extfieldsofstudy: Medicine Biology

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Upsetting the Balance: When Viruses Manipulate Cell Polarity Control

The central importance of cell polarity control is emphasized by the frequency with which it is targeted by many diverse viruses. It is clear that in targeting key polarity control proteins, viruses affect not only host cell polarity, but also influence many cellular processes, including transcription, replication, and innate and acquired immunity. Examination of the interactions of different virus proteins with the cell and its polarity controls during the virus life cycles, and in virally‐induced cell transformation shows ever more clearly how intimately all cellular processes are linked to the control of cell polarity.

Introduction
The control of cell polarity is a fundamental characteristic of multicellular organisms. Correct cell polarity leads to the ordered orientation of cells, both internally and with respect to one another, that is essential for the development of ordered systems, from intracellular organelles such as the endoplasmic reticulum, to the highly complicated networks of the brain. This being so, it is inevitable that the mechanisms and controls of cellular polarity will both affect and be affected by the presence of intracellular parasites such as viruses.
The control of normal cell polarity and tissue structure essentially defines most multicellular organisms, and its loss or misregulation essentially defines most tumors [1]. Cell polarity can be divided into four main types (shown in Fig. 1) that are highly conserved throughout evolution, with many vital regulatory elements being conserved from Drosophila melanogaster and Caenorhabditis elegans through to mice and humans [2]: apico-basal polarity (ABP), which describes the orientation of cells perpendicular to the basement membrane; planar cell polarity (PCP), describing their orientation parallel to it; migrating cell polarity (MCP), defining the leading and lagging edges of a migrating cell; and cell division polarity, defined by the difference between symmetric and asymmetric cell division in tissue differentiation.
Additional, more tissue-specific, forms of polarity control include neuronal cell polarity, defining the direction of neuronal signal transmission, and immune cell polarity (ICP) induced in activated cells of the immune system, such as T lymphocytes, macrophages or dendritic cells [3].
PCP is controlled by factors in the non-canonical Wnt/PCP pathway and by the interplay of a large number of signaling pathways in response to mechanical and biochemical forces [4], and PCP is established through the asymmetric and mutually exclusive localization of a highly conserved core set of proteins. These include the Scribble (Scrib) complex proteins, Scribble (Scrib) and Discs Large 1 (Dlg1), which are also critical regulators of ABP (see below), and which may represent the intersection of PCP and ABP controls [5]. PCP is essential for oriented cell division, collective cell migration and the development and function of many tissues [6].
ABP is a defining characteristic of epithelial cells and is also controlled by the intersection of several pathways, but at its core is a multi-macromolecular interaction network (Fig. 2) of three distinct protein complexes: the Scrib, Partitioning defective (Par) and Crumbs (Crbs) complexes [2,7,8]. These connect cell polarity with the control of cell-cell contacts via multiple scaffolding proteins at tight junctions (TJs) [9] and cadherins at adherens junctions (AJs) [10,11]. They also connect ABP with cell proliferation control through mitogen-activated signaling pathways [12,13], the control of cell size through the Hippo pathway [14,15], and directional cell migration and apoptosis through small GTPase signaling [16][17][18]. The function of this network is critically dependent on a number of factors, including precise regulation of the expression level and posttranslational modifications of the network components [2]. ABP regulation is fundamentally dependent on the mutually exclusive localization of different network components, which depends, at least in part, upon differential phosphorylation of the Scrib and Par complex proteins, some of which is regulated through the atypical protein kinase C [19,20]. This differential localization of components divides the cell into different zones, each defined by the presence of one of the three complexes of the pathway (Fig. 2 and other chapters in this volume); perturbation of the correct spatio-temporal localization of the individual components has profoundly deleterious effects on the functioning of the whole complex.
Studies in lower eukaryotes, such as Drosophila and C. elegans, show that perturbing any one of the proteins that function in this network can have very adverse effects on the overall functioning of the ABP pathway and have defined many of these proteins as potential tumor suppressors [21]. However, in higher eukaryotes, the picture is more confused; although expression of many polarity regulators is lost in tumors [22,23], it is often unclear whether this is a cause or a consequence of the tumor development.
However, in recent years, studies have shown that many human viruses, both oncogenic and not, target certain key polarity regulators, demonstrating the fundamental involvement of the ABP control network not only in different viral replication strategies, but also in in tumor suppression and tumorigenesis. Targeting of many of these cell polarity regulators by oncogenic viruses, such as human papillomaviruses (HPV), has focused attention on the cell proliferative regulating activities of these proteins, and how this is connected to the control of polarity. However, many non-oncogenic viruses target many of the same pathways as a means of avoiding immune detection, both through innate and acquired mechanisms. This therefore highlights the incredible multifunctional nature of these cell polarity regulators and emphasizes why viruses targeting these pathways can achieve multiple aims, all of which contribute to a successful virus infection: the generation of a replication-permissive environment, and the avoidance of immune detection. In rare cases, the combination of such perturbation of cellular homeostasis with a long-term persistent infection can give rise to events that ultimately result in malignancy.

Tumor Viruses and Polarity Control
Tumor viruses have been estimated to cause up to 12% of all human cancers [24,25]. These viruses, as stated above, are from widely differing evolutionary backgrounds and cause many different malignancies. However, they commonly attack the same pathways, targeting, either directly or indirectly, some of the central elements of polarity control [26,27] (see Fig. 2). The purpose of this targeting, from the point of view of the virus, is to render the cellular environment more conducive to viral replication, and any resulting malignancy is a rare event and often prevents an effective viral life-cycle. This emphasizes the importance of host cell polarity regulation to the replication strategy of the virus.
The majority of tumor viruses exhibit long-term persistent infections in the host, infecting precursor cells and using the cellular differentiation program for their replication. Examples include B-cell precursors infected by Epstein-Barr virus (EBV), endothelial precursors infected by Kaposi's sarcoma herpesvirus (KSHV), and epithelial precursors infected by the HPVs [28][29][30]. Long-term or persistent infections may continue for years and in some cases are lifelong, allowing the viral oncoproteins, or the oncogenic by-products of the viral life cycles, to interact with the cellular environment over long periods and to initiate the processes that lead eventually to malignant transformation and cancer.

HPV
HPVs are major carcinogens, being responsible for around 5% of all human cancers worldwide [31]. HPV-related malignancies are characterized by their addiction to the two viral oncoproteins, E6 and E7, which contribute directly to the development and maintenance of the tumor and of cells derived therefrom [32,33], even after many years in tissue culture [34]. They act together to immortalize keratinocytes [35], which are the natural target cells of the virus, and have been shown to cause tumors in transgenic mouse models [36][37][38]. Thus, E6 and E7 have the potential to be excellent therapeutic targets [39].
HPV replication occurs in terminally differentiating keratinocytes and is completely dependent upon the differentiation program of the epithelium [40]. The E6 and E7 proteins cooperate to reactivate the cell cycle in these cells; E7 induces cell cycle re-entry, while E6 abrogates the resulting pro-apoptotic responses [39,41,42]. While usually resulting in a productive infection of a few months' duration, this perturbation of cellular Cell polarity pathways are targeted by diverse human viruses. Interplay between the Scribble (SCRIB), PAR, and Crumbs (CRB) complexes is central to the control of cell polarity. These link to the TJ and AJs that control cell-cell contacts, and to the Wnt signaling pathway, through the SCRIB complex and cadherins. The CRB complex links to cell growth and proliferation through the Hippo signaling pathway and YAP/TAZ. The SCRIB complex also links to apoptotic pathways through β-PIX, and to growth factor signaling-induced proliferation through ERK, and to interferon-activated innate immune responses through STAT activation. The virus proteins are indicated by hexagons: red for cancerassociated virus proteins or blue for non-cancer causing. controls can initiate the steps that lead eventually to malignant transformation.

