Hepatocyte Growth Factor-regulated Tyrosine Kinase Research Articles

HRS mediates tumor immune evasion by regulating proteostasis-associated interferon pathway activation

By sorting receptor tyrosine kinases into endolysosomes, the endosomal sorting complexes required for transport (ESCRTs) are thought to attenuate oncogenic signaling in tumor cells. Paradoxically, ESCRT members are upregulated in tumors. Here, we show that disruption of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a pivotal ESCRT component, inhibited tumor growth by promoting CD8+ Tcell infiltration in melanoma and colon cancer mouse models. HRS ablation led to misfolded protein accumulation and triggered endoplasmic reticulum (ER) stress, resulting in the activation of the type I interferon pathway in an inositol-requiring enzyme-1α (IRE1α)/X-box binding protein 1 (XBP1)-dependent manner. HRS was upregulated in tumor cells with high tumor mutational burden (TMB). HRS expression associates with the response to PD-L1/PD-1 blockade therapy in melanoma patients with high TMB tumors. HRS ablation sensitized anti-PD-1 treatment in mouse melanoma models. Our study shows a mechanism by which tumor cells with high TMB evade immune surveillance and suggests HRS as a promising target to improve immunotherapy.

Abstract 108: Rejuvenation Potential Of Extracellular Vesicles From Renal Distal Tubular Cells - A Promising Strategy To Prevent Aging-associated Hypertension And Renal Function Impairment

Ageing is characterized by progressive multi-organ functional decline and disrupted homeostasis adaptation. Hypertension and kidney damage become increasingly prevalent as individuals age. Given its high metabolic activity and constant exposure to reactive oxygen species, the kidney is particularly susceptible to age-related deterioration. In our study, aged mice exhibited a notable decline in glomerular filtration rate (GFR) and urinary excretion of sodium, potassium, and calcium, alongside elevated urinary creatinine levels, indicating impaired renal function associated with ageing. Histologically, aged kidneys showed glomerular collapse, thickening of the glomerular basement membrane, and increased glomerular sclerosis. Cellular senescence plays a critical role in renal ageing. Senescent cells display an array of changes in gene and protein expression and thus also altered the contents packaged into extracellular vesicles (EVs), secreted into the extracellular milieu. The protein and RNA composition of EVs varied depending on the age and pathological status of the originating parent cells. Our data showed an increased expression of EV biogenesis protein HRS (hepatocyte growth factor-regulated tyrosine kinase substrate) in aged DCT cells (aDCTs). Consistently, with an increase in EV secretion in aDCTs. Administration of young DCT (yDCT)-derived EVs into aged kidneys resulted in a significant improvement of GFR and urinary ion excretion, accompanied by a reduction in age-associated elevation of systolic blood pressure (SBP). Conversely, when administering aDCT-derived EVs to young mice, we saw a significant decrease in GFR and an increase in SBP, indicating a detrimental effect of aDCT-EVs. Proteomic analysis revealed distinct protein expression profiles between yDCT-EVs and aDCT-EVs. These differentially expressed proteins are highly enriched in ageing regulatory pathways, underscoring the crucial role of DCT-EVs in kidney rejuvenation. Intriguingly, we identified a subset of proteins exclusively present in either yDCT-EVs or aDCT-EVs. Our future research will focus on identifying the key components of DCT-EVs responsible for driving kidney rejuvenation.

O-GlcNAcylation promotes tumor immune evasion by inhibiting PD-L1 lysosomal degradation

