Armin Akhavan

Platelet-derived growth factor-alpha receptor activation is required for human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus that can cause life-threatening disease in the fetus and the immunocompromised host. Upon attachment to the cell, the virus induces robust inflammatory, interferon- and growth-factor-like signalling. The mechanisms facilitating viral entry and gene expression are not clearly understood. Here we show that platelet-derived growth factor-α receptor (PDGFR-α) is specifically phosphorylated by both laboratory and clinical isolates of HCMV in various human cell types, resulting in activation of the phosphoinositide-3-kinase (PI(3)K) signalling pathway. Upon stimulation by HCMV, tyrosine-phosphorylated PDGFR-α associated with the p85 regulatory subunit of PI(3)K and induced protein kinase B (also known as Akt) phosphorylation, similar to the genuine ligand, PDGF-AA. Cells in which PDGFR-α was genetically deleted or functionally blocked were non-permissive to HCMV entry, viral gene expression or infectious virus production. Re-introducing human PDGFRA gene into knockout cells restored susceptibility to viral entry and essential viral gene expression. Blockade of receptor function with a humanized PDGFR-α blocking antibody (IMC-3G3) or targeted inhibition of its kinase activity with a small molecule (Gleevec) completely inhibited HCMV viral internalization and gene expression in human epithelial, endothelial and fibroblast cells. Viral entry in cells harbouring endogenous PDGFR-α was competitively inhibited by pretreatment with PDGF-AA. We further demonstrate that HCMV glycoprotein B directly interacts with PDGFR-α, resulting in receptor tyrosine phosphorylation, and that glycoprotein B neutralizing antibodies inhibit HCMV-induced PDGFR-α phosphorylation. Taken together, these data indicate that PDGFR-α is a critical receptor required for HCMV infection, and thus a target for novel anti-viral therapies.

The Signal Peptide of the Ebolavirus Glycoprotein Influences Interaction with the Cellular Lectins DC-SIGN and DC-SIGNR

ABSTRACT The C-type lectins DC-SIGN and DC-SIGNR (collectively referred to as DC-SIGN/R) bind to the ebolavirus glycoprotein (EBOV-GP) and augment viral infectivity. DC-SIGN/R strongly enhance infection driven by the GP of EBOV subspecies. Zaire (ZEBOV) but have a much less pronounced effect on infection mediated by the GP of EBOV subspecies. Sudan (SEBOV). For this study, we analyzed the determinants of the differential DC-SIGN/R interactions with ZEBOV- and SEBOV-GP. The efficiency of DC-SIGN engagement by ZEBOV-GP was dependent on the rate of GP incorporation into lentiviral particles, while appreciable virion incorporation of SEBOV-GP did not allow robust DC-SIGN/R usage. Forced incorporation of high-mannose carbohydrates into SEBOV-GP augmented the engagement of DC-SIGN/R to the levels observed with ZEBOV-GP, indicating that appropriate glycosylation of SEBOV-GP is sufficient for efficient DC-SIGN/R usage. However, neither signals for N-linked glycosylation unique to SEBOV- or ZEBOV-GP nor the highly variable and heavily glycosylated mucin-like domain modulated the interaction with DC-SIGN/R. In contrast, analysis of chimeric GPs identified the signal peptide as a determinant of DC-SIGN/R engagement. Thus, ZEBOV- but not SEBOV-GP was shown to harbor high-mannose carbohydrates, and GP modification with these glycans was controlled by the signal peptide. These results suggest that the signal peptide governs EBOV-GP interactions with DC-SIGN/R by modulating the incorporation of high-mannose carbohydrates into EBOV-GP. In summary, we identified the level of GP incorporation into virions and signal peptide-controlled glycosylation of GP as determinants of attachment factor engagement.