Polarity targeting by HPV E6
Of the nearly 200 known papillomavirus types, only a few high-risk HPV types are defined as cancer causing and, of these, HPV types 16 and 18 are the most clinically important. These viruses all have a PDZ (PSD-95/Dlg1/ZO1)-binding motif (PBM) at the C-terminus of their E6 proteins, which is largely absent from those of low-risk viruses, which cause only benign lesions. The high-risk E6 proteins are thus able to bind to a number of polarity control proteins that contain PDZ domains. These include core components of ABP cell polarity control-hDlg1, hScrib, and PATJ-and the TJ protein MAGI-1, which are targeted by E6 for ubiquitinmediated degradation at the proteasome, thus disrupting the stoichiometric equilibrium of these complexes [43][44][45][46]. However, it is becoming very clear that all HPV E6 PBMs are not equal, and that subtle differences in the exact amino acid sequence of the PBM can markedly alter the substrate selection, as can be seen from Table 1, the HPV E6 PBMs, whilst all having a core consensus PBM, have significant variation in the non-canonical amino acid residues. Indeed these differences can profoundly affect substrate selection, and the molecular basis for these differences has been extensively explored [47][48][49], showing the effect that even minor changes can have; for example, HPV-16 E6 was shown to preferentially interact with hScrib, and HPV-18 E6 with Dlg, and swapping the final amino acid also swaps these preferences [50]. More recently, the greater functional flexibility of the E6 PBM (i.e., the greater the number of different PDZ domain-containing proteins that it can bind) has been found to correlate with the degree of association of that HPV type with cervical cancer [51]. Interestingly, all of the PBMs tested in that study, including those from HPV types rarely (HPV-26, HPV-66), or never (HPV-40), found in cancer, were able to interact with hDlg, suggesting that this is a fundamental requirement for the replication of this group of HPV types. Interaction with hScrib and the TJ protein ZO2, however, was limited to the PBMs of virus types with a strong cancer association (HPV types 16,18,31,33,35,51), suggesting that the greater disruption of the Scrib complex caused by the combined loss of hScrib and hDlg1, particularly in the context of a long-term persistent infection, has a greater risk of disrupting cellular polarity in a manner that favors malignant transformation. Indeed, this has been recently confirmed in cellular transformation assays [52]. Human cervical keratinocytes exogenously expressing activated Ras, plus HPV-16 wild-type E6 and E7, are capable of anchorage-independent growth and are tumorigenic in nude mice. Expression in this system of an E6 defective in PDZ-binding prevents this, but the tumorigenic phenotype can be slightly rescued by the individual ablation of DLG1 or DLG4 (also known as PSD-95) and partially rescued by ablation of either hScrib, MAGI-1 or PAR3. However, ablation of hScrib plus DLG1 and DLG4, or hScrib plus MAGI1 and PAR3 in this system actually enhances the tumorigenic phenotype above that of cells expressing the wild-type HPV-16 E6 [52], demonstrating the pivotal importance of hScrib targeting in HPVinduced malignancy.
It is interesting to note that in the rhesus papillomavirus (MmPV1), which causes cervical cancer in rhesus macacques (Macaca mulata), E6 has no PBM but instead the E7 oncoprotein has a PBM [53]. This has a very different sequence from that of the HPV E6 PBMs and structurally would appear to be quite diverse [47,49]. Not surprisingly, proteomic and biochemical analyses showed that it binds to a different polarity regulator, the PAR3 protein [53], part of the PAR complex. Thus, MmPV1 targets the same overall polarity regulatory pathway as HPVs, and through the same mechanism, but using a different viral protein and with different cellular partners, demonstrating a marked evolutionarily conserved requirement for these viruses to target this pathway for viral replication, and most likely for their ability to cause cancer.
Evolutionary conservation of the polarity proteins is also illustrated in a new animal model for HPV E6 activity, where HPV E6 can bind to the Drosophila MAGI protein in a PBM-dependent fashion, and, if exogenously supplied with a human UBE3A (E6AP) ubiquitin-protein ligase, can induce its degradation [54]. Intriguingly, this model shows that a key factor in HPV-induced malignancy is the E6AP, which is used by E6 for substrate degradation, but is also required for E6 stability [55]. The Drosophila E6AP lacks the E6 interaction motif, and hence, human E6AP is required for E6 to exert a phenotype in flies. Notably, although both Dlg and MAGI would appear to be perturbed in this model by E6, it is MAGI that appears to play the critical role in the tumor phenotype [54].

Cell polarity and the HPV life cycle
The life cycle of HPVs is completely dependent on the differentiation program of the infected epithelium, and studies of infection in organotypic raft cultures show that viruses defective in binding the polarity proteins produce fewer progeny virus and their genomes are more unstable and prone to integration in the host DNA [56][57][58].
The reason for loss of correct virus genome segregation in the absence of the E6 PBM is as yet unknown, although it may be related to the levels of cell proliferation in the lesion. The E6 PBM plays a key role in expanding the number of proliferating cells (i.e., those capable of replicating viral DNA), by targeting the polarity proteins to uncouple the link between cell polarity control and cell proliferation control. The orderly asymmetric cell division seen in the normal differentiating epithelium is maintained by strict control of mitotic spindle orientation, maintenance of ABP, and correct formation of the cell-cell junctions [59,60]. The HPV E7 protein inappropriately stimulates cell cycle progression in the epithelial mid-layer, while E6 perturbs the Scrib and Par complexes to expand the population of infected cells capable of replicating the viral DNA. Expression of E7 alone has been shown to induce the formation of aberrant spindle poles [61,62], while disruption of Dlg and the Par complex also perturbs mitotic spindle orientation [63,64], all of which contributes to enhanced symmetrical cell division, thus expanding the population of replicationcompetent cells and accounting for the disordered epithelium observed in viral lesions.
As noted above, cell-cell communication through cell junctions is also affected in HPV-infected cells. MAGI-1 is targeted for degradation by high-risk HPV E6 proteins [65], possibly to counteract the signaling role of non-junctional MAGI-1 in the induction of apoptosis [66]. Indeed, when a mutant MAGI-1, which was no longer susceptible to E6-induced degradation, was re-expressed in HeLa cells, it was found to induce cell growth arrest and apoptosis [67]. On the other hand, E6 induces the stabilization of the TJ protein ZO-2, which appears to increase cell proliferation and enhance the wound healing ability of HeLa cells [68,51]. In addition, both E6 and E7 Table 1. The PBMs of viral proteins targeting cell polarity and their potential phosphocontrol. Many of the viral proteins that target cell polarity control do so by binding the PDZ domains of polarity control proteins through a PBM. The table shows the sequences of such viral PBMs, their cellular targets, and the potential of the PBM to be modified by phosphorylation. The T/S amino acid residues in green have been shown to be phosphorylated by the indicated kinase [76,263]; those in blue have consensus kinase recognition sites, but phosphorylation has not been formally demonstrated. target AJs by downregulation of E-cadherin, at least partly through the induction of Cdc6 [69][70][71][72], thus increasing proliferative signaling. Potentially, this disruption of cell junction control, combined with enhanced proliferation and decreased apoptotic signaling, could also contribute to the disordered epithelial structure characteristic of HPV lesions and, further, increase the risk of pro-oncogenic mutations arising in those lesions.

Regulation of PBM/PDZ binding
It is becoming very clear that PBM/PDZ binding, while allowing proteins a wide flexibility in choice of binding partners, is subject to a greater level of specificity and control than was originally thought [73]. The steric and electrostatic characteristics of the respective PBM and PDZ sequences give an irreducible level of specificity to the interactions of the papillomavirus PBMs [47][48][49]. However, the HPV PBMs are also bi-functional: there is a phosphoacceptor site embedded within the PBM, which, when phosphorylated, prevents PDZ binding [74] and instead confers affinity for proteins of the 14-3-3 family [75]. Examples of phospho-acceptor sites on the PBMs of diverse viral proteins are shown in Table 1. These proteins link to a variety of different pathways, including Hippo and p53 regulation, while kinases such as AKT and PKA have been implicated in regulating E6 interaction with the cell polarity regulators [76]. Recent data also suggest that the E6 PBM may be phosphorylated by DNA damage response kinases, allowing E6 to modulate p53's transcriptional transactivation activity [77]. It seems likely that this would reduce p53's apoptotic response, while allowing the cellular DNA damage repair enzymes to enhance viral genome replication [78,79]. Taken together with studies that show phospho-regulation of Dlg for E6 regulation [80,81], these findings highlight a complex pattern of phospho-regulation controlling the E6 PBM interactions. Perturbation of these controls is likely to have profound consequences for successful viral life cycles, and for the potential progression to malignancy.

HPV-induced malignancy and cell polarity
As mentioned above, the productive HPV life cycle occurs in the differentiating squamous epithelium: the virus infects cells in the basal layer of the stratified epithelium and progeny virions are shed as the surface layer of cells sloughs off. There is considerable debate surrounding the identity of the cells that are subject to malignant transformation: are they squamous cells in which a normally productive infection has stalled and become persistent? or are they cells that do not stratify, such as the columnar cells of the simple epithelium of the endocervix? It is clear that the majority of cervical (N 99%) and anal (N 90%) tumors occur at the transformation zone between the stratified and columnar epithelia [82], and many cervical tumor cells have an embryonic stem cell expression signature [83]. It has been suggested that many cervical tumors may have their origin in the cuboidal cells of the squamocolumnar junction: these cells are more accessible than the endocervical cells; they are highly susceptible to infection by HPV, but are unable to stratify and permit a productive infection; and they are located in an anatomical site that appears to be somewhat immuneprivileged. Thus, they appear to be convincing candidates for malignant transformation [84,85], and interestingly, a similar population of cells has been described at the anorectal junction [84], where HPV is also a potent carcinogen. However, other studies suggest that they arise in a population of stem cells, known as reserve cells, that maintain both stratified and columnar epithelia, depending upon tissue context [86,87,30]. Interestingly, while the majority of HPV-16-induced cervical cancers are derived from the squamous cells of the ectocervix, HPV-18 is found in a very high proportion of adenocarcinomas [88], which would be consistent with infection of the columnar epithelium of the endocervix. These differences may reflect the finding that E6 PBMs from different HPV types have distinct binding profiles with respect to their cellular PDZ-containing targets, many of which are polarity control proteins [51], and it would be interesting to further determine whether the E6 PBM has any role in the frequency with which different HPV types are found associated with adenocarcinomas.
Studies in transgenic mice have shown that the E6-PDZ interaction is required for the ability of E6 and E7 to induce hyperplasia and tumors in the skin and in the cervix [89,90], suggesting that deregulation of polarity control contributes to malignant transformation. This is further confirmed by the finding that E6 proteins mutated in the PBM lose the ability to induce an epithelial-to-mesenchymal transition (EMT) in keratinocytes and fail to cooperate with E7 in inducing cell transformation in certain in vitro settings [91]. E7 downregulates E-cadherin expression by epigenetic modification [70] and E6 also has a downregulatory effect, probably through the degradation of PDZ domain-containing polarity complex proteins leading to the perturbation of cellcell adhesion complexes [91,92].
Up to 20 different PDZ domain-containing targets of high-risk E6 proteins have been described [93], many of them targeted by E6 for ubiquitin-mediated degradation, and it is not clear which of these losses is/are associated with the induction of malignancy. Dlg1 was the first PDZ-containing target of HPV E6s to be identified [43] and seemed for some years to be a likely candidate, but recent work suggests that E6 PBMs from many HPV types can bind to Dlg1, regardless of the degree of association with cancer [51]. As shown in Fig. 3, this may point more to this association being an adaptation for the viral life cycle and replication at mucocutaneous anatomical sites, rather than to an association with malignancy [94], and suggests that the ability to target hDlg1 may be a necessary but not sufficient feature of cancercausing HPV types. Other E6 PBM degradation targets that have also been validated in vivo [44,95], such as MAGI-1 and hScrib, are also associated with polarity control, and the ability to bind to hScrib appears to be confined to those HPV types with highest cancer risk [49]. The key element here appears to be the degree of flexibility of the E6 PBM, with the ability to bind multiple PDZ-containing cell polarity regulators conferring the ability to target multiple signaling pathways.
Interestingly, it has recently been shown that hScrib expression in HeLa cells is required to maintain E6 expression levels, through hScrib regulation of mTORC signaling [96]. In the development of HPVinduced tumors, disruption of polarity control appears to drive malignancy, with both hDlg1 and hScrib being mislocalized, and often overexpressed, in medium-to high-grade lesions [97,98]. This suggests that in certain cellular locations, both hDlg1 and hScrib might have pro-oncogenic properties: studies that ablated each protein individually would seem to support this [96,99], and this effect has also been Fig. 3. The contribution of HPV E6 PBM target selection to HPV-induced carcinogenesis. The evolutionary acquisition of a PBM on HPV E6 proteins is probably associated with the colonizing of a new niche-and is associated with Dlg1 binding, but not with oncogenicity. Further adaptations of the PBM allow binding to more diverse polarity proteins, thus targeting more pathways to enhance the viral life cycle, but potentially also perturbing controls of epithelial differentiation, seen clinically as CIN I (cervical intraepithelial neoplasia, grade 1). Further adaptation of the PBM leads to a more flexible target binding profile, greater polarity perturbation, mislocalization or deletion of PDZ polarity proteins, and the acquisition of pro-oncogenic properties by Dlg1 and hScrib, manifest as CIN III (cervical intraepithelial neoplasia, grade 3) and its potential progression to cancer. Restoration of functional polarity proteins can lead to apoptosis of cervical cancer cells and may indicate a possible direction for therapeutic strategies. seen in non-HPV related tumor development [100][101][102]. However, in late-stage cancers, hDlg1, hScrib, and E-cadherin are often lost, which is consistent with data from various experimental settings showing that under certain conditions, they can act as tumor suppressors [97,100,103].

