are conserved and required for signaling
The dimeric architecture of TBK1 appears to be critically important for its function and is created by conserved interactions involving each of its domains ( Figure 3A, B). The crystallographic asymmetric unit contains a single copy of the TBK1 polypeptide chain, and the dimer lies on a two-fold symmetry axis in the P3 2 21 space group. Examination of the crystal lattice reveals a second possible dimer relationship in which the subunits could associate via a "head to head" contact between the kinase domains to yield an extremely elongated dimer (Supplementary Figure S3A). Visualization of the purified TBK1 protein by negative stain electron microscopy confirms the side-by-side dimer interpretation described here (Supplementary Figures S3B and S3C). Analysis of the oligomeric state of TBK1 in solution using SEC-MALS showed that both the full-length protein (residues 1-729) and the crystallized construct are dimeric (Supplementary Figure S3D). Thus the truncated CTD region, which contains the binding site for TANK and other adaptors, is not required for the structural integrity of the dimer.
The dimer is stabilized by contacts between the ULD and kinase domain with the SDD of the contralateral subunit ( Figure 3A). Although there is little direct contact between the SDD domains in the 2.4 and 2.5Å (form I) structures described here, we observe considerable interaction in an alternate crystal form obtained using 2-methyl-2,4-pentanediol (MPD) rather than polyethylene glycol as a crystallization agent (form II crystals, Supplementary Figure S3E,F). The ULD domain contributes its "EGR" sequence (residues 355-357) to the dimer interface. In each of the ULD domains, Glu355 forms a salt bridge with Arg444 and also hydrogen bonds with the indole nitrogen of Trp445 in the SDD of the opposite subunit. Gly356 packs against the dimer-related Gly356 residue, and Arg357 forms a salt bridge with Asp452 in the contralateral SDD ( Figure 3A).
Both the N-and C-lobes of the kinase domain participate in dimer contacts with the SDD of the opposite subunit ( Figure 3A). In the N-lobe, a salt-bridge is formed between Asp33 in the kinase and Lys589 in the SDD. In the C-lobe, the β7-β8 turn contacts the SDD of the opposing subunit. While the dimer interactions do not block the kinase active site, they do appear to "clamp" the relative orientations of its N-and C-lobes. Because kinase activity is thought to require flexibility between the lobes, this arrangement is suggestive of an autoinhibited state of TBK1. Thus we measured the specific activity of dimeric TBK1, including both the full-length protein and a 1-657 construct (Supplementary Table S2). Both are highly active in vitro, with specific activity similar to that of the isolated kinase domain (residues 1-310), which is monomeric. The dimer interaction does not block activation loop auto-phosphorylation in trans, and the Ser172-phosphorylated protein is also dimeric (Supplementary Figure S3D). These data indicate that TBK1 activation does not involve dissociation of the dimer. Furthermore, the similar N-and C-lobe orientations observed in the activated kinase domain (Supplementary Figure S1C) suggests that the dimer interactions of the kinase domain proper could be largely maintained upon activation.
Mapping of the evolutionary variation among vertebrate TBK1 and IKKε sequences onto the surface of the protein shows that the dimer contacts are generally well conserved, indicating that the dimer is functionally significant and that IKKε is very similarly organized ( Figure 3B). Residues involved in the N-lobe contact are particularly highly conserved. We note also that the N-lobe contact on the SDD is part of a more extensive conserved surface that wraps onto the opposite face of the SDD; the conservation of this surface is striking in contrast to the remainder of the exposed surface of the SDD ( Figure 3B, right panel). We tested the importance of the dimer interaction surfaces for TBK1 activation and signaling ( Figure 3C). In the ULD dimer interface, the E355A, R357A, and E355A/R357A double mutation abrogated phosphorylation of TBK1 on Ser172 as well as phosphorylation of IRF3. Mutations in the kinase/SDD dimer interface also impaired TBK1 activation and IRF3 phosphorylation (D33A in the kinase domain; R547D and K589D in the SDD domain). By contrast, mutation of kinase residue K251, which is not involved in dimer formation, was without effect. Mutations in each of the dimer contacts also impaired activation of both interferon-stimulated response element (ISRE) and NF-κB-activated luciferase reporters ( Figure 3D). Moreover, expression of these mutants failed to induce expression of the direct IRF3 target gene IFNB1, in addition to RANTES ( Figure 3D). We also tested the dimer interface mutants for their ability to form dimers in vitro. The R547D and E355A/R357A mutants were largely monomeric in solution ( Figure 3E). However, the D33A and E355A single point mutations were not sufficient to disrupt the dimer in vitro, at least at the relatively high protein concentrations required for analysis by mutli-angle light scattering. We further found that the R547D and E355A/R357A mutants impaired dimer formation in a cellular context as well, as assessed by co-immunoprecipitation of Flag-and V5-tagged constructs expressed in HEK293T cells ( Figure 3F).