SEA domain proteolysis determines the functional composition of dystroglycan

Post‐translational modifications of the extracellular matrix receptor dystroglycan (DG) determine its functional state, and defects in these modifications are linked to muscular dystrophies and cancers. A prominent feature of DG biosynthesis is a precursor cleavage that segregates the ligand‐binding and transmembrane domains into the noncovalently attached α‐and β‐subunits. We investigate here the structural determinants and functional significance of this cleavage. We show that cleavage of DG elicits a conspicuous change in its ligand‐binding activity. Mutations that obstruct this cleavage result in increased capacity to bind laminin, in part, due to enhanced glycosylation of α‐DG. Reconstitution of DG cleavage in a cell‐free expression system demonstrates that cleavage takes place in the endoplasmic reticulum, providing a suitable regulatory point for later processing events. Sequence and mutational analyses reveal that the cleavage occurs within a full SEA (sea urchin, enterokinase, agrin) module with traits matching those ascribed to autoproteolysis. Thus, cleavage of DG constitutes a control point for the modulation of its ligand‐binding properties, with therapeutic implications for muscular dystrophies. We provide a structural model for the cleavage domain that is validated by experimental analysis and discuss this cleavage in the context of mucin protein and SEA domain evolution. Akhavan, A., Crivelli, S. N., Singh, M., Lingappa, V. R., Muschler, J. L. SEA domain proteolysis determines the functional composition of dystroglycan. FASEB J. 22, 612–621 (2008)

Loss of Cell-Surface Laminin Anchoring Promotes Tumor Growth and Is Associated with Poor Clinical Outcomes

Perturbations in the composition and assembly of extracellular matrices (ECM) contribute to progression of numerous diseases, including cancers. Anchoring of laminins at the cell surface enables assembly and signaling of many ECMs, but the possible contributions of altered laminin anchoring to cancer progression remain undetermined. In this study, we investigated the prominence and origins of defective laminin anchoring in cancer cells and its association with cancer subtypes and clinical outcomes. We found loss of laminin anchoring to be widespread in cancer cells. Perturbation of laminin anchoring originated from several distinct defects, which all led to dysfunctional glycosylation of the ECM receptor dystroglycan. In aggressive breast and brain cancers, defective laminin anchoring was often due to suppressed expression of the glycosyltransferase LARGE. Reduced expression of LARGE characterized a broad array of human tumors in which it was associated with aggressive cancer subtypes and poor clinical outcomes. Notably, this defect robustly predicted poor survival in patients with brain cancers. Restoring LARGE expression repaired anchoring of exogenous and endogenous laminin and modulated cell proliferation and tumor growth. Together, our findings suggest that defects in laminin anchoring occur commonly in cancer cells, are characteristic of aggressive cancer subtypes, and are important drivers of disease progression.

Nuclear translocation of β-dystroglycan reveals a distinctive trafficking pattern of autoproteolyzed mucins

Dystroglycan (DG) is an extracellular matrix receptor implicated in muscular dystrophies and cancers. DG belongs to the membrane‐tethered mucin family and is composed of extracellular (α‐DG) and transmembrane (β‐DG) subunits stably coupled at the cell surface. These two subunits are generated by autoproteolysis of a monomeric precursor within a distinctive protein motif called sea urchin–enterokinase–agrin (SEA) domain, yet the purpose of this cleavage and heterodimer creation is uncertain. In this study, we identify a functional nuclear localization signal within β‐DG and show that, in addition to associating with α‐DG at the cell surface, the full‐length and glycosylated β‐DG autonomously traffics to the cytoplasm and nucleoplasm in a process that occurs independent of α‐DG ligand binding. The trafficking pattern of β‐DG mirrors that of MUC1‐C, the transmembrane subunit of the related MUC1 oncoprotein, also a heterodimeric membrane‐tethered mucin created by SEA autoproteolysis. We show that the transmembrane subunits of both MUC1 and DG transit the secretory pathway prior to nuclear targeting and that their monomeric precursors maintain the capacity for nuclear trafficking. A screen of breast carcinoma cell lines of distinct pathophysiological origins revealed considerable variability in the nuclear partitioning of β‐DG, indicating that nuclear localization of β‐DG is regulated, albeit independent of extracellular ligand binding. These findings point to novel intracellular functions for β‐DG, with possible disease implications. They also reveal an evolutionarily conserved role for SEA autoproteolysis, serving to enable independent functions of mucin transmembrane subunits, enacted by a shared and poorly understood pathway of segregated subunit trafficking.