Programmed-death ligand 1 (PD-L1) and its receptor programmed cell death 1 (PD-1) mediate T cell-dependent immunity against tumors. The abundance of cell surface PD-L1 is a key determinant of the efficacy of immune checkpoint blockade therapy targeting PD-L1. However, the regulation of cell surface PD-L1 is still poorly understood. Here, we show that lysosomal degradation of PD-L1 is regulated by O-linked N-acetylglucosamine (O-GlcNAc) during the intracellular trafficking pathway. O-GlcNAc modifies the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a key component of the endosomal sorting machinery, and subsequently inhibits its interaction with intracellular PD-L1, leading to impaired lysosomal degradation of PD-L1. O-GlcNAc inhibition activates T cell-mediated antitumor immunity invitro and in immune-competent mice in a manner dependent on HGS glycosylation. Combination of O-GlcNAc inhibition with PD-L1 antibody synergistically promotes antitumor immune response. We also designed a competitive peptide inhibitor of HGS glycosylation that decreases PD-L1 expression and enhances T cell-mediatedimmunity against tumor cells. Collectively, our study reveals a link between O-GlcNAc and tumor immune evasion, and suggests strategies for improving PD-L1-mediated immune checkpoint blockade therapy.

Astrocyte-derived exosomal nicotinamide phosphoribosyltransferase (Nampt) ameliorates ischemic stroke injury by targeting AMPK/mTOR signaling to induce autophagy

Acute ischemic stroke (AIS) is a global cerebrovascular disease with high disability and mortality, which has no effective therapy. Studies have demonstrated that astrocyte-derived exosomes (ADEXs) provided neuroprotection in experimental stroke models. Nevertheless, the role of exosomes derived from oxygen-glucose-deprivation/reoxygenation-stimulated astrocytes (OGD/R-stimulated astrocytes; OGD/R-ADEXs) in AIS remains largely unknown. Here, we found that OGD/R-ADEXs significantly reduced OGD/R-induced neuronal death and promoted neuronal autophagy. These effects were reversed when astrocytes were pretreated with GW4869, an exosome secretion inhibitor, or when hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) was knocked down. Neuroprotection was also observed during treatment with OGD/R-ADEXs in vivo. Further studies showed that Nampt, played a vital effect in the regulation of autophagy, was significantly increased in OGD/R-ADEXs. Knockdown of Nampt in astrocytes abolished the above-mentioned effects of OGD/R-ADEXs. Mechanistically, Nampt increased autophagy and decreased cell death by modulating AMPK/mTOR signaling, which recognized as a key signaling pathway of autophagy after AIS. Collectively, these results showed that Nampt released by OGD/R-ADEXs ameliorated acute ischemic stroke during neuronal injury by targeting AMPK/mTOR signaling to induce autophagy. Our study revealed a new key factor in the secretion of exosomes by OGD/R astrocytes, which regulated autophagy and induced neuroprotection in a mouse stroke model.

HRS Regulates Small Extracellular Vesicle PD-L1 Secretion and Is Associated with Anti-PD-1 Treatment Efficacy.

PD-L1 localized to immunosuppressive small extracellular vesicles (sEV PD-L1) contributes to tumor progression and is associated with resistance to immune-checkpoint blockade (ICB) therapy. Here, by establishing a screening strategy with a combination of tissue microarray (TMA), IHC staining, and measurement of circulating sEV PD-L1, we found that the endosomal sorting complex required for transport (ESCRT) member protein hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was the key regulator of circulating sEV PD-L1 in head and neck squamous cell carcinoma (HNSCC) patients. Increased HRS expression was found in tumor tissues and positively correlated with elevated circulating sEV PD-L1 in patients with HNSCC. The expression of HRS was also negatively correlated to the infiltration of CD8+ T cells. Knockdown of HRS markedly reduced PD-L1 expression in HNSCC cell-derived sEVs, and these sEVs from HRS knockdown cells showed decreased immunosuppressive effects on CD8+ T cells. Knockout of HRS inhibited tumor growth in immunocompetent mice together with PD-1 blockade. Moreover, a higher HRS expression was associated with a lower response rate to anti-PD-1 therapy in patients with HNSCC. In summary, our study reveals HRS, the core component of ESCRT-0, regulates sEV PD-L1 secretion, and is associated with the response to ICB therapy in patients with HNSCC, suggesting HRS is a promising target to improve cancer immunotherapy.

Hgs Deficiency Caused Restrictive Cardiomyopathy via Disrupting Proteostasis.