The larger conserved surface on the end of the SDD (including residues Tyr577, Glu580 and Ile582) also appears to be required for efficient IRF3 phosphorylation and expression of IFNB1 and RANTES mRNA ( Figure 3G). Strikingly, however, mutation of these residues does not impair TBK1 phosphorylation ( Figure 3G). These residues are not involved in dimerization or other interdomain contacts; thus, it will be of interest to test directly the role of these surfaces in recognition of TBK1 binding partners and substrates. TBK1 and IKKβ differ in their mode of dimerization and quaternary organization TBK1 and IKKβ are both dimers composed of sequential kinase, ULD, linker and SDD domains, but the sequence and conformations of their SDD domains are markedly divergent. Thus their mode of dimerization and overall quaternary organization is quite different. Interestingly, superposition of the ULD domains of TBK1 and IKKβ shows that both interact with the first long helix in the SDD domain, centered on a glycine residue that is conserved in primary sequence ( Figure 4A). This superposition supports the notion that the entire domain assembly of both TBK1 and IKKβ share a common evolutionary origin, and it also highlights the structural divergence in the SDD domains. Despite the observation that both share a "down-up-down" topology of the three mostly helical segments, their conformation is quite different and the domains overall cannot be superimposed along their entire length (Supplementary Figure S4). In IKKβ, the end of the final helical segment in the SDD mediates dimerization (Xu et al., 2011), while in TBK1 it is the end of the first helical segment that contributes to the SDD/SDD interface in the form II crystals (Supplementary Figure S3F, S4A). The divergence in the SDD domains begets larger differences in other domain interactions; the ULD domains in IKKβ extend away from the SDD domain of the opposite subunit, while in TBK1 they bridge between the two SDD domains ( Figure 4B).

K63-linked polyubiquitination of TBK1 on Lys30 and Lys401 is required for TBK1 activation and signaling
In the accompanying manuscript, Zhou et al. show that K63-linked polyubiquitination of IKKε at Lys30 and Lys401 is required for its catalytic activation. Mutation of Lys30 and Lys401 blocks IKKε kinase function, NF-κB activation and transformation. These modified lysine residues are conserved in TBK1 (with identical residue numbers, Figure S1A). Additionally, K63-linked polyubiquitination is required for viral activation of IRF3 (Zeng et al., 2009) and TBK1 associates with polyubiquitin-binding proteins including optineurin (Gleason et al., 2011). Thus we determined whether TBK1 is similarly modified by K63polyubiquitination. We co-transfected HEK293T cells with GST-TBK1 and either wild type HA-epitope tagged ubiquitin or a HA-tagged mutant ubiquitin that forms only K63-linked chains. When we isolated GST immune complexes and looked for HA-epitope tagged ubiquitin, we confirmed that TBK1 is also modified by K63-linked ubiquitination ( Figure  5A). This ubiquitination does not require TBK1 activity, as WT and a kinase-dead mutant TBK1 construct were similarly modified ( Figure 5B). Individual K30R and K401R point mutations exhibited decreased TBK1 ubiquitination, and we failed to observe ubiquitination of the TBK1 K30R/K401R double mutant ( Figure 5C). We next examined the effect of dimer-disrupting mutations on TBK1 ubiquitination. The E355A/R357D, R547D and D33A mutants exhibited markedly decreased K63-linked polyubiquitination in HEK293T cells ( Figure 5D). In contrast, the E355A and H459E/ N474A mutants that retained the ability to form dimers ( Figure 3E) were still ubiquitinated. We also found that the D33A mutant showed markedly decreased ubiquitination, despite retaining the ability to form dimers in solution. We note that Asp33 is proximal to the Lys30 ubiquitination site, thus this mutation may interfere with ubiquitination by affecting recognition of the modification site.