Human Herpesviruses
The human herpesviruses are large, enveloped, double-stranded DNA viruses that establish lifelong latent infections, which can be periodically reactivated by various stimuli to cause a productive lytic infection.

Tumor-associated herpesviruses
There are two human tumor viruses in the herpesvirus family: KSHV, which causes cancers only infrequently, and mainly in immunosuppressed patients; and EBV, which causes a large number of diverse tumor types. A number of the viral proteins that are expressed to maintain the latent phase of infection have oncogenic potential [24].
KSHV can infect a wide range of cells, including lymphoid cells, endothelial cells, and keratinocytes [24]; it also expresses a number of potentially oncogenic proteins [29]. These include LANA, which is essential for the establishment and maintenance of KSHV latency [24]; LANA expression upregulates Par3 and SNAIL, leading to the downregulation of E-cadherin and the promotion of EMT [104,105] In addition, the latency protein K15 upregulates miR-31 to inhibit the FAT4 cadherin [106], which has been implicated in the control of both apico-basal and planar polarity [107,108], and is a component of the Hippo pathway [109]; thus, expression of the K15 protein is likely to have wideranging effects on ABP and PCP cellular polarity.
EBV infection is generally found in B-lymphocytes, but the virus is also thought to infect polarized epithelium through the formation of direct adhesions between the B cells and the basolateral surface of epithelial cells [110,111], utilizing the endocytic recycling pathways of both donor and recipient cell to facilitate direct cell-cell transmission of the virus [112,113]. It has also been suggested that the virus exploits the endosomal machinery to cross the epithelial cells of the oral mucosa, without initiating an infection, to directly infect the B lymphocyes in the underlying dermis [112,114].
EBV, like all herpesviruses, persists latently for the life of the host in memory B cells and different latency states, characterized by specific gene-expression profiles, are associated with the development of specific tumor types [24]. None of the transforming proteins of EBV-EBNA1, LMP1, and LMP2a-has been detected in direct interaction with cell polarity regulators, but through upregulation of the TWIST, SNAIL, and SLUG transcription repressors, they severally downregulate expression of E-cadherin [115][116][117][118][119] and upregulate β-catenin [116,120,121]. In addition LMP1 stimulates CDC42 activation [122], leading to increased cell motility, and potentially to invasion, through CDC42's RhoGTPase activity, which is required for Scrib's ability to control polarized cell migration through actin remodeling [123][124][125]. Thus, although there is no direct interaction between KSHV or EBV proteins and the core cell polarity control proteins, they perturb many peripheral aspects of the pathway, leading to increased cell proliferation and an increased risk of malignant transformation.

Non-oncogenic herpesviruses
Herpes simplex viruses (HSV1 and HSV2), cytomegalovirus, and the varicella zoster virus (VZV) infect epithelial cells where the lytic, or productive, virus life cycle occurs and it is not clear whether the polarity of these cells is involved in the virus replication cycle. However, in plaque assays, HSV infection has been shown to induce polarized cell migration (MCP) in uninfected keratinocytes, which migrate toward the site of infection, and become infected in their turn, [126], while VZV glycoprotein E expression colocalizes with the ZO1 TJ protein and can increase cell-cell contacts and enhance TJ formation in low calcium, presumably to allow cell-tocell spread of the virus particles [127]. The cytomegalovirus gpUS9 protein has similarly been reported to colocalize with cell junction components such as ZO1 and E-cadherin in polarized epithelial cells [128], although this finding has been disputed [129]. In addition to their lytic cycle, HSV and VZV form latent infections in the dorsal ganglia, forming reactivated lesions at the original site under certain stimuli, which argues a very sophisticated interaction with the neuronal cell polarity determinants of the neuronal cell. The glycosylated US9 and gE/gI proteins of both HSV and VZV, and also the rodent pseudorabies virus (PsRV) have been implicated in the microtubule-mediated ferrying of the virus capsid within the axon [130][131][132].
Thus, it appears that effects on the core regulators of cell polarity are seen unequivocally only with the tumorigenic herpesviruses, confirming again that perturbation of the correct functioning of polarity control is one of the primary features of malignancy.

Hepatitis Viruses
Hepatitis viruses are the major cause of hepatitis worldwide (World Health Organization) and chronic infections of hepatocytes with hepatitis B virus (HBV; a double-stranded DNA hepadnavirus) and hepatitis C virus (HCV; a single-stranded RNA flavivirus) are associated with over 80% of hepatocellular carcinomas (HCCs) [24]. Hepatocytes are polygonal multipolar cells, with multiple apical and basal surfaces and hepatic function is dependent upon the maintenance of hepatocyte PCP and the correct functioning of the hepatocyte's numerous TJs [133]. The high rates of cell division and double-strand DNA breaks characteristic of chronic hepatitis are thought to be the means by which HBV DNA integrates into the cellular genome, where it is found in more than 85% of HBV-induced HCCs [134]. Studies suggest that the integration site is random, but may promote the inappropriate activation of neighboring cellular genes, with potentially pro-oncogenic sequelae.
HBV expresses a protein, X (Hbx), that contributes to tumorigenesis through its effects on many cellular mechanisms, including polarity control pathways. The virus uses the various activities of Hbx to optimize viral replication through epigenetic modification and transcriptional reprogramming of the cell. It suppresses E-cadherin expression though epigenetic modification [135], as well as upregulating SNAIL expression and downregulating miR-373 expression [136], while also perturbing the β-catenin interaction with the APC tumor suppressor, thus increasing β-catenin levels [137], which, in turn, further downregulates E-cadherin. Hbx also upregulates YAP expression, thus stimulating cell proliferation [138]. The resulting perturbation of PCP promotes the EMT, which, in combination with the increased cell division, thus contributes to the development of HCC.
HCV is a very different virus from HBV, but given the similarity of their replicative environments and clinical outcomes, it is not surprising that they target a number of similar cellular pathways [139]. It is clear that HCV targets certain proteins of the TJ, including claudin, occludin, and the interferon-induced transmembrane protein 1 [140][141][142], during virus entry into the basolateral membrane of the hepatocyte [143]. In contrast, intact TJs have also been shown to be instrumental in defending the cell against HCV entry [144,145]; however, virus envelope components have been shown to relocalize TJ components, possibly to prevent superinfection of the cell, but potentially leading to the compromise of junctional integrity [146,147].
During infection, the HCV core protein has been shown to inhibit the SHIP2 phosphatase, leading to downregulation of Dlg1 and Scrib at the basolateral membrane. This disrupts both apico-basal and PCP and morphology and causes the formation of multilumen cysts, which compromise hepatocyte function [148,149]. The core protein also induces methylation of the E-cadherin promoter, leading to its downregulation [150], while the viral NS5A protein activates PI3 kinase activity, leading to increased β-catenin stability and upregulating c-Myc expression [151,152], these activities together tending to promote EMT-associated changes in cell polarity. Such changes are further promoted by the synergy between NS5A activation of TWIST2 [153], while the viral core and Env proteins induce increased TGF-β signaling [154]. It has recently been reported that the NS4B protein binds to Scrib, through at least three PDZ domains, and induces its proteasome-mediated degradation [155]. This appears to protect the infected cell from Scrib-mediated apoptosis, a result that also appears to contribute directly to an increased likelihood of cell transformation. Thus, the virus uses multiple ways to manipulate host cell polarity control, which, in combination with chronic hepatic inflammation and hence accelerated cell division, can contribute to drive malignant transformation.