The molecular mechanisms underlying restrictive cardiomyopathy (RCM) are not fully understood. Hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) is a vital element of Endosomal sorting required for transport (ESCRT), which mediates protein sorting for degradation and is crucial for protein homeostasis (proteostasis) maintenance. However, the physiological function and underlying mechanisms of HGS in RCM are unexplored. We hypothesized that HGS may play vital roles in cardiac homeostasis. Cardiomyocyte-specific Hgs gene knockout mice were generated and developed a phenotype similar to human RCM. Proteomic analysis revealed that Hgs deficiency impaired lysosomal homeostasis in cardiomyocytes. Loss of Hgs disrupted cholesterol transport and lysosomal integrity, resulting in lysosomal storage disorder (LSD) with aberrant autophagosome accumulation and protein aggregation. Suppression of protein aggregation by doxycycline treatment attenuated cardiac fibrosis, and diastolic dysfunction in Hgs-knockout mice. These findings uncovered a novel physiological role of HGS in regulating cardiac lysosomal homeostasis and proteostasis, suggesting that the deficient HGS contributes to LSD-associated RCM-like cardiomyopathy.

The ESCRT-0 subcomplex component Hrs/Hgs is a master regulator of myogenesis via modulation of signaling and degradation pathways

BackgroundMyogenesis is a highly regulated process ending with the formation of myotubes, the precursors of skeletal muscle fibers. Differentiation of myoblasts into myotubes is controlled by myogenic regulatory factors (MRFs) that act as terminal effectors of signaling cascades involved in the temporal and spatial regulation of muscle development. Such signaling cascades converge and are controlled at the level of intracellular trafficking, but the mechanisms by which myogenesis is regulated by the endosomal machinery and trafficking is largely unexplored. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery composed of four complexes ESCRT-0 to ESCRT-III regulates the biogenesis and trafficking of endosomes as well as the associated signaling and degradation pathways. Here, we investigate its role in regulating myogenesis.ResultsWe uncovered a new function of the ESCRT-0 hepatocyte growth factor-regulated tyrosine kinase substrate Hrs/Hgs component in the regulation of myogenesis. Hrs depletion strongly impairs the differentiation of murine and human myoblasts. In the C2C12 murine myogenic cell line, inhibition of differentiation was attributed to impaired MRF in the early steps of differentiation. This alteration is associated with an upregulation of the MEK/ERK signaling pathway and a downregulation of the Akt2 signaling both leading to the inhibition of differentiation. The myogenic repressors FOXO1 as well as GSK3β were also found to be both activated when Hrs was absent. Inhibition of the MEK/ERK pathway or of GSK3β by the U0126 or azakenpaullone compounds respectively significantly restores the impaired differentiation observed in Hrs-depleted cells. In addition, functional autophagy that is required for myogenesis was also found to be strongly inhibited.ConclusionsWe show for the first time that Hrs/Hgs is a master regulator that modulates myogenesis at different levels through the control of trafficking, signaling, and degradation pathways.

The ubiquitin ligase UBE4B regulates amyloid precursor protein ubiquitination, endosomal trafficking, and amyloid β42 generation and secretion

The extracellular accumulation of amyloid β (Aβ) fragments of amyloid precursor protein (APP) in brain parenchyma is a pathological hallmark of Alzheimer's disease (AD). APP can be cleaved into Aβ on late endosomes/multivesicular bodies (MVBs). E3 ubiquitin ligases have been linked to Aβ production, but specific E3 ligases associated with APP ubiquitination that may affect targeting of APP to endosomes have not yet been described. Using cultured cortical neurons isolated from rat pups, we reconstituted APP movement into the internal vesicles (ILVs) of MVBs. Loss of endosomal sorting complexes required for transport (ESCRT) components inhibited APP movement into ILVs and increased endosomal Aβ42 generation, implying a requirement for APP ubiquitination. We identified an ESCRT-binding and APP-interacting endosomal E3 ubiquitin ligase, ubiquitination factor E4B (UBE4B) that regulates APP ubiquitination. Depleting UBE4B in neurons inhibited APP ubiquitination and internalization into MVBs, resulting in increased endosomal Aβ42 levels and increased neuronal secretion of Aβ42. When we examined AD brains, we found levels of the UBE4B-interacting ESCRT component, hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs), were significantly decreased in AD brains. These data suggest that ESCRT components critical for membrane protein sorting in the endocytic pathway are altered in AD. These results indicate that the molecular machinery underlying endosomal trafficking of APP, including the ubiquitin ligase UBE4B, regulates Aβ levels and may play an essential role in AD progression.