We next tested the effect of point mutations in the ubiquitination sites on TBK1-driven gene expression and on TBK1 activation. While the individual TBK1 K30R and K401R mutants induced IFNB1 and RANTES mRNA levels to a similar degree as WT TBK1, the TBK1 K30R/K401R mutant failed to induce IFNB1 and RANTES mRNA levels ( Figure 6A). Addition of the "phosphomimetic" S172E mutation did not bypass the requirement for ubiquitination, but this mutation has been previously shown to be ineffective in activating TBK1 (Kishore et al., 2002). Consistent with their effects on IFNB1 and RANTES expression, we observed that the single point mutants retained constitutive phosphorylation of Ser172, whereas the K30R/K401R double mutant was not phosphorylated, even when expressed at similar levels ( Figure 6B). It is unlikely that the K30R and K401R mutations affect folding or stability of TBK1, as both residues are exposed on the surface of the dimer ( Figure 6C). In addition, we note that the individual K30R and K401R mutants retained activity and at least some degree of K63-linked polyubiquitination and also supported TBK1 phosphorylation ( Figure 5C). We further observed that while dimerization and K63polyubiquitination mutants exhibited impaired TBK1 activation and signaling in cells, these mutants do not abolish TBK1 kinase activity in vitro (Supplementary Figure S5).
To determine the requirements for dimerization and ubiquitination in a physiologic setting, we reconstituted TBK1 −/− mouse embryo fibroblasts (MEFs) with wild type or mutant TBK1 constructs and tested the effect of poly I:C stimulation on interferon-β expression ( Figure 6D). Stimulation increased IFNB1 mRNA levels in cells reconstituted with wild type TBK1 at 2 and 4 hours, as expected. This response was ablated in cells reconstituted with dimerization defective TBK1 mutants E355A/R357A and R547D, and in the K30R/K401R polyubiquitination mutant ( Figure 6D). We conclude that like IKKε (Zhou et al. accompanying manuscript), TBK1 undergoes K63-linked polyubiquitination on conserved Lysines 30 and 401. Polyubiquitination on at least one of these two sites and dimer formation are required for phosphorylation of Ser172 in the kinase activation loop and regulation of IRF-responsive genes.

Discussion
TBK1 is engaged by a diversity of stimuli, yet how it is activated in these contexts remains incompletely characterized. In the setting of cytosolic dsDNA, STING acts as a scaffold that recruits IRF3 and TBK1 to its C-terminal tail (Tanaka and Chen, 2012). Oligomerization of STING and ER localization upon cytosolic DNA detection recruits both TBK1 and IRF3, promotes TBK1 Ser172 phosphorylation, and thus facilitates specific activation of IRF3. A similar mechanism may facilitate phosphorylation of STAT6 by TBK1 in the setting of antiviral innate immunity . Recent work has also demonstrated that viral infection and RLR-mediated MAVS activation induces large functional aggregates on the mitochondrial membrane that involve K63-linked polyubiquitination and recruitment of TRAF2 and TRAF6 (Hou et al., 2011). Recruitment of NEMO to these MAVS complexes may bind TANK and TBK1 to promote its activation (Zeng et al., 2009). A similar model was proposed for MYD88-dependent TBK1 activation that occurs downstream of IL1 or specific TLRs (Clark et al., 2011b). In this setting K63-linked polyubiquitin chains formed by TRAF6 are proposed to recruit a complex including NEMO and TANK that links TAK1 and the canonical IKK complex to TBK1/IKKε Ser172 phosphorylation and activation.
