Vesicular Trafficking Proteins Research Articles

Retinal cells derived from patients with DRAM2-dependent CORD21 dystrophy exhibit key lysosomal enzyme deficiency and lysosomal content accumulation

Biallelic mutations in DRAM2 lead to an autosomal recessive cone-rod dystrophy known as CORD21, which typically presents between the third and sixth decades of life. Although DRAM2 localizes to the lysosomes of photoreceptor and retinal pigment epithelium (RPE) cells, its specific role in retinal degeneration has not been fully elucidated. In this study, we generated and characterized retinal organoids (ROs) and RPE cells from induced pluripotent stem cells (iPSCs) derived from two CORD21 patients. Our investigation revealed that CORD21-ROs and RPE cells exhibit abnormalities in lipid metabolism, defects in autophagic flux, accumulation of aberrant lysosomal content, and reduced lysosomal enzyme activity. We identified potential interactions of DRAM2 with vesicular trafficking proteins, suggesting its involvement in this cellular process. These findings collectively suggest that DRAM2 plays a crucial role in maintaining the integrity of photoreceptors and RPE cells by regulating lysosomal function, autophagy, and potentially vesicular trafficking.

Modeling the cell biology of monogenetic intestinal epithelial disorders.

Monogenetic variants are responsible for a range of congenital human diseases. Variants in genes that are important for intestinal epithelial function cause a group of disorders characterized by severe diarrhea and loss of nutrient absorption called congenital diarrheas and enteropathies (CODEs). CODE-causing genes include nutrient transporters, enzymes, structural proteins, and vesicular trafficking proteins in intestinal epithelial cells. Several severe CODE disorders result from the loss-of-function in key regulators of polarized endocytic trafficking such as the motor protein, Myosin VB (MYO5B), as well as STX3, STXBP2, and UNC45A. Investigations of the cell biology and pathophysiology following loss-of-function in these genes have led to an increased understanding of both homeostatic and pathological vesicular trafficking in intestinal epithelial cells. Modeling different CODEs through investigation of changes in patient tissues, coupled with the development of animal models and patient-derived enteroids, has provided critical insights into the enterocyte differentiation and function. Linking basic knowledge of cell biology with the phenotype of specific patient variants is a key step in developing effective treatments for rare monogenetic diseases. This knowledge can also be applied more broadly to our understanding of common epithelial disorders.

Rab7-dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier, coupled with microbial dysbiosis, has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability, and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7-dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

Rab7-dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis.

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier, coupled with microbial dysbiosis, has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability, and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7-dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

Integrative Multi-Omics Analysis Identifies Transmembrane p24 Trafficking Protein 1 (TMED1) as a Potential Prognostic Marker in Colorectal Cancer.

Several TMED protein family members are overexpressed in malignant tumors and associated with tumor progression. TMED1 belongs to the TMED protein family and is involved in protein vesicular trafficking. However, the expression level and biological role of TMED1 in colorectal cancer (CRC) have yet to be fully elucidated. In this study, the integration of patient survival and multi-omics data (immunohistochemical staining, transcriptomics, and proteomics) revealed that the highly expressed TMED1 was related to the poor prognosis in CRC. Crystal violet staining indicated the cell growth was reduced after knocking down TMED1. Moreover, the flow cytometry results showed that TMED1 knockdown could increase cell apoptosis. The expression of TMED1 was positively correlated with other TMED family members (TMED2, TMED4, TMED9, and TMED10) in CRC, and the protein-protein interaction network suggested its potential impact on immune regulation. Furthermore, TMED1 expression was positively associated with the infiltration levels of regulatory T cells (Tregs), cancer-associated fibroblasts (CAFs), and endothelial cells and negatively correlated with the infiltration levels of CD4+ T cells, CD8+ T cells, and B cells. At last, the CTRP and GDSC datasets on the GSCA platform were used to analyze the relationship between TMED1 expression and drug sensitivity (IC50). The result found that the elevation of TMED1 was positively correlated with IC50 and implied it could increase the drug resistance of cancer cells. This research revealed that TMED1 is a novel prognostic biomarker in CRC and provided a valuable strategy for analyzing potential therapeutic targets of malignant tumors.

Myosin Va: Capturing cAMP for synaptic plasticity.