Merkel Cell Polyomavirus
MCPyV is the first polyomavirus found to be associated with a human cancer [156,157], and over 80% of Merkel cell carcinomas have been found to harbor viral DNA sequences [156]. Although MCPyV infection is widespread in normal skin, a number of characteristics define it as a probable causative agent: clonal integration of the viral genome into the host cell DNA appears to precede tumor development [158,159]; the tumor cells require persistent expression of the viral small T antigen (sT) to survive [160,161]; and the association with immunesuppression strongly supports a viral etiology for the cancer [158].
Interactome analysis has shown that the MCPyV sT binds to a wide range of cellular proteins, most relevantly including CD44 and emerin [162]. CD44 is a widely expressed protein, involved in stabilizing the TJ, thus regulating epithelial barrier function and cell-cell communication [163]. Knockout of CD44 has been shown to result in mislocalization of Par3 to the cell membrane and disruption of the TJ barrier function in mouse skin. Emerin is a component of the LINC complex, which connects the nuclear lamina to the actin cytoskeleton [164]; it binds β-catenin, restricting its nuclear entry [165] and, at least in certain cell types, constraining the Wnt/β-catenin signaling [166] that is required for the MCPyV life cycle in human dermal fibroblasts [167]; and it therefore seems probable that the sT interaction with emerin would relieve this inhibition.
HIV-1 is defined as tumorigenic owing to the tumors that develop as a direct result of its immunosuppressive impact on the host, but although tumors are common in HIV patients, HIV-1 is not a tumor virus in the classic sense. The cancers most commonly associated with HIV-1-infection are induced by other viruses: Kaposi's sarcoma, induced by KSHV; anogenital carcinomas induced by HPV; and lymphomas, induced mainly by EBV.
To further its life cycle, HIV-1 impinges upon several aspects of the cellular polarity control machinery. The virus envelope protein gp120 has been reported to decrease the ZO-1 levels at the TJ, and this may contribute to efficient virus entry [168].
Normal T-cell activation depends on cell polarity pathways, with Rac1, cdc42 and the Scrib/β-Pix complex signaling downstream to PAK at the immune synapse [169]. However, in HIV-1-infected T cells, the viral Nef protein activates PAK independently [170], inducing a pseudo-activated state that is favorable for viral replication [171], but which also induces anti-apoptotic signals [172], leading to increased survival of the infected cell and enhanced viral pathogenesis [173]. Late in infection, the viral Vpu protein has been shown to sequester β-TrcP in the cytoplasm [174], preventing its E3 ligase activity and thereby stabilizing Snail and β-catenin [175], destabilizing E-cadherin, and enhancing the release of progeny virus particles [176]. β-TrcP is similarly targeted by the Vaccinia virus A49 protein [177] and by the Rotavirus NSP1 protein [178]; these conserved virus strategies indicating a key point at which viral activity can redirect host cell functions to serve the virus's end. Fresh cycles of virus infection may also be enhanced by secreted HIV-1 Tat protein from already-infected cells, which can also result in decreased ZO-1 expression at the TJ in uninfected cells [179,180].
HIV-1 thus perturbs ICP cellular polarity to enhance the efficiency of virus infection, leading to host immunosuppression and increased susceptibility to cancers induced by other viruses.
Unlike HIV-1, HTLV-1 is a true tumor virus, being a direct cause of adult T-cell leukemia, and maintenance of the malignancy, as with HPV-induced tumors, depends on the continued expression of a viral oncogene, in this case the viral HBZ gene. The Tax oncoprotein, although lost in many later-stage T-cell leukemia cells, is required for viral replication and is implicated in many of the steps that lead to tumor development. It has a C-terminal PBM, through which, like HPV E6, it binds a number of proteins involved in cell polarity control, including Dlg, hScrib, MAGI-1, MAGI-3, and Lin7 [181][182][183][184][185][186]. Tax binding to these proteins results, at least in part, in their aggregation or mislocalization within the cell [184,187,188]. Since Tax competes with PTEN for binding to Dlg [189], this attenuates PTEN-mediated restriction of the PI3K/Akt pathway [190], increasing Akt activation and enhancing cell proliferation. As with HPV E6, the Tax PBM has been shown to be required for persistent viral infection [191], for IL2-independent growth of HTLV-1-infected T cells [192], and also for its transforming potential in cooperation with NF-κB [193,194]. The importance of the PBM to the carcinogenic phenotype of HTLV-1 is underlined by its absence from the Tax protein of nononcogenic HTLV-2 [182].
The viral envelope glycoprotein Env also has a PBM, through which it binds hDlg1 [195] and recruits it to the virological synapse that forms between neighboring T cells, and which is thought to be a means of transmitting virus directly from cell to cell. It is thought that the recruitment of hDlg1 may stabilize these synapses to enhance viral transmission [196]. This process is also enhanced by Tax, which is closely associated with the microtubule organizing center (MTOC) on the cell surface, to which it recruits intercellular adhesion molecule 1 (ICAM-1) [197,198], thus activating MEK/ERK signaling and inducing the reorganization of MTOC polarity to form the virological synapse [199]. The involvement of ERK signaling may also suggest a role for hScrib in this process [13]. Thus, the virus exploits the normal cell polarity machinery to enhance its ability to enter cells directly, while evading any risk of immune detection [200].

Non-tumorigenic Viruses
Adenoviruses Adenoviruses (AdVs) are large DNA viruses with linear double-stranded genomes; they usually cause self-limiting, lytic respiratory or gastrointestinal infections in humans. AdV infection occurs through the virion binding to the coxsackie and adenovirus receptor (CAR), which is located on the basolateral cell surface and is essential for TJ integrity [201]. CAR has a C-terminal PBM that mutational studies suggest is not involved in CAR's basolateral targeting, but is required for interactions with other PDZ domain-containing TJ proteins: MAGI-1, ZO-1, and DLG4, amongst others [202]. Thus, the initial steps of AdV infection involve interaction with proteins essential in maintaining correct cell-cell interactions and polarity.
AdVs are not usually associated with any tumorigenic activity in humans; however, the human adenovirus 9 (AdV9) has, uniquely, been long been known to have transformation potential in animal cells and can cause tumors in laboratory rodents [203]. In human cells, it has no such effect, but it has been used as a model for virus-induced tumorigenesis [204].
The main oncoprotein of AdV 9 is the earlyexpressed E4-ORF1 protein, which has at its Cterminus the first virus PBM to be discovered [181,205]. Through this, E4-ORF1, like high-risk HPV E6, binds to Dlg, MUPP, MAGI-1, ZO-2, and PATJ [45,181,[206][207][208]. Unlike E6, E4-ORF1 does not induce the degradation of its PDZ-containing targets, but sequesters or relocates them. The E4-ORF1 protein exists in either monomeric or trimeric states, with different binding specificities. Monomeric E4-ORF1 binds to its PDZ-containing targets associated with the TJ (MUPP1, MAGI-1, and ZO-2), and with the Crumbs cell polarity complex (MUPP1 and PATJ), sequestering them in insoluble complexes within the cytoplasm, thus disrupting ABP control and compromising the TJ barrier function [209]. In contrast, the trimeric form of E4-ORF1 binds only to Dlg, relocating it to the plasma membrane and, as with the HTLV-1 Tax/Dlg interaction [189], resulting in the constitutive activation of the PI3K signaling pathway [210,211]. Thus, under certain circumstances, perturbation of Dlg by E4-ORF1 turns Dlg into a pro-oncogenic factor [212,213], resulting in increased cell cycle turnover, and thus probably an enhanced environment for AdV9 replication; however, it is likely to also increase the potential for EMT and the risk of malignant transformation. Thus, in enhancing the virus life cycle, the AdV9 E4-ORF1 can compromise both cell junction integrity and cell polarity control. There is a clear parallel here with the early stages of HPV-induced cervical cancer [98], where mislocalization and upregulation of Dlg may also play a pro-oncogenic role. Both of these cases thus underline the multifunctional nature of the polarity control proteins, and the contribution that the complex interplay of their function and location makes to the determination of cell fate.

Orthomyxoviruses
These are negative-strand RNA viruses, of which the best-studied is Influenza A virus, which infects the respiratory epithelia of many birds and mammals [214]. Indeed, the frequent transmission of different influenza virus serotypes between host species renders it a major global health risk for many species of economic importance, such as chickens and pigs, as well as humans.
The influenza A virus non-structural protein NS1 is a multifunctional virulence factor that has a number of protein interaction domains, including an SH3 domain, through which it binds and activates PI3K [215][216][217], leading to Akt activation and enhanced cell growth, and also inhibiting pro-apoptotic factors, thus extending the life-span of the infected cell to allow enhanced viral replication [218][219][220].
The vast majority of NS1 proteins analyzed also have a C-terminal PBM, the presence and sequence of which correlate with viral virulence, since it is often mutated or lost in attenuated viruses [221][222][223]. Interestingly, the avian and human subtypes of influenza A virus have quite different and characteristic PBM sequences (ESEV and RSKV, respectively), which differ in their target specificities, and with their virulence in mammalian cells [224,225]. The avian-type PBM binds strongly to the cellular polarity proteins Scrib, Dlg, MAGI-1, MAGI-2, MAGI-3, PDLIM2, and PSD-95 (Dlg4) [226][227][228][229], while the human-type PBM binds more weakly or not at all. The interaction with Scrib relocalizes Scrib from the plasma membrane to cytoplasmic puncta, protecting infected cells from Scrib's pro-apoptotic functions [226], and reducing interferon-induced STAT activation [227], an effect also seen with the Tick-Borne Encephalitis virus (TBEV) NS5 protein [230]. Dlg has also been shown to localize with Scrib in these cytoplasmic puncta, disrupting cell-cell junctions and ABP and PCP polarity control [231]. The NS1 PBM binding also colocalizes MAGI-1 in these puncta, potentially perturbing its regulation of interferon-β signaling [232]. The Dlg/Scrib interaction has also recently been suggested to be involved in dendritic cell maturation, and to be targeted there by NS1, thus potentially leading to evasion of both innate and acquired immune systems [219,233].