In addition to these recruitment models, direct K63-linked polyubiquitination of TBK1 by TRAF3, NDRP1, or MIB1 E3 ligases may be involved in its innate-immune mediated activation of TBK1 Parvatiyar et al., 2010;Wang et al., 2009). CYLDmediated deubiquitination of TBK1 has also been shown to inhibit the TBK1-driven IRF3 response (Friedman et al., 2008). A recent report identifies Lys69, Lys154 and Lys372 as K63-linked polyubiquitination sites in TBK1 (Wang et al., 2012). From a structural perspective, two of these sites appear unlikely to represent regulatory modification sites. Lys154 forms key structural interactions, and is near-universally conserved among all protein kinases. Mutation or modification of this residue can be expected to disrupt the fold of the kinase domain. Lys372 is among the most variable residues in vertebrate TBK1 and IKKε sequences, and access to this residue appears to be impeded in the TBK1 dimer. Lys69, however, is relatively exposed for modification and lies near the Lys30 and Lys401 sites we have identified. In the accompanying manuscript, Zhou et al. have dissected the essential role of K63-linked polyubiquitination in the regulation of IKKε in the context of both cell transformation and immune signaling. Interestingly, the two essential residues for IKKε K63-linked polyubiquitination, Lys30 and Lys401, are conserved with TBK1. We demonstrate here that K63-linked polyubiquitination of TBK1 at Lys30 and Lys401 is essential for its downstream activity. Our structural work reveals that these two residues are found on opposing faces of the TBK1 monomers but are in close proximity in the context of the catalytically active TBK1 dimer ( Figure 6C). Furthermore, mutation of both sites disrupts signaling to IFN-β following TBK1 overexpression in HEK293T cells, or upon poly I:C stimulation in TBK1-null MEFs. TBK1 Ser172 phosphorylation is also disrupted in the K30R/K401R mutant, suggesting that ubiquitination is directly required for TBK1 activation in a cellular milieu.
The mechanistic role of K63-linked ubiquitination at these sites remains to be fully elucidated. The active sites of the two kinase domains lie on opposite faces of the TBK1 dimer, and therefore the isolated dimer cannot autoactivate. Thus K63-linked ubiquitination may regulate recruitment of another kinase that promotes Ser172 phosphorylation, and it may promote assembly of TBK1 into higher order complexes that allow its autoactivation in trans. In support of this model, NEMO has been shown to bind to polyubiquitinated TBK1 via its ubiquitin binding domains (Wang et al., 2012). Furthermore, TBK1 binds the NEMOrelated protein optineurin as well as adaptor NDP52, both of which recognize K63-linked polyubiquitinated proteins (Morton et al., 2008;Thurston et al., 2009;Wild et al., 2011). Mutation of the ubiquitin recognition domain of optineurin impairs activation of TBK1 in response to the TLR agonists LPS and polyI:C (Gleason et al., 2011). While a polyubiquitindependent recruitment or clustering mechanism appears to be an essential component of TBK1 activation, we cannot rule out the possibility that ubiquitintation could also play an allosteric role by releasing or modifying intramolecular contacts of the kinase N-and Clobes to promote TBK1 activation.
Taken together, these observations reveal a multi-step mechanism for TBK1 activation in which K63-linked polyubiquitination is a pre-requisite for kinase activation by Ser172phosphorylation in cells. The dimeric, multidomain architecture of TBK1 is critical for orchestrating these regulatory events, and for subsequent phosphorylation of IRF3. Dimer interface mutations in the kinase (D33A), ULD (E355A/R357A) or SDD domains (R547D) block K63-linked polyubiquitination, and thus also prevent Ser172 phosphorylation. Our biophysical characterization of the monomeric proteins shows that they are not simply misfolded, and we find that even the isolated (monomeric) kinase domain retains kinase activity in vitro. It is unclear whether kinase activation is the only role of polyubiquitination, because the S172E phosphomimetic mutant does not effectively activate TBK1 (Kishore et al., 2002). In addition to its roles in mediating dimer formation and as a site of polyubiquitination, the SDD domain is also required for proper substrate phosphorylation in cells -the conserved surface surrounding Glu580 is required for IRF3 phosphorylation and reporter activation, despite the fact that it is dispensable for TBK1 activation per se. The SDD is not required for efficient IRF3 phosphorylation in vitro, as the isolated kinase domain and intact TBK1 phosphorylate IRF3 with similar kinetics (Ma et al., 2012). We speculate that this surface may be a site of interaction with a substrate-recruiting adapter such as STING (Tanaka and Chen, 2012).