Cytomegalovirus Immediate-Early Proteins Promote Stemness Properties in Glioblastoma

Glioblastoma (GBM) is the most common and aggressive human brain tumor. Human cytomegalovirus (HCMV) immediate-early (IE) proteins that are endogenously expressed in GBM cells are strong viral transactivators with oncogenic properties. Here, we show how HCMV IEs are preferentially expressed in glioma stem-like cells (GSC), where they colocalize with the other GBM stemness markers, CD133, Nestin, and Sox2. In patient-derived GSCs that are endogenously infected with HCMV, attenuating IE expression by an RNAi-based strategy was sufficient to inhibit tumorsphere formation, Sox2 expression, cell-cycle progression, and cell survival. Conversely, HCMV infection of HMCV-negative GSCs elicited robust self-renewal and proliferation of cells that could be partially reversed by IE attenuation. In HCMV-positive GSCs, IE attenuation induced a molecular program characterized by enhanced expression of mesenchymal markers and proinflammatory cytokines, resembling the therapeutically resistant GBM phenotype. Mechanistically, HCMV/IE regulation of Sox2 occurred via inhibition of miR-145, a negative regulator of Sox2 protein expression. In a spontaneous mouse model of glioma, ectopic expression of the IE1 gene (UL123) specifically increased Sox2 and Nestin levels in the IE1-positive tumors, upregulating stemness and proliferation markers in vivo. Similarly, human GSCs infected with the HCMV strain Towne but not the IE1-deficient strain CR208 showed enhanced growth as tumorspheres and intracranial tumor xenografts, compared with mock-infected human GSCs. Overall, our findings offer new mechanistic insights into how HCMV/IE control stemness properties in GBM cells.

Endocytic trafficking of laminin is controlled by dystroglycan and is disrupted in cancers

ABSTRACT The dynamic interactions between cells and basement membranes serve as essential regulators of tissue architecture and function in metazoans, and perturbation of these interactions contributes to the progression of a wide range of human diseases, including cancers. Here, we reveal the pathway and mechanism for the endocytic trafficking of a prominent basement membrane protein, laminin-111 (referred to here as laminin), and their disruption in disease. Live-cell imaging of epithelial cells revealed pronounced internalization of laminin into endocytic vesicles. Laminin internalization was receptor mediated and dynamin dependent, and laminin proceeded to the lysosome through the late endosome. Manipulation of laminin receptor expression revealed that the dominant regulator of laminin internalization is dystroglycan, a laminin receptor that is functionally perturbed in muscular dystrophies and in many cancers. Correspondingly, laminin internalization was found to be deficient in aggressive cancer cells displaying non-functional dystroglycan, and restoration of dystroglycan function strongly enhanced the endocytosis of laminin in both breast cancer and glioblastoma cells. These results establish previously unrecognized mechanisms for the modulation of cell–basement-membrane communication in normal cells and identify a profound disruption of endocytic laminin trafficking in aggressive cancer subtypes.

N- and O-linked glycosylation coordinate cell-surface localization of a cardiac potassium channel