Flaviviruses
These are single-stranded RNA viruses that are mainly zoonotic and transmitted to mammals by insect vectors. TBEV causes a severe encephalitis in humans, with a high mortality rate (20%-30%). The TBEV NS5 protein, and that of the closely related West Nile Virus (WNV), is an RNA-dependent RNA polymerase that is essential for replication of the virus genome, but it also has a rare internal PBM, through which it binds to Scrib and relocates to the plasma membrane. Binding Scrib blocks interferon-mediated JAK/STAT signaling, presumably to evade the host's innate immune response [230]. TBEV and WMV NS5 also bind to ZO-1, apparently stabilizing it at the TJ, and also binds to the RIMS2 protein [234][235][236], a PDZcontaining neurone-specific protein, [237] reflecting the viruses' neuronal etiology. However, it is not yet clear what role this NS5/ZO-1/RIMS complex may play in the virus infectious cycle.
Dengue virus (DENV), which causes the severe febrile illness Dengue fever, is also insect-borne and has an NS5 protein very similar to the TBEV NS5, and which also binds ZO-1 through its internal PBM [234], but in the case of DENV, ZO-1 appears to be sequestered in the nuclear or perinuclear regions of the infected endothelial cell. DENV infection also increases the expression of macrophage inhibitory factor, probably to evade the host immune response and this may also displace ZO-1 from the TJ [238]. The resulting TJ impairment is probably responsible for the vascular leakage that is a hallmark of severe Dengue infection [239,240].
These studies highlight unexpected roles for ABP cell polarity regulation in the control of immune signaling, and it seems likely that some of the wellstudied human oncogenic viruses which target these cell polarity regulators are also taking advantage of the immune avoidance opportunities that these interactions provide, and not just promoting an increase in cell proliferation.

Coronaviruses
Coronaviruses are large single-and positivestranded enveloped RNA viruses which infect the gastrointestinal and respiratory tracts of birds and mammals. The most studied human coronavirus, SARS-CoV, causes severe acute respiratory syndrome (SARS) and its original major outbreak is thought to have originated as a zoonosis from a bat coronavirus. Like most coronaviruses, SARS-CoV infects polarized epithelium specifically through the apical cell membrane [241][242][243]. The virus envelope protein (E) has a C-terminal PBM, through which it binds to the cellular PALS protein [244], a component of the Crumbs polarity complex that defines the apical domain, and controls the establishment of apicobasal polarity and the formation of TJs. In infected cells, PALS relocalizes specifically to the perinuclear regions of the Golgi, where virus assembly occurs. Although the location of other TJ proteins, such as ZO-1, does not appear to be affected, the formation of TJs was shown to be aberrant in SAR-CoV-infected cells, and apicobasal polarity is defective in cyst formation of MDCK cells ectopically expressing E, but not EΔPBM. It is not clear whether SARS-CoV requires the E/PALS interaction for virus entry, but the interaction may contribute to virus budding. The importance of the Crumbs complex [245,246] in the control of polarity is shown clearly here; the mislocalization of just one Crm component completely disrupting the epithelial integrity, which may, in turn, be one cause of the particularly severe pathology of SARS.
The MERS (Middle East respiratory syndrome)-CoV, in contrast, can enter and exit cells through both apical and basolateral membranes [247], and viral release occurs through inducing apoptosis in the infected cell, but no specific interactions with the polarity proteins have been reported.

Paramyxoviruses
These are enveloped, negative-, and single-strand RNA viruses, which include the highly contagious measles, mumps and (now-eradicated) Rinderpest viruses. The measles virus (MV) is spread by aerosols, but curiously does not directly infect the respiratory epithelium. Instead, in the alveoli it infects activated, and hence polarized, myeloid cells, through binding of the virus H glycoprotein to the signaling lymphocyte activation molecule on the cell surface [248]. The virus then replicates extensively in myeloid cells in the lymphatic organs, and ultimately the circulating infected myeloid cells deliver the viral genomes directly to the columnar epithelial cells of the upper airway. Making use of the ABP of these cells, the virus enters via their basolateral surfaces, through binding to the AJ protein Nectin 4 [249,250]. The Nectin 4 connection, via Afadin, to the actin cytoskeleton is then required for MV infection to spread directly from cell to cell through the formation of pores in the lateral surfaces of adjacent epithelial cells [251]. This results in the formation of large infectious centers in the tracheal epithelium, which are thought to eventually detach and be expelled in aerosols, facilitating transmission of the virus. Thus, the life cycle of MV uses both the ICP of activated myeloid cells in the lungs to infect the host and the ABP of tracheal epithelium as a means of egress and further cycles of contagion.
The mumps virus (MuV) differs from MV, as its receptor, sialic acid, is expressed on both apical and basolateral membranes, allowing its initial infection via the apical surface of airway epithelia, and its secondary infection in the viremic phase through the basolateral surfaces of the epithelia of the various target organs of MuV. Virus release, however, occurs solely from the apical surface of epithelial cells and requires the Rab11 protein, a protein found in recycling endosomes and characteristic of differentiated epithelium [252,253], again showing virus exploitation of normal cellular mechanisms.

Rhabdoviruses
Rabies virus (RaV) is a small single-and negativestrand RNA virus that infects the neuronal tissue of a very wide host range, causing an acute, and usually fatal, encephalitis. The virus infects the peripheral nervous tissue from wounds or bites, and travels along the axon to the central nervous system. The RV glycoprotein (G) has a C-terminal PBM [254] that appears to be a virulence factor, depending upon its amino acid sequence, similar to that of the Influenza A virus NS1. The PBM of virulent RaV strains (QTRL) has only one known binding partner: it binds with very high specificity to the MAST2 serinethreonine kinase. MAST2 is a normal binding partner of PTEN, which it phosphorylates, allowing phospho-PTEN to enter the nucleus and inhibit the PI3kinase/ Akt pathway, limiting neurosurvival. The virulent RaV G protein binds to MAST2 preventing PTEN phosphorylation and leading to Akt activation, thus promoting neuronal cell survival [255]. The PBM of attenuated RaV (ETRL) is more promiscuous in its binding: it does not compete with PTEN for MAST2 binding, instead it binds to a number of PDZcontaining proteins, including Dlg2 (PSD-93) and MUPP1. It also binds to the tyrosine phosphatase PTPN4, which binding promotes apoptosis and neuronal cell death [256,257]. Interestingly, in the absence of any binding ligand, the PDZ domain of PTPN4 inhibits the catalytic activity of the PTP domain, while ligand binding activates the PTP activity [258]. This lends further weight to the notion that certain PDZ domains may not be simply molecular interactors, but might also act in the dynamic regulation of signaling pathways, suggesting yet another means by which viruses may exert control over specific cellular processes.
The identification of the differences in virus targets between virulent and non-virulent strains of RaV has practical applications: studies are ongoing to identify peptides that could be used therapeutically, either binding MAST2 to promote neuro-regeneration or binding PTPN4 as oncolytic therapies in neuronal tumors [259,260]. The apoptotic bodies induced by non-virulent RaV are strong potentiators of the antiviral immune response [261], which may potentially contribute to vaccine design [256]. In addition, they are being examined as predictors of neurovirulence in the safety testing of live viral vaccines, such as those against yellow fever, polio, or measles [262].

Conclusions
The central importance of cell polarity control is emphasized by the frequency with which it is targeted by many diverse viruses. Figure 4 shows an overview of the role of polarity protein targeting in persistent and lytic virus infections. It is clear that in targeting key polarity control proteins, viruses not only affect host cell polarity, but also influence many cellular processes, including transcription, replication, and innate and acquired immunity. Examination of the interactions of different virus proteins with the cell and its polarity controls during the virus life cycles, and in virallyinduced cell transformation, shows ever more clearly how intimately all cellular processes are linked to the control of cell polarity. Viruses act as signposts, indicating the key cellular pathways and controls that maintain cellular and tissue homeostasis.