Host ESCRT factors are recruited during chikungunya virus infection and are required for the intracellular viral replication cycle

Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins.

Hepatocyte growth factor-regulated tyrosine kinase substrate is essential for endothelial cell polarity and cerebrovascular stability.

AimsHepatocyte growth factor-regulated tyrosine kinase substrate (Hgs), a key component of the endosomal sorting complex required for transport (ESCRT), has been implicated in many essential biological processes. However, the physiological role of endogenous Hgs in the vascular system has not previously been explored. Here, we have generated brain endothelial cell (EC) specific Hgs knockout mice to uncover the function of Hgs in EC polarity and cerebrovascular stability.Methods and resultsKnockout of Hgs in brain ECs led to impaired endothelial apicobasal polarity and brain vessel collapse in mice. We determined that Hgs is essential for recycling of vascular endothelial (VE)-cadherin to the plasma membrane, since loss of Hgs blocked trafficking of endocytosed VE-cadherin from early endosomes to recycling endosomes, and impaired the motility of recycling endosomes. Supportively, overexpression of the motor kinesin family member 13A (KIF13A) restored endosomal recycling and rescued abrogated polarized trafficking and distribution of VE-cadherin in Hgs knockdown ECs.ConclusionThese data uncover a novel physiological function of Hgs and support an essential role for the ESCRT machinery in the maintenance of EC polarity and cerebrovascular stability.

Multivesicular body sorting and the exosomal pathway are required for the release of rat hepatitis E virus from infected cells

Recent reports have shown that rat hepatitis E virus (HEV) is capable of infecting humans. We also successfully propagated rat HEV into human PLC/PRF/5 cells, raising the possibility of a similar mechanism shared by human HEV and rat HEV. Rat HEV has the proline-rich sequence, PxYPMP, in the open reading frame 3 (ORF3) protein that is indispensable for its release. However, the release mechanism remains unclear. The overexpression of dominant-negative (DN) mutant of vacuolar protein sorting (Vps)4A or Vps4B decreased rat HEV release to 23.9 % and 18.0 %, respectively. The release of rat HEV was decreased to 8.3 % in tumor susceptibility gene 101 (Tsg101)-depleted cells and to 31.5 % in apoptosis-linked gene 2-interacting protein X (Alix)-depleted cells. Although rat HEV ORF3 protein did not bind to Tsg101, we found a 90-kDa protein capable of binding to wild-type rat HEV ORF3 protein but not to ORF3 mutant with proline to leucine mutations in the PxYPMP motif. Rat HEV release was also decreased in Ras-associated binding 27A (Rab27A)- or hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-depleted cells (to 20.1 % and 18.5 %, respectively). In addition, the extracellular rat HEV levels in the infected PLC/PRF/5 cells were increased after treatment with Bafilomycin A1 and decreased after treatment with GW4869. These results indicate that rat HEV utilizes multivesicular body (MVB) sorting for its release and that the exosomal pathway is required for rat HEV egress. A host protein alternative to Tsg101 that can bind to rat HEV ORF3 should be explored in further study.