The plus-end directed actin-dependent motor protein, myosin Va, is of particular relevance for outward vesicular protein trafficking and for restraining specific cargo vesicles within the actin cortex. The latter is a preferred site of cAMP production, and the specificity of cAMP signaling is largely mediated through the formation of microdomains that spatially couple localized metabotropic receptor activity and cAMP production to selected effectors and downstream targets. This review summarizes the core literature on the role of myosin Va for the creation of such a cAMP microdomain at the mammalian nerve-muscle synapse that serves the activity-dependent recycling of nicotinic acetylcholine receptors (nAChRs)-a principal ligand-gated ion channel which is imperative for voluntary muscle contraction. It is discussed that i) the nerve-muscle synapse is a site with a unique actin-dependent microstructure, ii) myosin Va and protein kinase A regulatory subunit Iα as well as nAChR and its constitutive binding partner, rapsyn, colocalize in endocytic/recycling vesicles near the postsynaptic membrane, and iii) impairment of myosin Va or displacement of protein kinase A regulatory subunit Iα leads to the loss of nAChR stability. Regulation of this signaling process and underlying basic pieces of machinery were covered in previous articles, to which the present review refers.

Arabidopsis Dynamin-Related Protein AtDRP2A Contributes to Late Flg22-Signaling and Effective Immunity Against Pseudomonas syringae Bacteria.

In eukaryotes, dynamins and dynamin-related proteins (DRPs) are high-molecular weight GTPases responsible for mechanochemical fission of organelles or membranes. Of the six DRP subfamilies in Arabidopsis thaliana, AtDRP1 and AtDRP2 family members serve as endocytic accessory proteins in clathrin-mediated endocytosis. Most studies have focused on AtDRP1A and AtDRP2B as critical modulators of plant pattern-triggered immunity (PTI) against pathogenic, flagellated Pseudomonas syringae pv. tomato DC3000 bacteria and immune signaling in response to the bacterial flagellin peptide flg22. Much less is known about AtDRP2A, the closely related paralog of AtDRP2B. AtDRP2A and AtDRP2B are the only classical, or bona fide, dynamins in Arabidopsis, based on their evolutionary conserved domain structure with mammalian dynamins functioning in endocytosis. AtDRP2B but not AtDRP2A is required for robust ligand-induced endocytosis of the receptor kinase FLAGELLIN SENSING2 for dampening of early flg22 signaling. Here, we utilized Arabidopsis drp2a null mutants to identify AtDRP2A as a positive contributor to effective PTI against P. syringae pv. tomato DC3000 bacteria, consistent with reduced PATHOGEN RELATED1 (PR1) messenger RNA accumulation. We provide evidence that AtDRP2A is a novel modulator of late flg22 signaling, contributing positively to PR1 gene induction but negatively to polyglucan callose deposition. AtDRP2A has no apparent roles in flg22-elicited mitogen-activated protein kinase defense marker gene induction. In summary, this study adds the evolutionary conserved dynamin AtDRP2A to a small group of vesicular trafficking proteins with roles as non-canonical contributors in immune responses, likely due to modulating one or both the localization and activity of multiple different proteins with distinct contributions to immune signaling. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The ESCRT-III Protein Chmp1 Regulates Lipid Storage in the Drosophila Fat Body.

Defects in how excess nutrients are stored as triglycerides can result in several diseases including obesity, heart disease, and diabetes. Understanding the genes responsible for normal lipid homeostasis will help understand the pathogenesis of these diseases. RNAi screens performed in Drosophila cells identified genes involved in vesicle formation and protein sorting as important for the formation of lipid droplets; however, all of the vesicular trafficking proteins that regulate lipid storage are unknown. Here, we characterize the function of the Drosophila Charged multivesicular protein 1 (Chmp1) gene in regulating fat storage. Chmp1 is a member of the ESCRT-III complex that targets membrane localized signaling receptors to intralumenal vesicles in the multivesicular body of the endosome and then ultimately to the lysosome for degradation. When Chmp1 levels are decreased specifically in the fly fat body, triglyceride accumulates while fat-body-specific Chmp1 overexpression decreases triglycerides. Chmp1 controls triglyceride storage by regulating the number and size of fat body cells produced and not by altering food consumption or lipid metabolic enzyme gene expression. Together, these data uncover a novel function for Chmp1 in controlling lipid storage in Drosophila and supports the role of the endomembrane system in regulating metabolic homeostasis.

Elucidating the Role of Chmp1 Overexpression in the Transport of Polyamines in Drosophila melanogaster.