Acknowledgments
We are most grateful to all members of the Banks Lab and other colleagues for many fruitful discussions. L.B. acknowledges research support provided by the Associazione Italiana per la Ricerca sul Cancro (Grant No. 18578).


s2fieldsofstudy: Biology Medicine

text:
Lasertherapy as A Strategy for Treatment Healing under Caloric Restriction – Study in Rats

Complications in the healing process are challenging, especially in clinical situations of caloric restriction (CR). The lasertherapy becomes an important strategy that aids the repair, especially in CR. Thus, it is important to investigate the InGaAlP-660 nm laser as an strategy to repair cutaneous wounds in rats submitted to 30% of CR and to understand the tissue repair in clinical situations of CR. Thirty-six male Wistar rats were used, of which half were fed with 30% less ration, and half with ad libitum diet, for 21 days. Then, punch lesions of 1.5 cm in diameter were made on the animals backs, which were divided into: NR (no-restricted), R (restricted)—both before lesion; C (control), RC (restricted-control), L (laser), RL (restricted-laser)—after lesion. Samples of the skin/lesion/scar were collected on the 2nd, 7th and 14th days post-injury for histological, biochemical and molecular analyses. The R group showed reduction of body mass, epidermal/dermal thickness, inflammation, angiogenesis, fibroplasia and collagenesis. The RL group showed control of inflammation, oxidative damage and increase of antioxidants than RC, which probably favored angiogenesis, collagenesis and reepithelialization, similar to C and L. Thus, 30% of CR impaired the skin (before lesion). In the lesion, lasertherapy has shown to be effective in tissue repair mainly in CR status, being thus, the laser clinically important strategy to tissue repair in critical situations of caloric restriction.

Introduction 
Wound healing involves celular events, cytokines and growth factors production on wound, aiming the complete reepithelialization. In inflammatory phase, mediators promote chemotaxis, mainly of neutrophils and macrophages, which are involved in tissue debridement and development of oxidative stress [1]. This is characterized by imbalance between oxidants and antioxidants, due to the excessive production of ROS (reactive oxygen) and RNS (nitrogen) species and the reduced neutralization of these by the antioxidants. Then, oxidative damage is aggravated, mainly when associated with caloric restriction (CR), causing difficulties on tissue healing [2]. Moreover, angiogenesis improves nutritional and oxygen supply in the wound bed. The collagen and extracellular matrix production by fibroblasts recompose the damaged tissue, promoting the reepithelialization [3].
In this view, it is noted the healing process requires adequate nutritional status, since it consumes a good part of the body's reserve. In CR, the level of nutrients provided is comparable to ad libitum consumption, but with a reduction in calories consumed, i.e., undernutrition without malnutrition [4].
Moreover, there are impacts of CR on the immune system [5] and, consequently, on the wound healing, because the immune system is expensive in terms of demands for energy [6] being it the first systems whose sacrifices might be made at the beginning of CR [5]. In these ways, CR was found to have adverse D DAVID PUBLISHING effects on wound healing [7] and resulted in decreased of fibroblasts function and collagen accumulation [8], limiting the granulation tissue.
The sirtuin (SIRT) regulates activity of many proteins that are related to energy metabolism, cell survival and longevity [9]. CR increases the expression of SIRT-1 in multiple tissues and it is increased in response to nutrient deprivation in cultured cells [10]. SIRT-1 controls the gluconeogenic/glycolytic pathways in the liver in response to fasting or food restriction [11]. Moreover, SIRT activation by CR has been implicated in changes in the immune system, because the immune activation is energetically expensive [12]. It has been shown that activation of the immune system significantly increases mortality in nutrient-limited settings [13]. Thus, the inhibitory effect of SIRT-1 on NF-κB activity is consistent with its role in regulating the available nutrients during times of energy crisis, diverting calories away from the immune system to spare them for essential survival processes [12], which probably can cause deficit in the healing process.
In this context, there is an intense difficulty in the treatment of some types of cutaneous wounds, especially when associated with nutritional scarcity. Low-intensity lasertherapy has been noted for its important healing activity [14]. Moreover, there is evidence that laser has been shown important healing effects on tissues due to its physiological and therapeutic effects depending on the absorption of photons by chromophores in the target tissue. Studies have shown that the photons emitted by the laser are transformed in ATP and used by the cell to power needed metabolic activities, such as cell proliferation, and collagen synthesis. All of them, accelerate the tissue repair in chronic wounds [15,16]

Animals
All surgical and experimental procedures used in The ration was daily weighed for five days to evaluate the animals intake-average on ad libitum diet. For CR establishment, it was offered to 18 animals (R group-before lesion = RC, and RL groups, after lesion) 30% less ration from the intake-average, during 21 days. The others 18 rats were the control groups (NR groups-before lesion = C, and L groups, after lesion), which remained on ad libitum diet during 35 days of experiment.
On the 21st day of CR, wounds were performed on the all animals dorsum, which have been followed-up by 2nd, 7th and 14th days (n = 3 rats/group/time of follow-up) [18].

Caloric Restriction Confirmation
The blood samples were collected directly from the heart-left ventricle of last euthanasia on 14 th day post-injury (35th day of CR). Serums were subsequently stored at -20 °C for analysis of glucose, cholesterol, triglycerides, protein and albumin according to the manufacturer's standard instructions (Laborlab, Guarulhos-SP-Brazil). In addition, the animals were weighed on 1st, 7th, 14th, 21st and 35th days of CR (35th day of CR was similar to 14th day post-injury) and also analyzed for SIRT-1 expression by Western blotting [18].

Surgical Procedure and Experimental Groups
The rats were anesthetized by the combination of ketamine (3.0 mL/kg) with xylazine (1.0 mL/kg) for skin injuries performance. Then, trichotomy was performed on the skin of the dorsum of all animals. For a greater use (refinement) and a reduction on the animal's amount, two excisions were performed on the dorsal region of each rat using a histological punch of 1.5 cm diameter, reaching the dermo-epidermal region until the exposure of the muscular fascia [16,[19][20][21]. These both skin samples obtained were used to investigate the skin changes caused by 30% of CR in the skin before lesion (without treatment), obtaining the groups (n = 6 animals/group): NR (no-restricted-on ad libitum diet) and R (restricted-under 30% of CR).
After performing the two wounds, all animals were divided into four distinct experimental groups (each group with n = 3 animals/group/time of follow-up): C (control: wounds treated topically with laser switched off and ad libitum diet); RC (restricted-control: treated with laser switched off and 30% of CR); L (laser: wounds with topically lasertherapy and ad libitum diet); RL (restricted-laser: lasertherapy and 30% of CR).
Then, both lesions from each rat received the same specific treatment and no occlusive dressing was used. The animals were placed in individual cages and received 10 drops of analgesic (dipyrone) diluted in 250 mL of water for 72 h.

Lasertherapy
It was used Photon Lase III (DMC ® -São Carlos-SP-Brazil) InGaAlP (indium-gallium-aluminum-phosphor): 660 nm wavelength (visible-red), 3.63 W/cm² of irradiance, 100 mW of power, 134.54 J/cm² of energy density, 3.7 kJ of energy/point, 14.8 J of total energy dose, 0.0275 cm² of beam covering area application, 37 seconds under continuous mode in 4 points at the wound extremities, ±2.0 mm of distance from each wounds by 90° of angle between wound and applicator probe. The treatments were started soon after the experimental wound and performed 3×/week (at the same time).

Sample Harvesting
After 2nd, 7th and 14th days post-wound, the animals were euthanized with anesthetic deepening (ketamine: 3.0 mL/Kg and xylazine: 1.0 mL/Kg, 300 μL/Kg animal body weight) and cervical dislocation. Then, both skin/wound/scar were harvested and divided in parts for histologicals, biochemicals and moleculars studies (n = 3 part of wound/scar of rats/group/time of follow-up).

Evaluation of Wounds Reepithelialization
On the 0, 2nd, 7th and 14th days, both wounds of each animal were photographed in basic mode, without flash, without zoom and using the same camera. To standardize the distance from the camera to the wound, the camera was attached to a support 30 cm apart and perpendicular to the rat wound. A ruler, arranged beside the animal and along the wounds, was used to standardize the unit area of the wounds in 2 cm and also to serve as a known measure in the calibration of the ImageJ software when calculating the area of the wound.
ImageJ software was used to determinate the wound area to calculate the Wound Healing Rate: WHR (wound healing rate) = (initial area -final area) / initial area. The initial area corresponds to the day of the surgical procedure (0 day) and the final area corresponds to the day of euthanasia (2nd, 7th or 14th days, post-injury). WHR values greater than zero represent a decrease in wounded area (reepithelialization) and smaller than zero represent an increase in wounded area [16,20].

Histomorphometry analysis
The skin/wound/scar (n = 3/group/time of follow-up) biopsies were conditioned for 48 hours in 10% buffered formaldehyde solution and followed by paraffin-histological processing (Hematoxylin-Eosin) for simultaneous quantification of inflammatory infiltrate, fibroblasts and blood vessels using the "Cell Counter" Plugin of ImageJ software. It was used 5 images (magnification: 400×) and the quantification of inflammatory infiltrate, fibroblasts and blood vessels were double-blind. Results were expressed as number of cells' average [21].
For measuring epidermal and dermal thickness, histological images (400× of magnification) were taken in sequence from the epidermis to the subcutaneous cellular tissue. These images were automatically grouped using the Photomerge function of the Adobe Photoshop CC 2018. By the ruler on Photoshop, 3 lines/image was traced on epidermis and dermis and then, Photoshop supplied the thickness (cm) [16].

Myeloperoxidase (MPO)
The MPO assay was performed for the determination of the neutrophilic infiltrate by the macerate supernatant samples of the skin/wound/scar of the rats during the follow-up and treatments. Samples were homogenized by Polytron ® homogenizer (Kinematica, USA). After centrifugation, 5.0 µL of supernatant was diluted in 45 µL 0.08 M NaPO 4 . MPO activity was detected using 25 µL 1.6 mM tetramethylbenzidine (Sigma-Aldrich ® , USA) and read at 450 nm (SpectraMax ® , USA) [21].
2.2.10 Hydroxyproline (HO-Pro) HO-Pro assay was performed to total collagen determiantion. After hydrolysis in 6 N HCl at 110 °C, the samples were treated with 1.41% chloramine T and 15% p-dimethylaminobenzaldehyde, followed by incubation at 60 °C and read at 550 nm [23].