Effects of Obesity and Acute Resistance Exercise on Skeletal Muscle Intercellular Communication Pathways

Obesity (OB) disrupts cellular communication consistent with lower skeletal muscle capillarization. Exosomes, small microvesicles, transport and deliver mRNA, miRNA, and proteins in an endocrine manner and are released by muscle during aerobic exercise. The effects of resistance exercise (REx) on exosome biogenesis is unknown. PURPOSE: Investigate if resistance exercise increases skeletal muscle exosome biogenesis pathways and if this response is impaired in obesity. METHODS: Lean (LN) and obese (OB) (n=8/group) sedentary men and women performed 3 sets of 8-12 repetitions/set of acute, single leg knee extension resistance exercise at 80% of 1-RM. Vastus lateralis biopsies were obtained at rest and at 15 min, and 3 hr post-exercise. Muscle mRNA, protein expression, fiber typing, and capillary staining were measured. RESULTS: The gene expression of the exosome biogenesis components hepatocyte growth factor-regulated tyrosine kinase (HGS) and vacuolar protein sorting mutant (VPS4a) were lower in OB than LN at rest (~25%) and at 15 min post- (~20%), but not 3 hr post-exercise. Expression of exosome surface markers apoptotic linked gene-2 interacting protein X (Alix) was lower (OB ~35% and LN ~20% 15min post-exercise) and tumor susceptibility gene-101 (TSG-101) was higher (OB ~50% and LN ~40% 3hr post-exercise) in response to REx in both groups. Acute resistance exercise increased vascular endothelial growth factor (VEGF) mRNA similarly in LN and OB. Interestingly, anti-angiogenic thrombospondin-1 (TSP-1) mRNA was increased by acute REx only in OB (~230% 3hr post-exercise). miR-130a (angiogenesis), miR-206 (myoblast to myotube differentiation) and miR-503 (repressor of cell proliferation) were increased in OB at rest and following exercise. Type II fiber size was greater and capillary density was lower in OB. CONCLUSION: Obesity alters skeletal muscle exosome biogenesis, angiogenic, and muscle differentiation pathways possibly contributing to greater muscle fiber size and lower muscle capillarization. Resistance exercise alters skeletal muscle exosome marker expression similarly in both lean and obese.

Syk and Hrs Regulate TLR3-Mediated Antiviral Response in Murine Astrocytes.

Toll-like receptors (TLRs) sense the presence of pathogen-associated molecular patterns. Nevertheless, the mechanisms modulating TLR-triggered innate immune responses are not yet fully understood. Complex regulatory systems exist to appropriately direct immune responses against foreign or self-nucleic acids, and a critical role of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), endosomal sorting complex required for transportation-0 (ESCRT-0) subunit, has recently been implicated in the endolysosomal transportation of TLR7 and TLR9. We investigated the involvement of Syk, Hrs, and STAM in the regulation of the TLR3 signaling pathway in a murine astrocyte cell line C8-D1A following cell stimulation with a viral dsRNA mimetic. Our data uncover a relationship between TLR3 and ESCRT-0, point out Syk as dsRNA-activated kinase, and suggest the role for Syk in mediating TLR3 signaling in murine astrocytes. We show molecular events that occur shortly after dsRNA stimulation of astrocytes and result in Syk Tyr-342 phosphorylation. Further, TLR3 undergoes proteolytic processing; the resulting TLR3 N-terminal form interacts with Hrs. The knockdown of Syk and Hrs enhances TLR3-mediated antiviral response in the form of IFN-β, IL-6, and CXCL8 secretion. Understanding the role of Syk and Hrs in TLR3 immune responses is of high importance since activation and precise execution of the TLR3 signaling pathway in the brain seem to be particularly significant in mounting an effective antiviral defense. Infection of the brain with herpes simplex type 1 virus may increase the secretion of amyloid-β by neurons and astrocytes and be a causal factor in degenerative diseases such as Alzheimer's disease. Errors in TLR3 signaling, especially related to the precise regulation of the receptor transportation and degradation, need careful observation as they may disclose foundations to identify novel or sustain known therapeutic targets.