The conserved Glu580 surface of the TBK1 SDD domain, as well as its dimer contacts and polyubiquitination sites are preserved in IKKε, but not in the IKKα and IKKβ. Furthermore, the SDD-and ULD-mediated contacts with the N-and C-lobes of the kinase are not found in the IKKβ structure, which dimerizes in an essentially unrelated manner. Although the canonical IκB kinases and the IKK-related kinases TBK1 and IKKε share some homology, the dramatic structural divergence between these classes of IKKs reflects their different biology and regulation. Indeed, although TBK1 and IKKε were discovered vis-à-vis their intersections with NF-κB signaling, it is increasingly clear that many of their major functions are quite distinct from this pathway and from those of canonical IKKs. The present data provide a structural foundation for understanding the regulation and interactions of both TBK1 and IKKε, and for further dissection of participation of TBK1 in diverse processes including viral clearance, autophagy (Weidberg and Elazar, 2011;Wild et al., 2011), and survival of KRAS-dependent cancers (Barbie et al., 2009;Ou et al., 2011;Xie et al., 2011). Additionally, the structures described here will facilitate the development of small molecule inhibitors that may be useful as anti-cancer or anti-inflammatory agents.

Experimental Procedures Expression Constructs
The pCMV6-XL5-TBK1-WT and pCMV6-XL5-TBK1-K38M constructs were purchased from Origene and PCR subcloned using Gateway technology into the pLEX980 vector. Sequencing of pLEX980-TBK1-WT and pLEX980-TBsK1-K38M confirmed that these sequences were correct. TBK1 mutants of the pLEX980-TBK1-WT construct were generated using standard PCR mutagenesis, Gateway cloned back into the pLEX980 or the closely related pLEX304 vector, and sequence verified. HA-ubiquitin and HA-Ub K63-only constructs were used as described (Abbott et al., 2004;Boehm et al., 2007).

Protein Expression, purification and crystallization
Constructs spanning residues 1-310, 1-657 or full length (1-729) of human TBK1bearing the wild-type sequence or D135N mutation were expressed as 6xHis plus glutathione-Stransferase (GST) fusion proteins in Hi5 insect cells using the pTriEx transfer vector (Novagen) and BacVector-3000 Baculoviral DNA (Novagen). Details of purification by Nickel-NTA and glutathione sepharose chromatography are given in Supplemental Methods online. The TBK1(1-657) D135N protein protein was concentrated to 8 mg/ml for, for crystallization. MRT67307 or BX-795 was added to the TBK1 protein solution to 0.25 mM immediately prior to crystallization. Crystals of form I were obtained in 4% (w/v) PEG8000 and 100 mM Tris pH 8.7. Crystals of form II grew in 5% MPD and 100 mM Hepes pH 7.5. The SeMet TBK1 protein was crystallized at 7 mg/ml with BX795 added to 0.5 mM in hanging drops over a reservoir containing 5% PEG6000, 2.5% MPD, 100 mM Hepes pH 7.5, and 10 mM TCEP.

Structure determination
The structure was determined by SAD phasing with a 3.65 Å SeMet derivate dataset collected on the NE-CAT beamline at Argonne National Laboratory. The TBK1/BX795 structure was refined to a final R-value of 19.7% (Rfree=25.5%) at 2.5Å resolution, and the TBK1/MRT67307 to a final R-value of 19.9% (Rfree=23.6%) at 2.4 Å resolution. The form II crystal structure was refined to an Rvalue of 22% (Rfree=27.9%) at 3.3Å resolution. Crystallographic data collection and refinement statistics are presented in Supplementary  Table S1, and further details of structure determination and refinement are provided in Supplemental Methods online.

In-vitro enzyme kinetics and other biophysical studies
Kinase assays were carried out in triplicate using the ATP/NADH coupled assay system in a 96-well format as described (Yun et al., 2007). Further details and experimental procedures for IP kinase assays, in vitro phosphorylation of kinase-dead TBK1 proteins, SEC-MALS, and Electron Microscopy are provided in Supplemental Methods online.