Oxidative stress and antioxidants
The lipoperoxidation products were analyzed by measuring the formation of thiobarbituric acid reactive species (TBARS) [24]. Then 100 µL tissue homogenates was deproteinized with 20% trichloroacetic acid. The supernatant was exposed to 0.7% thiobarbituric acid, heated to 95 °C and read at 535 nm.

Statistical Analysis
Data were expressed as mean ± standard error. All data were passed for Kolmogorov-Smirnov normality test. For the data fitting the normality curve, the statistical test chosen was t-Student test for before lesion groups (comparing NR/R groups) and ANOVA Two-Way (Multiple-comparisons) and Tukey post-test to groups after lesion (comparing C/RC/L/LR groups). For the data not fitting in the normality curve (glucose/cholesterol/protein/albumin) the statistical test was Mann-Whitney (for triglycerides was t-Student test), all were performed in Statistic 7.0 and GraphPad Prism 6.0 softwares. The pre-established level of significance was p < 0.05.

Caloric Restriction Status and Implications on Healing
Nutritional deficiency remains one of the most common causes of morbidity and mortality among children, also prevalent among elderly people [26] and hospitalized patients specially in underdeveloped countries. Even in industrialized countries, people are vulnerable to protein malnutrition, which can accompany weight control, lifestyle-related disease and aging. Diets in these populations are frequently deficient in macronutrients, micronutrients, or both [27][28][29]. The nutritional deficit is usually found in these at-risk populations and is a critical factor in susceptibility to infection [30]. Numerous nutrients are known to be important for optimal wound healing. CR is associated with immunological changes, making wound healing inadequate due to inflammatory phase prolongation and fibroplasia impairment, reduction in collagen and extracellular matrix synthesis, angiogenesis and wound remodeling [31,32].
As a treatment of these immunocompromised wounds, lasertherapy has been an important alternative because the phototherapy depends on the absorption of photons by chromophores in the target tissue, accelerating the tissue repair [15] mainly as in pathological conditions such as in CR status. Then, wounds associated with nutritional scarcity heal slowly or not at all, and the lasertherapy could benefit the treatment in a 30% of CR animal model. In this way, it becomes relevant to investigate the effects of lasertherapy as a strategy in the repair of cutaneous lesions in rats submitted to 30% of caloric restriction.
To confirm CR-status, R group showed important reduction of body weight relation to NR from 7th to 35th days of CR. Only NR presented important increase of body mass during the follow-up of CR relation to previous days (Fig. 1A). This finding corroborates with Hunt et al. [4] on 40% of CR.
About the biochemical analyses, there was no difference between NR and R for glucose, protein and albumin (Fig. 1C, 1F and 1G) on the 35th day of CR. However, about cholesterol and triglycerides ( Fig. 1D and 1E) the R group was lower than NR, corroborating to Amaral et al. [18]. Low body weight, hypocholesterolemia and hypotriglyceridemia contents are features usually found in animals submitted to CR, indicating the adequacy of the animal model used in this work [18]. The Caloric-Restriction-Society/CRS consists of a group of humans individuals who practice CR with optimum nutrition (CRON). The subjects of the study were 18 volunteers (the CRONies) that have been practicing the restricted-diet for 3-15 years. Classical signs of CR adaptations were evident in the CRONies, showing significantly lower blood glucose levels, corroborating to our results, insulin levels, and blood pressure. Lipid profiles of the CRONies were significantly healthier than the height-matched controls. Specifically, total cholesterol, LDL, and triglycerides were all reduced, corroborating to our results, and HDL was elevated compared to controls [33]. Regarding to our similar results of serum protein and albumin between both groups, it is possibly due to the slow and late reduction in CR [34].
CR induces the expression of SIRT-1 that participates as signaling of nutritional scarcity [35]. Serum was collected from humans after CR increased SIRT-1 expression. Among the changes related to SIRT-1 are increased hepatic gluconeogenesis, decreased glycolysis and increased hypothalamic signaling of hunger [36]. In our results SIRT-1 was increased, mainly on 7th and 14th days and in LR its expression was superior to the other groups (Fig. 1B). In addition, the increase of SIRT-1 in the L and LR compared to C and CR on 14th day, might suggest laser associated with CR can increase SIRT-1 expression on tissues. Besides SIRT-1 being a CR marker, it has shown SIRT-1 is required for efficient wound healing when it was used on mice with epidermis-specific SIRT-1 deletion. SIRT-1 deficiency in this model inhibits the repair of epidermis and dermis, altering the production of many cytokines, inhibited the recruitment of macrophages, neutrophils and mast cells, increased oxidative stress and reduced the angiogenesis in granulation tissue, similar to our results (Figs. 2 and 3). SIRT-1 when inhibited, decreased wound healing, while it moderately increased proliferation, suggesting that SIRT-1's role in wound healing is a complex well-coordinated process and proliferation alone is not sufficient for wound repair. Possibly other functions of epidermal SIRT-1 including regulating of cell migration, inflammation and stress response, are the more important determinants for SIRT1's role in wound healing [37].

Inflammation, Oxidative Stress, Antioxidants and Angiogenesis Profiles
The question that emerges from these observations is why natural selection would favor a system whose inflammation is regulated by nutrient availability. In the case of CR, the explanation for this is that immune activation is energetically expensive and increases energy consumption even in nutrient-limited settings [13]. Thus, the inhibitory effect of SIRT-1 on NF-κB activity is consistent with its role in regulating of available nutrients during times of energy crisis, diverting calories away from the immune system to spare them for essential survival processes [12]. We showed that R group reduced inflammatory infiltrate, TGF-β1, NAG, blood-vessels and VEGF (Fig. 1B, 1C, 1E, 1F and 1G) relation to NR, This corroborates with the findings of Yeung et al. [38] that CR inhibits NF-κB expression by reducing inflammation and consequently, the impairment of angiogenesis. On the 2nd day, RC and RL showed lower recruitment of inflammatory infiltrate and TGF-β1 than C. On the 7th day, L remained the level of inflammatory infiltrate similar to 2nd day and higher relation to the other groups, while C group remained superior TGF-β1 relating to other groups and similar to 2nd day ( Fig.  2B and 2C) suggesting the reduction on inflammation in CR and more evidenced when associated with laser.
Regarding to MPO, RC was higher than other groups on the 2nd day (Fig. 2D). About NAG on the 2nd day, RC and L were lower than C, while these were lower than C and RL on the 14th day (Fig. 2E). The blood-vessel was higher on RL than other groups for all times of follow-up (Fig. 2F). VEGF was more pronunciated on L and RL on 2nd day than C and RC. On the 14th day, this expression reduced considerably, but the C group maintained superior to other groups (Fig. 2G). Similar to our finding, lasertherapy has demonstrated its effects on the decrease of inflammatory cells and the improvement of neovascularization. The laser photobiomodulation is able to alternate cellular metabolism, especially by interfering with mitochondrial-membrane potential and ATP synthesis. Furthermore, such modality of laser therapy is also effective in stimulating the cellular cycle and therefore cellular proliferation. Through such capability of biostimulation, laser therapy may induce a decisive impact on the course of biological events that take place during wound healing. The laser treatment depresses the exudative phase (anti-inflammatory effect) at the same time that enhances the proliferative phase during acute and chronic inflammation, activating the local blood circulation and stimulating proliferation of endothelial cells [39].
Regarding to oxidative stress, TBARS on 2nd day was lower on L and LR relating to C and RC. However, TBARS on 14th day was higher relating to 2nd day, but RC and RL remain lower than C (Fig.  3A). About the antioxidants, catalase was more expressed on RL than other groups (Fig. 3C) suggesting the association of CR and lasertherapy improved antioxidants. About SOD1 and SOD2, the RL group was under expression relation to other groups. Similar to our findings, it is considerable CR might increase the resistance of cells to various types of stress [40]. In this view, CR is associated with increased DNA repair capacity, decreased production of superoxide, and decreased levels of damaged DNA, lipids, and proteins [41].

Tissue Formation
The considerable stimuli of inflammatory phase on 2nd day and modulation of inflammatory response   during the follow-up, associated to oxidative damage control by higher antioxidants production and pronounced angiogenic stimuli, suggest the improvement on reepithelialization on LR superior to RC and similar to L and C (Fig. 4A). The nutritional deficit on RC could have become difficult for the wound healing, but when associated with lasertherapy RL was effective on repair. The CR seems to have also negatively influenced fibroplasia, mainly on RL on 7th and 14th days (Fig. 4B). However, this seems not to influence the collagenesis: RL showed superior HO-Pro than RC/L on 7th day and than L on 14th day (Fig. 4C) and superior collagen III/I than other groups ( Fig. 4D and 4E).
The scarcity of nutrients on R was capable to make changes on fibroplasia, HO-Pro and collagen I relation to NR (Fig. 4B-4E). Furthermore, the CR was capable to become the skin (epidermis/dermis) thinner relating to NR (Fig. 4F-4H), suggesting the considerable protein catabolism due to nutrient scarcity [11]. In these ways, CR has shown adverse effects on wound healing and resulted in decreased collagen accumulation and crosslinks [8]. Impaired wound healing was observed in CR in mice but a short period of ad libitum feeding enhanced their capacity to undergo wound repair. In our study, certainly this wound healing capacity was enhanced by lasertherapy. The cells from CR animals preserved proliferative, biosynthetic, and contractile capacities, which was only evident once an adequate source of nutrients became available [42]. Therefore, cell function may be limited by the scarcity of nutrients and limit the growth of granulation tissue.
The use of laser associated with CR of 30% was effective in healing, reducing inflammation and controlling oxidative damage by increasing antioxidants. This favored reepithelialization by stimulating the formation of granulation tissue, fibroplasia and collagenesis.