Abstract P3-08-15: Proteomic profile of PAM50 intermediate risk early breast cancers

Abstract Background Breast cancer is a heterogeneous disease with a wide range of outcomes that are not fully predicted by routine clinical and pathologic features. The risk of recurrence of hormone receptor positive early-stage breast cancer, the most frequent tumor subtype, increases continuously over time. Genomic-based signatures have been developed to categorize patients according to their risk of recurrence and guide therapeutic decisions in this tumor subtype. However, the main genomic-based assays used in clinical practice classify up to 40% of the patients in an intermediate risk group. Many interrogations remain about the optimal strategy in this group. The aim of this study was to refine the molecular characterization of the intermediate risk group and determine the proportion of shared features with the low or high-risk groups using a mass spectrometry based proteomic approach. Methods Tumors with available routine PAM50 assay (PROSIGNA) results were selected from a cohort of breast cancer patients treated at Oscar Lambret Cancer Center (France). Fifteen tumors evenly split between PAM50 low, intermediate and high-risk groups were analyzed to determine the proteomic profiles of both cancer cells and stroma using MALDI mass spectrometry imaging combined with microproteomics, a spatially resolved proteomic technology. Results PAM50-Intermediate risk tumors had a distinctive proteomic profile compared to low and high-risk tumors. Heterogeneous nuclear ribonucleoproteins, 4-aminobutyrate aminotransferase, and pleckstrin homology-like domain family A member 1 are discriminating proteins between intermediate risk and low risk tumors. Differences were observed in expression of integrin beta-1, DNA replication licensing factors, splicing factors and interleukin enhancer-binding factor 2 between intermediate and high-risk tumors. Proteomic profiles of stroma according to tumor risk groups also showed differential protein expressions mainly between intermediate and high-risk groups. Breast cancer markers such as nuclear mitotic apparatus protein 1(NUMA1), C-1-tetrahydrofolate synthase (MTHFD1), cystatin-C (CST3), and T-cell immune regulator 1 (TCIRG1) were identified in high-risk tumors. Specific protein profiles were identified in stroma versus tumor. Immunoglobulin kappa chain, IGHG1, IGHM, IGHM, MMP2, ORM1 & ORM2, podocan, asprorin, immunoglobulin superfamily containing leucine-rich repeat protein (ISLR) were detected in stroma. By contrast, squamous cell carcinoma antigen recognized by T-cells 3 (SART3), shootin-1, mitotic checkpoint protein BUB3, XRCC5 &XRCC6, membrane-associated progesterone receptor component 2 (PGRMC) and hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) were specifically detected in tumor. Further analyses on an expanded cohort of patients will be presented. Conclusion MALDI mass spectrometry proteomics reveal distinctive tumor and microenvironment profiles in PAM50 intermediate risk early breast cancers. Citation Format: Hajjaji N, Aboulouard S, Robin YM, Bertin D, Fournier I, Bonneterre J, Salzet M. Proteomic profile of PAM50 intermediate risk early breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-08-15.

A Critical Role for Sorting Nexin 1 in the Trafficking of Metabotropic Glutamate Receptors.

Group I metabotropic glutamate receptors (mGluRs) function as modulators of neuronal physiology and they have also been implicated in various neuropsychiatric disorders. Trafficking of mGluRs plays important roles in controlling the precise localization of these receptors at specific region of the cell, as well as it regulates the activity of these receptors. Despite this obvious significance, we know very little about the cellular machineries that control the trafficking of these receptors in the CNS. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of few cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. Using "molecular replacement" approach in hippocampal neurons derived from mice of both sexes, we show here that SNX1 plays critical role in the trafficking of mGluR1, a member of the group I mGluR family. Overexpression of dominant-negative SNX1 or knockdown of endogenous SNX1 resulted in the rapid recycling of the receptor. Importantly, recycling via the rapid recycling route, did not allow the resensitization of the receptors. Our data suggest that both, N-terminal and C-terminal region of SNX1 play critical role in the normal trafficking of the receptor. In addition, we also show here that SNX1 regulates the trafficking of mGluR1 through the interaction with Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate), a protein that has been implicated in both signaling and vesicular trafficking. Thus, these studies reveal a mechanistic role of SNX1 in the trafficking of group I mGluRs and its physiological implications.SIGNIFICANCE STATEMENT Group I mGluRs are activated by the neurotransmitter glutamate in the CNS, and play various important roles in the brain. Similar to many other receptors, trafficking plays crucial roles in controlling the precise localization as well as activity of these receptors. Despite this obvious significance very little is known about the cellular machineries that control the trafficking of these receptors. We demonstrate here, that SNX1 plays a critical role in the trafficking of mGluR1, a member of the group I mGluR family. SNX1-mediated trafficking is critical for the resensitization of the receptor. SNX1 controls the trafficking of the receptor through the interaction with another protein, Hrs. The results suggest a role for SNX1 in the regulation of group I mGluRs.