Polyamines are small organic cations that are essential for many biological processes such as cell proliferation and cell cycle progression. While the metabolism of polyamines has been well studied, the mechanisms by which polyamines are transported into and out of cells are poorly understood. Here, we describe a novel role of Chmp1, a vesicular trafficking protein, in the transport of polyamines using a well-defined leg imaginal disc assay in Drosophila melanogaster larvae. We show that Chmp1 overexpression had no effect on leg development in Drosophila, but does attenuate the negative impact on leg development of Ant44, a cytotoxic drug known to enter cells through the polyamine transport system (PTS), suggesting that the overexpression of Chmp1 downregulated the PTS. Moreover, we showed that the addition of spermine did not rescue the leg development in Chmp1-overexpressing leg discs treated with difluoromethylornithine (DFMO), an inhibitor of polyamine metabolism, while putrescine and spermidine did, suggesting that there may be unique mechanisms of import for individual polyamines. Thus, our data provide novel insight into the underlying mechanisms that are involved in polyamine transport and highlight the utility of the Drosophila imaginal disc assay as a fast and easy way to study potential players involved in the PTS.

Proteomic Identification of an Endogenous Synaptic SUMOylome in the Developing Rat Brain.

Synapses are highly specialized structures that interconnect neurons to form functional networks dedicated to neuronal communication. During brain development, synapses undergo activity-dependent rearrangements leading to both structural and functional changes. Many molecular processes are involved in this regulation, including post-translational modifications by the Small Ubiquitin-like MOdifier SUMO. To get a wider view of the panel of endogenous synaptic SUMO-modified proteins in the mammalian brain, we combined subcellular fractionation of rat brains at the post-natal day 14 with denaturing immunoprecipitation using SUMO2/3 antibodies and tandem mass spectrometry analysis. Our screening identified 803 candidate SUMO2/3 targets, which represents about 18% of the synaptic proteome. Our dataset includes neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins as well as vesicular trafficking and cytoskeleton-associated proteins, defining SUMO2/3 as a central regulator of the synaptic organization and function.

Transcriptome analysis using patient iPSC-derived skeletal myocytes: Bet1L as a new molecule possibly linked to neuromuscular junction degeneration in ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease in which patients gradually become paralyzed due to loss of motor function. Many genetically inheritable mutations have been linked to ALS; however, the majority of ALS patients are considered sporadic. Therefore, there is a need for a common therapy that is effective for all ALS patients. Although there is evidence of the disease beginning in the periphery at the neuromuscular junction (NMJ), the specific processes involved in skeletal muscle and at the NMJ are still largely unknown. To study common disease mechanisms in ALS skeletal muscle, we performed RNA sequencing of skeletal myocytes differentiated from induced pluripotent stem cells (iPSCs) derived from familial ALS (with C9ORF72, SOD1, or TARDBP mutations) and sporadic ALS patients. Compared to healthy control lines, the myocytes from all ALS lines showed downregulation of four genes: BET1L, DCX, GPC3, and HNRNPK. We next measured the expression levels of these four genes in hind limb muscle samples from a rat model of familial ALS (SOD1G93A transgenic) and found that only the Bet1L gene, which encodes Bet1 Golgi Vesicular Membrane Trafficking Protein Like, was commonly downregulated. Bet1L protein appeared to be localized to the basal lamina of the NMJ, with decreased expression over time in SOD1G93A transgenic rats. Importantly, the expression levels began to decrease early in the disease process. Our results indicate that loss of Bet1L at the NMJ could be of interest for better understanding ALS disease progression.

Hepatic Vps33b deficiency aggravates cholic acid-induced cholestatic liver injury in male mice.

Vacuolar protein sorting 33B (VPS33B) is important for intracellular vesicular trafficking process and protein interactions, which is closely associated with the arthrogryposis, renal dysfunction, and cholestasis syndrome. Our previous study has shown a crucial role of Vps33b in regulating metabolisms of bile acids and lipids in hepatic Vps33b deficiency mice with normal chow, but it remains unknown whether VPS33B could contribute to cholestatic liver injury. In this study we investigated the effects of hepatic Vps33b deficiency on bile acid metabolism and liver function in intrahepatic cholestatic mice. Cholestasis was induced in Vps33b hepatic knockout and wild-type male mice by feeding 1% CA chow diet for 5 consecutive days. We showed that compared with the wild-type mice, hepatic Vps33b deficiency greatly exacerbated CA-induced cholestatic liver injury as shown in markedly increased serum ALT, AST, and ALP activities, serum levels of total bilirubin, and total bile acid, as well as severe hepatocytes necrosis and inflammatory infiltration. Target metabolomics analysis revealed that hepatic Vps33b deficiency caused abnormal profiles of bile acids in cholestasis mice, evidenced by the upregulation of conjugated bile acids in serum, liver, and bile. We further demonstrated that the metabolomics alternation was accompanied by gene expression changes in bile acid metabolizing enzymes and transporters including Cyp3a11, Ugt1a1, Ntcp, Oatp1b1, Bsep, and Mrp2. Overall, these results suggest a crucial role of hepatic Vps33b deficiency in exacerbating cholestasis and liver injury, which is associated with the altered metabolism of bile acids.