Conclusions
The 30% of CR was able to impair the skin (before lesion). Laser therapy has shown to be effective in tissue repair mainly in CR status, being thus the laser clinically important strategy to tissue repair in critical situations of CR. Future studies about others clinical situations are important to investigate the action of laser therapy on tissue repair.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Sorting Out the Role of α-Synuclein in Retromer-Mediated Endosomal Protein Sorting

Retromer is a phylogenetically conserved, multisubunit coat complex that controls endosomal protein trafficking and sorting. Mutations in the retromer gene VPS35 cause late-onset Parkinson disease, suggesting that trafficking defects cause neurodegeneration. Sorting nexins assist retromer to guide cell surface proteins to their assigned destinations, and our interest here is sorting nexin 3 (Snx3). Snx3 binds to membranes via a phox homolog (PX) domain that binds phosphatidylinositol 3-phosphate (PI3P), and in human cells its cargo proteins are the transferrin and Wnt receptors and the divalent metal ion transporter, whereas in yeast the best characterized cargo is the iron permease Ftr1. We recently discovered that α-synuclein inhibits Snx3-retromer recycling of Ftr1 in an unexpected way: α-synuclein, which avidly binds to negatively charged lipids, blocks the association of Snx3 to early endosomes. Here, we discuss mechanisms by which α-synuclein can disrupt Snx3-retromer–mediated recycling.

cytotoxic, and insoluble amyloid fibers. Elevated levels of αsynuclein due to the multiplication of the SNCA locus or from diminished lysosomal function and/or posttranslation modification such as phosphorylation can promote α-synuclein aggregation/self-association. Functionally, α-synuclein is thought to regulate synaptic vesicle fusion with the presynaptic membrane. Emerging evidence indicates that α-synuclein is also intimately involved in endocytosis, which is the focus of this Commentary.

α-Synuclein Blocks Endoplasmic Reticulum-to-Golgi Traffic
A pioneering genetic screen revealed that α-synuclein blocks endoplasmic reticulum (ER)-to-Golgi protein traffic in yeast and that overexpression of Rab1 GTPase in yeast, flies, worms, and human neurons rescues the trafficking defect. 4 The specific trafficking defect in yeast was that α-synuclein inhibits the trafficking of alkaline phosphatase (ALP) and carboxypeptidase (CPY) from the ER to the Golgi. These 2 proteins normally transit with their receptor, Vps10, en route to their final destination, the vacuole. Given that α-synuclein blocks the trafficking of ALP and CPY, we infer that α-synuclein probably also blocks the ER-to-Golgi trafficking of Vps10. The human ortholog of Vps10 is the cation-independent mannose-6-phosphate receptor (CI-MPR), and, curiously, CI-MPR mislocalizes in cells that overexpress α-synuclein.

Mutations in Retromer Cause Late-Onset PD
Most (>90%) of the PD cases are sporadic in that there are no known genetic causes (other than the aggregation of α-synuclein), whereas a small percentage (<10%) of cases are due to mutations 2 Journal of Experimental Neuroscience in a variety of genes. Missense mutations in DJ-1, PINK1, LRRK2, PRKN, or SNCA cause early-onset PD, whereas mutations in VPS35 cause late-onset PD. 5 That mutations in VPS35 cause late-onset PD strongly suggests that defects in retromermediated protein trafficking are a cause of PD. Moreover, further illustrating the importance of endocytosis vis-à-vis α-synuclein, overexpression of endocytosis-related genes 6 rescues α-synuclein aggregation and toxicity in cell models of PD.
In the retrograde endosomal protein trafficking pathway, upon being endocytosed, cell surface receptors and transporters have 2 fates: they are either degraded in the lysosome or recycled back to the plasma membrane. The retromer complex, 7 which localizes to early endosomes, retrieves cell surface proteins from the degradative pathway and recycles them either to the trans-Golgi for redelivery to the plasma membrane or the plasma membrane directly. Retromer, which is composed of a trimer and dimer, coats the cytosolic face of early endosomes. The trimer is composed of Vps26-Vps29-Vps35 (Vps26, Vps29, and Vps35 in yeast) and functions in cargo selection. The dimer is composed Snx1/Snx2 (Vps5 in yeast) and Snx5/Snx6 (Vps17 in yeast), and this subcomplex senses and induces curvature and membrane tubulation. The sorting nexins Snx1 and Snx2 (and Snx5/Snx6) each contain 2 membrane-binding domains: a PX domain, which binds PI3P, and a BIN/Amphiphysin/Rvs (BAR) domain that senses membrane curvature and induces vesicle tubulation. Other sorting nexins, such as Snx3, can also bind cargo proteins and physically interact with the retromer machinery. Snx3 is an adapter that binds the endocytic recycling sequence of the yeast permease Ftr1 and the human iron importer divalent metal ion transporter. For cargo to be retrieved from the degradative pathway, the multimeric retromer complex must bind to the endocytic recycling sequence on the cargo in early endosomes. This is followed by the formation of endosomal tubules from which retromer-cargo vesicles bud off and then transit to the trans-Golgi for recycling to the plasma membrane.

α-Synuclein Inhibits Snx3-Retromer-Mediated Endosomal Trafficking of Fet3-Ftr1
We recently reported that α-synuclein inhibits Snx3-retromermediated recycling of Fet3-Ftr1 in yeast by blocking the association of the 2 PX-domain proteins, ie, Snx3 and Vps17, to the surface of the early endosomes. 8 Specifically, α-synuclein, which binds to negatively charged lipids, appears to inhibit the binding of the Snx3 PX domain to PI3P, which is negatively charged. This was the first paper to report such a novel activity for α-synuclein. Although this finding was made in yeast, the ability of α-synuclein to inhibit the association of PX proteins with PI3P will likely translate to human cells. Yeast Snx3 binds to PI3P-containing artificial membranes with a K d of 2 µM. If human Snx3 (and other PX proteins) bind to PI3P with a similar affinity, then α-synuclein should also block the association of these human PX domain proteins to the surface of vesicles.
Factors other than PI3P content that will determine whether α-synuclein binds to vesicles are PS content and the presence of lipid rafts, which are composed of cholesterol and sphingolipids. Collectively, we propose that neuronal pathology in PD is instigated by some species of α-synuclein-be it monomer, dimer, or oligomer-blocking ER-to-Golgi traffic, by αsynuclein disrupting retromer components, or by mutations in retromer components.

Chlamydia trachomatis Protein IncE Binds to and Subverts the Function of the Human PX-BAR Sorting Nexin and Retromer Component Snx5
Chlamydia trachomatis is a sexually transmitted bacterial pathogen that colonizes host cells. On entry into the host cells, the bacteria become enclosed in a membrane that is referred to as an inclusion. The membrane-enclosed pathogen expresses proteins that interact with host proteins, with a goal of blocking retrograde transport of the membraneenclosed pathogens to the lysosome for degradation. In essence, this bacterium subverts or hijacks the host's retrograde transport system to avoid detection, gain nutrients, and reproduce. Until recently, the mechanisms by which C trachomatis subvert the host's retrograde transport pathway were unknown.
A breakthrough report revealed that the C trachomatis effector protein IncE suppresses retromer-mediated recycling of CI-MPR by specifically binding to a conserved patch on Snx5 ( Figure 1A). 10 Snx5, which is a retromer component, binds to an endosome-to-TGN (trans-Golgi network) sorting motif on CI-MPR. IncE is a membrane-bound protein that loops through the membrane enclosure that surrounds the pathogen such that the N-and C-termini of IncE are on the cytoplasmic face of the inclusion membrane. The C-terminus of IncE also specifically binds to Snx5, Snx6, and Snx32 but not to Snx1. In essence, IncE recruits these 3 PX-BAR nexins to the surface of the C trachomatis inclusions, where they probably reshape the membrane to promote tubule formation. The question is how does the recruitment of Snx5, Snx6, and Snx32 to the pathogen inclusion membranes affect retromer? The retromer dimer is composed of combinations of Snx1 or Snx2 and Snx5 or Snx6. For example, the functional retromer dimer may be Snx1-Snx5, Snx1-Snx6, or Snx2-Snx5, and so on. By binding to Snx5 on the pathogen membranes, IncE depletes host cells of this molecule, consequently fewer molecules of Snx5 are available to bind to CI-MPR; thus, IncE disrupts retromer-mediated trafficking of CI-MPR. In addition, the IncE peptide directly inhibits the interaction of CI-MPR with Snx5. This example of a bacterial pathogen subverting a human host cell's vesicular trafficking machinery is germane to this discussion of vesicular trafficking and PD, as discussed below.
α-Synuclein interacts with a network of proteins involved in 2 cellular pathways: the endocytic pathway and RNA metabolism. 11 This conclusion was based on a recent study that used a cross-linking technique in conjunction with mass spectroscopy Patel and Witt 3 to uncover proteins in the vicinity of α-synuclein. The identified α-synuclein-protein interaction network components were further evaluated by co-immunoprecipitation and a membrane-2-hybrid assay to test for physical interactions. Focusing on the endocytic pathway, curiously, α-synuclein specifically binds to the retromer trimer proteins Vps26 and Vps29 and to the retromer dimer protein Snx1. 11 The authors concluded that "the retromer emerges as a multi-protein complex that Source: Adapted from Sun et al. 9