The biogenesis and processing of membrane and secretory proteins takes place at the endoplasmic reticulum (ER) where they are integrated into or translocated across the ER membrane. A remarkable element implicated in biogenesis of this class of protein is that they are almost always processed by various cotranslational and posttranslational modifications which ultimately determine protein stability, localization and function. Defaults in biogenesis, processing or trafficking of membrane proteins are often the direct cause of various human diseases. In particular, some forms of cardiac arrhythmias, such as the long QT syndrome (LQTS), are caused by aberrant processing and defective trafficking of ion channels to the plasma membrane (Anderson et al. 2006). The LQTS is a congenital heart disease characterized by an abnormally long QT interval measured with a body surface electrocardiogram and increase risk of torsade de pointes, a fatal cardiac arrhythmia. Several forms of LQTS arise from mutations in genes encoding main or auxiliary subunits of potassium channels implicated in the repolarization phase of cardiac action potential. Some of these mutations generate dysfunctional ion channels, thus delaying the repolarization phase of the cardiac action potential and prolonging the QT interval. In contrast, other mutations have no or little effect on the function of potassium channels but instead result in misprocessing and defective trafficking of the channel protein to the plasma membrane where they normally function (Anderson et al. 2006). In human heart, KCNE1 and KCNQ1 co-assemble to form the slow outward current (IKs) of the repolarization phase of the action potential. Homozygous mutations in KCNE1 or KCNQ1 genes gives rise to the Jervell and Lange–Nielsen syndrome (JLNS), a form of LQTS associated with congenital deafness (Splawski et al. 1997). In a recent issue of The Journal of Physiology, Chandrasekhar et al. (2011) investigated the molecular mechanisms by which one mutation in KCNE1 diminish the contribution of IKs to the repolarization phase of the cardiac action potential, giving rise to JLNS. They found a novel posttranslational modification in the KCNE1 subunit and they suggest that abolishing this modification by a natural mutation is associated with defective trafficking of the channel protein to the plasma membrane. One common type of modification of ion channels involves addition of oligosaccharide chains to asparagine (N) residues, a process referred to as N-linked glycosylation. Several cardiac ion channels undergo N-linked glycosylation and mutations in consensus N-linked glycosylation sites have been identified in potassium channels implicated in LQTS (Anderson et al. 2006). In contrast, much less if known about processing of cardiac ion channels by O-linked glycosylation. In eukaryotic cells, several types of O-linked glycosylation exist but they all involve addition of carbohydrate moieties to serine (S) or threonine (T) amino acids. In their study, Chandrasekhar et al. (2011) pointed to multiple consensus sites for O-linked glycosylation in KCNE1 and experimentally identified the actual T residue that undergoes glycosylation. They showed KCNE1 undergoes both N- and O-linked glycosylation when heterologously expressed with KCNQ1 in cells. By exploring a transgenic mouse model stably expressing concatenated KCNE1–KCNQ1 fusion protein, they found that the cardiac IKs complex acquires O-linked glycosylation in vivo. Mutational analysis identified a T at position 7 (T7) as the sole site for O-linked glycosylation. Interestingly, this same site is critical for N-linked glycosylation at N5, allowing abrogation of both modifications with a single mutation. Indeed, one such mutation exists in patients with LQTS and it severely compromises the glycosylation and trafficking of KCNE1–KCNQ1 channel complexes (Bas et al. 2001). In contrast, mutations that eliminate either N- or O-linked glycans separately have minimal effect on the functional activity, biogenesis and trafficking of the channel proteins. Notably, the latter conclusions were based on investigations of glycosylation sites that were created by mutagenesis. The authors argue that the consensus N- and O-linked glycosylation sites overlap in native KCNE1 sequence, thus making it difficult to investigate these glycosylation types separately by mutational analysis. However, in the absence of additional information, it is unclear as to why it is not possible to create mutants that would allow separate analysis of native N- and O-linked glycosylation sites. Theoretically, mutation of N5 should abrogate N-linked glycosylation without affecting O-linked glycosylation, thereby enabling investigations aimed at identifying the consequence of N-linked glycosylation on biogenesis and function of the KCNE1–KCNQ1 complex. Another alternative to separately investigate the consequence of N- and O-linked glycosylation in biogenesis and trafficking of KCNE1 involves pharmacological treatments which inhibit distinct glycosylation types. For example, treatment of live cells with tunicamycine specifically abolishes N-linked glycosylation, thereby allowing investigation of this defect without interference from defects associated with O-linked glycans. One important novelty of the studies reported by Chandrasekhar et al. (2011) is the discovery that the cardiac IKs complex is modified by O-linked glycans. However, the precise consequence of this modification for the function and biogenesis of the ion channel remains unknown. The authors suggest that this modification is potentially important for subcellular localization of the IKs complex in cardiac cells. This hypothesis can be tested by investigating the localization of mutant channels in cardiac cell lines (such as the HL-1 cell line) or in a transgenic mouse model similar to the one described by Chandrasekhar et al. (2011). An alternative function for this modification is to allow regulation of IKs based on physiological demand. It is well known that the IKs channel is modulated by phosphorylation as a result of adrenergic stimuli. Thus, specific protein modifications can allow interaction with molecular machinery involved in regulation of IKs or the specific targeting of the ion channel to the proximity of adrenergic receptors (Marx et al. 2002). Undoubtedly, a better understanding of the implication of O-linked glycosylation for IKs function requires investigations in native cardiac cells and under physiological conditions.