A potent and selective small molecule inhibitor of myoferlin attenuates colorectal cancer progression.

As a pivotal vesicular trafficking protein, Myoferlin (MYOF) has become an attractive target for cancer therapy. However, the roles of MYOF in colorectal cancer invasion remain enigmatic, and MYOF‐targeted therapy in this malignancy has not been explored. In the present study, we provided the first functional evidence that MYOF promoted the cell invasion of colorectal cancer. Furthermore, we identified a novel small molecule inhibitor of MYOF (named YQ456) that showed high binding affinity to MYOF (KD = 37 nM) and excellent anti‐invasion capability (IC50 = 110 nM). YQ456 was reported for the first time to interfere with the interactions between MYOF and Ras‐associated binding (Rab) proteins at low nanomolar levels. This interference disrupted several vesicle trafficking processes, including lysosomal degradation, exosome secretion, and mitochondrial dynamics. Further, YQ456 exhibited excellent inhibitory effects on the growth and invasiveness of colorectal cancer. As the first attempt, the anticancer efficacy of YQ456 in the patient‐derived xenograft (PDX) mouse model indicated that targeting MYOF may serve as a novel and practical therapeutic approach for colorectal cancer.

Selective Aster inhibitors distinguish vesicular and nonvesicular sterol transport mechanisms

The Aster proteins (encoded by the Gramd1a-c genes) contain a ligand-binding fold structurally similar to a START domain and mediate nonvesicular plasma membrane (PM) to endoplasmic reticulum (ER) cholesterol transport. In an effort to develop small molecule modulators of Asters, we identified 20α-hydroxycholesterol (HC) and U18666A as lead compounds. Unfortunately, both 20α-HC and U18666A target other sterol homeostatic proteins, limiting their utility. 20α-HC inhibits sterol regulatory element-binding protein 2 (SREBP2) processing, and U18666A is an inhibitor of the vesicular trafficking protein Niemann-Pick C1 (NPC1). To develop potent and selective Aster inhibitors, we synthesized a series of compounds by modifying 20α-HC and U18666A. Among these, AI (Aster inhibitor)-1l, which has a longer side chain than 20α-HC, selectively bound to Aster-C. The crystal structure of Aster-C in complex with AI-1l suggests that sequence and flexibility differences in the loop that gates the binding cavity may account for the ligand specificity for Aster C. We further identified the U18666A analog AI-3d as a potent inhibitor of all three Aster proteins. AI-3d blocks the ability of Asters to bind and transfer cholesterol in vitro and in cells. Importantly, AI-3d also inhibits the movement of low-density lipoprotein (LDL) cholesterol to the ER, although AI-3d does not block NPC1. This finding positions the nonvesicular Aster pathway downstream of NPC1-dependent vesicular transport in the movement of LDL cholesterol to the ER. Selective Aster inhibitors represent useful chemical tools to distinguish vesicular and nonvesicular sterol transport mechanisms in mammalian cells.

RAB39B is redistributed in dementia with Lewy bodies and is sequestered within aβ plaques and Lewy bodies.