HRS-WASH axis governs actin-mediated endosomal recycling and cell invasion.

Transmembrane proteins in the sorting endosome are either recycled to their point of origin or destined for lysosomal degradation. Lysosomal sorting is mediated by interaction of ubiquitylated transmembrane proteins with the endosomal sorting complex required for transport (ESCRT) machinery. In this study, we uncover an alternative role for the ESCRT-0 component hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) in promoting the constitutive recycling of transmembrane proteins. We find that endosomal localization of the actin nucleating factor Wiscott-Aldrich syndrome protein and SCAR homologue (WASH) requires HRS, which occupies adjacent endosomal subdomains. Depletion of HRS results in defective constitutive recycling of epidermal growth factor receptor and the matrix metalloproteinase MT1-MMP, leading to their accumulation in internal compartments. We show that direct interactions with endosomal actin are required for efficient recycling and use a model system of chimeric transferrin receptor trafficking to show that an actin-binding motif can counteract an ubiquitin signal for lysosomal sorting. Directed receptor recycling is used by cancer cells to achieve invasive migration. Accordingly, abrogating HRS- and actin-dependent MT1-MMP recycling results in defective matrix degradation and invasion of triple-negative breast cancer cells.

Effect of Acute Exercise on Skeletal Muscle Exosome Biogenesis

Exercise training promotes a wide range of beneficial adaptations. Skeletal muscle is now considered an endocrine organ. Exosomes, small microvesicles, are produced by and participate in the endocrine function of skeletal muscle. Exosome biogenesis is regulated in part by components of the multivesicular body (MVB) processing pathway: hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), signal transducing adapter molecule 1 (STAM), VTA1 homolog (VTA1), and vacuolar protein sorting-associated protein 4A (VPS4a). PURPOSE: Determine if exercise induces skeletal muscle exosome biogenesis. METHODS: Twelve lean, young men completed acute aerobic cycling at 55% VO2Max for 45 minutes followed immediately by single leg knee extensor resistance exercise (3 sets, 8-12 reps, 55% 1-RM). Vastus lateralis biopsies were obtained prior to (PRE) and 1-hour post aerobic (AEX) and aerobic+resistance (A+REX) exercise. Gene expression for proteins in the MVB pathway was analyzed using rt- PCR. RESULTS: There was no effect of exercise on STAM (PRE: 1.0; AEX: 1.03; A+REX: 1.28, AU), VTA1 (PRE: 1.0; AEX: 0.93; A+REX: 1.14, AU), or VPS4a (PRE: 1.0; AEX: 0.93; A+REX: 1.06, AU) . There was a trend for an increase in HGS at 1 hr post-exercise (PRE: 1.0; AEX: 0.97; A+REX: 1.27, AU). CONCLUSIONS: There was no effect of acute exercise on the gene expression of components of the exosome biogenesis pathway. However, activation of exosome biogenesis may be evident at different time points post exercise or with greater exercise intensities.