Carbonic Anhydrase I Is Recognized by an SOD1 Antibody upon Biotinylation of Human Spinal Cord Extracts

We recently reported the presence of a novel 32 kDa protein immunoreactive to a copper, zinc superoxide dismutase (SOD1) antibody within the spinal cord of patients with amyotrophic lateral sclerosis (ALS). This unique protein species was generated by biotinylation of spinal cord tissue extracts to detect conformational changes of SOD1 specific to ALS patients. To further characterize this protein, we enriched the protein by column chromatography and determined its protein identity by mass spectrometry. The protein that gave rise to the 32 kDa species upon biotinylation was identified as carbonic anhydrase I (CA I). Biotinylation of CA I from ALS spinal cord resulted in the generation of a novel epitope recognized by the SOD1 antibody. This epitope could also be generated by biotinylation of extracts from cultured cells expressing human CA I. Peptide competition assays identified the amino acid sequence in carbonic anhydrase I responsible for binding the SOD1 antibody. We conclude that chemical modifications used to identify pathogenic protein conformations can lead to the identification of unanticipated proteins that may participate in disease pathogenesis.

Motorized traffic of a cardiac ion channel: implication of conventional kinesin in transport of Kv1.5 channels to the plasma membrane

As primary conductors of ionic charge across virtually every cell in our body, ion channels play a fundamental role in various aspects of mammalian physiology. The role of ion channels in physiology is particularly evident in the function of the heart and the brain which operate primarily by conducting electrical impulses. Not surprisingly, investigations of mechanisms associated with ion channel function have dominated basic science research in areas of cardiology and neurology. However, we know very little about the mechanisms involved in intracellular delivery and the localization of ion channels. A mislocalized ion channel is not only physiologically worthless, its altered localization may be detrimental to the cell and the overall survival of the organism. In a recent issue of The Journal of Physiology, Zadeh et al. (2009) investigated the molecular mechanisms implicated in the delivery of a cardiac voltage-gated potassium channel (Kv1.5) to its final destination, the plasma membrane. They found that a particular isoform (Kif5b) of the kinesin superfamily is involved in the transport of the Kv1.5 channels to the cell surface. Prior to this study, it was well known that kinesin motor proteins are critical for the transport of ion channels across lengthy distances in axons of nerve cells (Rivera et al. 2007). However, the study of Zadeh et al. (2009) suggests that kinesins are equally involved in the transport of ion channels in cardiac cells. Kinesins and dyneins are motor proteins which deliver a large cargo to their final cellular destination by moving along the microtubule tracks. As depicted in Fig. 1, in general, kinesins move from the centre of the cell towards the periphery (or the plus end of microtubules) and dyneins move in the reverse direction. Kinesin-1 (also known as conventional kinesin) is perhaps the most prominent member of the kinesin superfamily. Kinesin-1 is a tetrameric complex composed of two heavy chains (KHC) and two light chains (KLC). The amino terminal end of KHC interacts with the microtubule and hydrolyses ATP to generate the force for movement. Binding to cargo is mediated by either KHC or KLC (reviewed in Hirokawa & Noda, 2008). Figure 1 Synthesis and transport of Kv1.5 channels Zadeh et al. (2009) focused their investigation on Kif5b, the only KHC subunit of kinesin-1 which is ubiquitously expressed (Hirokawa & Noda, 2008). They found that overexpression of Kif5b results in a significant increase in functional expression of Kv1.5, presumably because more channels are delivered to the plasma membrane. The delivery of Kv1.5 channels by Kif5b is expected to take place between the Golgi and the plasma membrane (as shown in Fig. 1). Therefore, manipulations that block transit to Golgi should lessen or eliminate the increase in