Loss of function mutations within the vesicular trafficking protein Ras analogy in brain 39B (RAB39B) are associated with rare X‐linked Parkinson’s disease (PD). Physiologically, RAB39B is localized to Golgi vesicles and recycling endosomes and is required for glutamatergic receptor maturation but also for alpha‐Synuclein (aSyn) homeostasis and the inhibition of its aggregation. Despite evidence linking RAB39B to neurodegeneration, the involvement of the protein in idiopathic neurodegenerative diseases remains undetermined. Here, analysis of the spatial distribution and expression of RAB39B was conducted in post‐mortem human brain tissue from cases of dementia with Lewy bodies (DLB, n = 10), Alzheimer’s disease (AD, n = 12) and controls (n = 12). Assessment of cortical RAB39B immunoreactivity using tissue microarrays revealed an overall reduction in the area of RAB39B positive gray matter in DLB cases when compared to controls and AD cases. Strikingly, RAB39B co‐localized with beta‐amyloid (Aβ) plaques in all cases examined and was additionally present in a subpopulation of Lewy bodies (LBs) in DLB. Biochemical measures of total RAB39B levels within the temporal cortex were unchanged between DLB, AD and controls. However, upon subcellular fractionation, a reduction of RAB39B in the cytoplasmic pool was found in DLB cases, alongside an increase of phosphorylated aSyn and Aβ in whole tissue lysates. The reduction of cytoplasmic RAB39B is consistent with an impaired reserve capacity for RAB39B‐associated functions, which in turn may facilitate LB aggregation and synaptic impairment. Collectively, our data support the involvement of RAB39B in the pathogenesis of DLB and the co‐aggregation of RAB39B with Aβ in plaques suggests that age‐associated cerebral Aβ pathology may be contributory to the loss of RAB39B. Thus RAB39B, its associated functional pathways and its entrapment in aggregates may be considered as future targets for therapeutic interventions to impede the overall pathological burden and cellular dysfunction in Lewy body diseases.

Identification of p115 as a novel ACSL4 interacting protein and its role in regulating ACSL4 degradation

Long-chain acyl-CoA synthetase 4 (ACSL4) is an ACSL family member that exhibits unique substrate preference for arachidonic acid. ACSL4 has a functional role in hepatic lipid metabolism, and is dysregulated in non-alcoholic fatty liver disease. Our previous studies demonstrated AA-induced ACSL4 degradation via the ubiquitin-proteasomal pathway (UPP). To characterize this unique mechanism, we applied proteomic approaches coupled with LC-MS/MS and identified the intracellular general vesicular trafficking protein p115 as the prominent ACSL4 interacting protein in HepG2 cells. Importantly, we found that AA greatly enhanced p115-ACSL4 association. Combined AA treatment with p115 knockdown suggested an additive role for p115 in AA-driven ACSL4 degradation. Furthermore, in vivo studies revealed a significant upregulation of p115 protein in the liver of mice fed a high fat diet that has been previously reported to induce downregulation of ACSL4 protein expression. This new finding has revealed a novel inverse correlation between ACSL4 and p115 proteins in the liver. p115 is crucial for ER-Golgi trafficking and Golgi biogenesis. Thus far, p115 has not been reported to interact with UPP proteins nor with FA metabolism enzymes. Overall, our current study provides a novel insight into the connection between ER-Golgi trafficking and UPP machinery with p115 as a critical mediator. SignificanceACSL4 is uniquely regulated by its own substrate AA, and in this study, we have found that AA leads to an enhanced interaction of ACSL4 with a novel interacting partner, the intracellular vesicle trafficking protein p115. The latter is crucial for Golgi biogenesis and ER-Golgi transport and is not known to be associated with the ubiquitin-proteasome machinery or protein stability regulation until now. This study is the first report of a possible coordination of the protein secretion pathway and the UPP in regulating a key metabolic enzyme. Our study lays the foundation to this unique crosstalk between the two major cellular pathways- secretion and protein degradation and opens up a new avenue to explore this partnership in controlling hepatic lipid metabolism. Overall, the complete elucidation of the AA-mediated ACSL4 regulation will help identify key targets in participating pathways that can be further studied for the development of therapeutics against diseases such as NAFLD, NASH and hepatocarcinoma, which are associated with dysregulated ACSL4 function.

Role and mechanisms of callose priming in mycorrhiza-induced resistance.

Mycorrhizal plants display enhanced resistance to several pathogens. However, the molecular mechanisms regulating mycorrhiza-induced resistance (MIR) are still elusive. We aim to study the mechanisms underlying MIR against Botrytis cinerea and the role of callose accumulation during this process. Mycorrhizal tomato plants inoculated with Rhizoglomus irregularis displayed callose priming upon B. cinerea infection. The callose inhibitor 2-deoxy-d-glucose abolished MIR, confirming the relevance of callose in the bioprotection phenomena. While studying the mechanisms underlying mycorrhiza-induced callose priming, we found that mycorrhizal plants display an enhanced starch degradation rate that is correlated with increased levels of β-amylase1 transcripts following pathogen infection. Starch mobilization in mycorrhizal plants seems coordinated with the increased transcription of sugar transporter and invertase genes. Moreover, the expression levels of genes encoding the vesicular trafficking proteins ATL31 and SYP121 and callose synthase PMR4 were higher in the mycorrhizal plants and further boosted by subsequent pathogen infection. All these proteins play a key role in the priming of callose accumulation in Arabidopsis, suggesting that callose priming is an induced resistance mechanism conserved in different plant species. This evidence highlights the importance of sugar mobilization and vesicular trafficking in the priming of callose as a defence mechanism in mycorrhiza-induced resistance.