Reporter Assays and Quantitative Reverse-Transcriptase PCR
HEK293T cells were seeded in 6 well plates at 7 × 10 5 cells/well, and reverse transfected using Fugene 6 Reagent. Cells were transfected with 1µg pLEX980-EGFP, pLEX980-TBK1-WT, TBK1-K38M, or the indicated TBK1 mutants together with 500 ng NFκB(1) Firefly luciferase reporter vector (Panomics) or 500 ng ISRE Firefly luciferase reporter vector (Panomics), and 500 ng pRL-TK Renilla luciferase control vector. At 24 h posttransfection cells were trypsinized and seeded at a density of 2500 cells/well in 384-well plates, with 64 replicate wells per condition. At 48 h post-transfection, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega) or RNA was prepared for QRT-PCR, as described in Supplemental Methods.

Dimer Immunoprecipitation
N-terminal FLAG and C-terminal V5-tagged pLEX-TBK1-WT, pLEX-TBK1-E355A/ R357A, and pLEX-TBK1-R547D constructs were transiently transfected into HEK-293T cells and 48h later cells were lysed in RIPA buffer. FLAG immunoprecipitation was performed in RIPA buffer using FLAG conjugated agarose beads (Sigma). Immunoblotting of whole cell extracts and immunoprecipitated material was performed with a mouse anti-FLAG antibody (Sigma) or mouse anti-V5 antibody (Invitrogen).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Highlights
• TBK1 forms a dimer structurally distinct from IKKβ • Crystal structure of TBK1 with inhibitor MRT67307 reveals basis for specificity versus canonical IKKs • TBK1 is K63 polyubiquitinated on Lys30 in the kinase domain and Lys401 in the SDD domain • K63-linked ubiquitination of dimerized TBK1 is required for kinase activation and signaling    (A) Superposition of TBK1 with IKKβ (magenta). Structures are superimposed using the ULD, which highlights the general similarity in the manner in which the ULD anchors the kinase domains (KD) to the SDD in both proteins. This structural alignment also superimposes a glycine residue (black dot) in the ULD/SDD interface (Gly442 in TBK1 and Gly450 in IKKβ) that is conserved in both proteins and occurs in the same position in their primary sequences, despite the overall divergence in their SDD domains (see also Supplementary Figure S4E, F). Note also that the orientation of the kinase domains in the two structures is quite different relative to the ULD and SDD domains. (B) Surface views of TBK1 (top panels) and IKKβ (lower panels), with corresponding domains of IKKβ colored as in TBK1. In TBK1, the ULD domains bridge between dimer-related SDD domains, while in IKKβ they extend away from the opposite SDD domain. Likewise, the kinase domains in IKKβ are differently oriented and do not form dimer contacts. The IKKβ structure is drawn from PDB code 3QA8 (Xu et al., 2011). (A) TBK1 is K63-linked polyubiquitinated. HA-tagged wild type or K63-only ubiquitin were cotransfected with GST-TBK1 into HEK293T cells. GST immune complexes (TBK1) were isolated followed by immunoblotting with the indicated antibodies. 5% of the whole cell lysate was loaded for comparison (input). (B) Wild type and kinase dead TBK1 are ubiquitinated equally. HA-tagged K63-only ubiquitin were cotransfected with wild type (WT) and kinase dead (KD) GST-TBK1 into HEK293T cells. GST immune complexes (TBK1) were isolated followed by immunoblotting with the indicated antibodies. 5% of the whole cell lysate was loaded for comparison (input). (C) TBK1 undergoes K63-linked polyubiquitination at Lys30 and Lys401. HEK293T cells were transfected as indicated. V5 immune complexes (TBK1) were isolated followed by immunoblotting with the indicated antibodies. 5% of the whole cell lysate was loaded for comparison (input). (D) TBK1 dimerdeficient mutants do not undergo K63-linked ubiquitination. HEK293T cells were transfected as indicated. V5 immune complexes (TBK1) were isolated followed by immunoblotting with the indicated antibodies. 5% of the whole cell lysate was loaded for comparison (input). ubiquitination mapped on TBK1 structure. One TBK1 monomer is in green, the other in cyan. Note that Lys401 from monomer "a" is close to Lys30 of monomer "b" across the dimer interface. (D) qRT-PCR measurement of IFNB1 and TBK1 mRNA following TBK1 reconstitution in TBK1−/− MEFs and stimulation with polyIC. Cells were nucleofected with