\u03b2-defensin 1 expression in HCV infected liver/liver cancer: an important role in protecting HCV progression and liver cancer development

β-defensin family plays a role in host defense against viral infection, however its role in HCV infection is still unknown. In this study, we demonstrated that β-defensin 1 was significantly reduced in HCV-infected liver specimens. Treatment with interferon and ribavirin upregulated β-defensin-1, but not other β-defensin tested, with the extent and duration of upregulation associated with treatment response. We investigated β-defensin family expression in liver cancer in publicly available datasets and found that among all the β-defensins tested, only β-defensin 1 was significantly downregulated, suggesting β-defensin 1 plays a crucial role in liver cancer development. Further analysis identified E-cadherin as the top positive correlated gene, while hepatocyte growth factor-regulated tyrosine kinase substrate as the top negative correlated gene. Expression of two proteoglycans were also positively correlated with that of β-defensin 1. We have also identified small molecules as potential therapeutic agents to reverse β-defensin 1-associated gene signature. Furthermore, the downregulation of β-defensin 1 and E-cadherin, and upregulation of hepatocyte growth factor-regulated tyrosine kinase substrate, were further confirmed in liver cancer and adjacent normal tissue collected from in-house Chinese liver cancer patients. Together, our results suggest β-defensin 1 plays an important role in protecting HCV progression and liver cancer development.

The Role of Ubiquitination and Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate in the Degradation of the Adrenomedullin Type I Receptor

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 2 (RAMP2) comprise a receptor for adrenomedullin (AM). Although it is known that AM induces internalization of CLR•RAMP2, little is known about the molecular mechanisms that regulate the trafficking of CLR•RAMP2. Using HEK and HMEC-1 cells, we observed that AM-induced activation of CLR•RAMP2 promoted ubiquitination of CLR. A mutant (CLRΔ9KR), lacking all intracellular lysine residues was functional and trafficked similar to the wild-type receptor, but was not ubiquitinated. Degradation of CLR•RAMP2 and CLRΔ9KR•RAMP2 was not dependent on the duration of AM stimulation or ubiquitination and occurred via a mechanism that was partially prevented by peptidase inhibitors. Degradation of CLR•RAMP2 was sensitive to overexpression of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), but not to HRS knockdown, whereas CLRΔ9KR•RAMP2 degradation was unaffected. Overexpression, but not knockdown of HRS, promoted hyperubiquitination of CLR under basal conditions. Thus, we propose a role for ubiquitin and HRS in the regulation of AM-induced degradation of CLR•RAMP2.

HRS plays an important role for TLR7 signaling to orchestrate inflammation and innate immunity upon EV71 infection.

Enterovirus 71 (EV71) is an RNA virus that causes hand-foot-mouth disease (HFMD), and even fatal encephalitis in children. Although EV71 pathogenesis remains largely obscure, host immune responses may play important roles in the development of diseases. Recognition of pathogens mediated by Toll-like receptors (TLRs) induces host immune and inflammatory responses. Intracellular TLRs must traffic from the endoplasmic reticulum (ER) to the endolysosomal network from where they initiate complete signaling, leading to inflammatory response. This study reveals a novel mechanism underlying the regulation of TLR7 signaling during EV71 infection. Initially, we show that multiple cytokines are differentially expressed during viral infection and demonstrate that EV71 infection induces the production of proinflammatory cytokines through regulating TLR7-mediated p38 MAPK, and NF-κB signaling pathways. Further studies reveal that the expression of the endosome-associated protein hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is upregulated and highly correlated with the expression of TLR7 in EV71 infected patients, mice, and cultured cells. Virus-induced HRS subsequently enhances TLR7 complex formation in early- and late-endosome by interacting with TLR7 and TAB1. Moreover, HRS is involved in the regulation of the TLR7/NF-κB/p38 MAPK and the TLR7/NF-κB/IRF3 signaling pathways to induce proinflammatory cytokines and interferons, respectively, resulting in the orchestration of inflammatory and immune responses to the EV71 infection. Therefore, this study demonstrates that HRS acts as a key component of TLR7 signaling to orchestrate immune and inflammatory responses during EV71 infection, and provides new insights into the mechanisms underlying the regulation of host inflammation and innate immunity during EV71 infection.