Sorting nexin 11 knockout mice exhibit enhanced thermosensing behaviour

Temperature sensing is an important adaptive mechanism for warm-blooded animals such as humans. ThermoTRP ion channels are activated by distinct but overlapping physiological temperatures. Our previous research demonstrated that sorting nexin 11 (SNX11) regulates lysosomal degradation of plasma membrane TRPV3, one of ThermoTRP ion channel proteins. Here, we found that SNX11, a vesicular trafficking protein, modulates mouse behaviour in response to temperature changes. Snx11-knockout mice exhibit a stronger preference for mild temperatures along with enhanced sensitivity to harmful heat. Mechanistically, keratinocytes from Snx11-knockout mice exhibit a larger temperature-gated TRPV3 membrane current and have enhanced thermoTRPV3 expression in the plasma membrane compared to wild-type keratinocytes. Additionally, Snx11-knockout mice show higher endogenous TRPV3 protein levels in skin tissues than wild-type mice do. Therefore, our results indicate that SNX11 may regulate thermal perception via alteration of functional thermoTRPV3 on the plasma membrane of thermally sensitive cells, which is the first link between vesicular trafficking and thermal transduction.

The GTPase Rab39a promotes phagosome maturation into MHC-I antigen-presenting compartments.

For CD8 T lymphocytes to mount responses to cancer and virally-infected cells, dendritic cells must capture antigens present in tissues and display them as peptides bound to MHC-I molecules. This is most often accomplished through a pathway called antigen cross-presentation (XPT). Here, we report that the vesicular trafficking protein Rab39a is needed for optimal cross-presentation by dendritic cells invitro and cross-priming of CD8 T cells invivo. Without Rab39a, MHC-I presentation of intraphagosomal peptides is inhibited, indicating that Rab39a converts phagosomes into peptide-loading compartments. In this process, Rab39a promotes the delivery of MHC-I molecules from the endoplasmic reticulum (ER) to phagosomes, and increases the levels of peptide-empty MHC-I conformers that can be loaded with peptide in this compartment. Rab39a also increases the levels of Sec22b and NOX2, previously recognized to participate in cross-presentation, on phagosomes, thereby filling in a missing link into how phagosomes mature into cross-presenting vesicles.

Enhancing the copy number of Ldrab6 gene in Leishmania donovani parasites mediates drug resistance through drug‐thiol conjugate dependent multidrug resistance protein A (MRPA)

Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan Leishmania donovani parasite which may be fatal if left untreated. While drug-sensitive parasites are able to live and multiply within the host macrophages, they develop resistance to drugs used against them for survival and multiplication in the infected patients undergoing routine treatment. Development of new agents devoid of such drug resistance potential is achievable by identifying new drug targets in the parasite. One such target is the key regulator of intracellular vesicular trafficking protein, RabGTPase which belongs to the Ras GTPase superfamily. We recently elucidated whole genome sequence (WGS) of L. donovani (clinical Indian isolate; BHU 1220, GenBank: AVPQ00000000.1) and identified Ldrab6 gene. We now provide experimental evidence for this gene's ability to impart drug-resistant phenotype to wild-type (sensitive) Leishmania upon transfection. trans-Dibenzalacetone (DBA), a synthetic analog of curcumin, was used to determine its antileishmanial activity in wild-type parasites and parasites transfected with Ldrab6 gene. Dose-response study showed that DBA had no effect on transfected parasites at 20 µg/mL dose, whereas wild-type promastigotes showed 50% inhibition (IC50) at the same dose. This indicates the development of resistant mechanism in the transfected parasites due to enhancement of the copy number of Ldrab6 gene in L. donovani parasites. Flow cytometric analysis revealed elevated level of thiols in transfectants when compared to wild-type parasites treated with DBA. To assess the functional activity of multidrug resistance-associated protein (MRP) pump in transfectants, the accumulation of calcein, a known MRP pump substrate and probenecid, a known MRP pump regulator, were analyzed. The results indicate that Ldrab6 gene in Leishmania conferred resistance by the well-established mechanism of drug-thiol conjugation and sequestration by ABC transporter multidrug resistance-protein A (MRPA). Accordingly, Leishmania parasites transfected with Ldrab6 gene can be used as an experimental cell line for the screening of new lead molecules for their propensity to develop drug resistance.