functional expression of Kv1.5 by Kif5b. Zadeh et al. (2009) show that blocking the exit of Kv1.5 channels from the endoplasmic reticulum by brefeldin A (BFA) attenuates the increase in functional expression of Kv1.5 by Kif5b. In order to more directly test the specific role of Kif5b, in several of their experiments the authors compared the effect of overexpressing Kif5bDN, a dominant negative construct of Kif5b. As expected, the presence of BFA had marginal effect on the influence of Kif5bDN on the current density of Kv1.5. In addition, in a tetracycline-inducible expression system, the authors demonstrate that induced expression of Kv1.5 subsequent to expression of Kif5bDN significantly diminishes the magnitude of currents generated by Kv1.5 channels, presumably due to a block of channel delivery to the plasma membrane. Peculiarly, however, when investigated at steady-state level, coexpression of Kif5bDN and Kv1.5 resulted in a significant increase in the function of Kv1.5 channels. Zadeh et al. (2009) argue that the increase in surface expression of Kv1.5 upon coexpression of Kif5bDN is mediated through a distinct mechanism unrelated to that observed with coexpression of Kif5b. A previous study by the same group has demonstrated that inhibition of dynein increases the surface expression of Kv1.5 (Choi et al. 2005), prompting the authors to suggest that the increase in the current density of Kv1.5 at steady-state level by Kif5bDN is mediated by the inhibition of dynein function. Indeed, Zadeh et al. (2009) show that several manipulations that reduce channel internalization, potentially by interfering with dynein function, diminish the effect of Kif5bDN on the functional expression of Kv1.5. The effect of Kif5b, however, persisted even after inhibiting channel internalization. Zadeh et al. (2009) have concluded that Kif5b is essential for the delivery of Kv1.5 channels to the plasma membrane in cardiac cells, which is perhaps the most likely and the simplest conclusion that can be drawn from their data. Several issues, however, remain to be addressed and some important experiments are needed to strengthen the conclusion of this study. In particular, the authors clearly demonstrate a dramatic increase in current density in two different cell lines upon coexpression of Kv1.5 and Kif5b. However, as evident from their data, the cell lines that they use express endogenous channels, the traffic of which may also be influenced by Kif5b. Therefore the contribution of Kv1.5 channels to the increase in current density observed upon expression of Kif5b is unclear. Since the endogenous channels appear to have similar kinetics to Kv1.5, it is particularly important to subtract their contribution from electrophysiological measurements in order to accurately and quantitatively estimate changes in Kv1.5 function. Furthermore, the authors note that they have been unable to demonstrate a direct interaction between Kv1.5 and Kif5b. It is therefore possible that the increase in current density associated with overexpression of Kif5b is due to increased surface expression of a ubiquitously expressed putative auxiliary subunit required for the function of Kv1.5. Thus, it is critical to quantitatively and directly measure the surface expression of exogenously expressed Kv1.5 channels before and after overexpression of Kif5b. Similarly, it is equally important to define the specificity of the effect of Kif5b on cardiac ion channel trafficking. Are Kv1.5 channels unique in their dependence on Kif5b or do other cardiac channels also require Kif5b for their trafficking to the plasma membrane? Is KLC a player in the delivery of Kv1.5 channels to the plasma membrane? What is the amino acid sequence in Kv1.5 that is required for enhanced functional expression by Kif5b and is that sequence conserved in any other voltage-gated potassium channel? Answers to these questions, amongst others, are central to solidifying the implication of kinesin-1 in the delivery of ion channels to the plasma membrane of cardiac cells. In addition, these answers may help in identifying novel therapeutic targets for cardiac disorders arising from deficient delivery of ion channels to the plasma membrane.