Search Results (65)

Drug resistance is still one of the main challenges for the treatment of colorectal cancer (CRC). Whilst some resistance mechanisms are well known, from the static therapy success rate, clearly, still much is undiscovered. Intracellular transport mechanisms have attracted attention as having a possible role in drug resistance, and here, the Endosomal Sorting Complex Required for Transport (ESCRT) protein family is studied as a source of drug resistance modulation using human CRC cell lines and clinical material. From an initial screening of ESCRT proteins in a panel of 10 CRC wild-type cell lines using immunoblotting, Vacuolar Protein Sorting-Associated Protein A4 (VPS4A) was identified as being consistently highly expressed, and it was selected for further investigation. Immunohistopathological evaluation in a small panel of CRC patient samples demonstrated high expression in the tumor epithelium compared to normal intestinal epithelium. The knockdown of VPS4A resulted in enhanced sensitivity of cells to oxaliplatin, and it was subsequently seen that oxaliplatin-resistant sublines had significantly higher VPS4A expression than their wild-type variants. In addition, it was demonstrated that a small molecule inhibitor of VPS4A, aloperine, could interact synergistically with oxaliplatin to enhance its sensitivity in an oxaliplatin-resistant cell line. We hypothesize from initial RNA sequencing analysis that the mechanism of action of VPS4A modulation is through depleting levels of the drug efflux transporter MRP2 in the cell, preventing oxaliplatin egress and increasing cell exposure to the drug. The evidence presented here thus indicates that ESCRT machinery, specifically VPS4A, may act as a modulator of oxaliplatin resistance in CRC. Full article

Endosomal dysfunction is one of the earliest cellular signs in Alzheimer’s disease. Tumor susceptibility gene 101 protein (TSG101) is a component of the endosomal sorting complex required for transport (ESCRT)-I, which plays a key role in sorting ubiquitinated cell surface proteins and lipids onto intraluminal vesicles of multivesicular bodies for trafficking to lysosomes or autophagosomes for degradation, or to the plasma membrane for exosomal secretion. TSG101-dependent trafficking has been implicated in the propagation and spread of misfolded proteins associated with neurodegenerative diseases. We used transgenesis mice to study the in vivo consequences of disrupting TSG101-dependent trafficking in adult neurons. Mice lacking Tsg101 in forebrain neurons (Tsg101^ck2-null) showed rapid loss of hippocampal neurons and progressive forebrain atrophy. Astrogliosis was apparent in the dentate gyrus within 1 week of deleting Tsg101, followed by apoptosis of hippocampal CA3 neurons and accumulation of the autophagy adapter P62/SQSTM1 and ubiquitinated proteins. Failure to detect lipidated LC3 indicated autophagy was impaired rather than upregulated. Endosomal markers (RAB5 and RAB7) and amyloid protein also accumulated in hippocampal neurons of Tsg101^ck2-null mice. Our data establish a critical role for TSG101 in neuronal survival and demonstrate the importance of the in vivo assessment of gene and protein functions. Full article

Background: Choroid plexus is a complex structure in the human brain that is responsible for the secretion of extracellular vesicles (EVs) in cerebrospinal fluid. Few studies to date have generated choroid plexus (ChP) organoids differentiated from human induced pluripotent stem cells (hiPSCs) and analyzed their secreted EVs. The scalable Vertical-Wheel bioreactors (VWBRs) provide low shear stress and a controlled environment. Methods: This study utilized VWBRs for the differentiation of hiPSCs into ChP organoids and generation of the secreted EVs compared to a static culture. Additionally, this study loaded curcumin into ChP organoid-derived EVs, performed EV lyophilization, and determined the ability of the re-hydrated EVs to alleviate neuro-inflammation. Results: The results demonstrated that the VWBR culture exhibited more aerobic metabolism and active glucose and glutamine consumption than the static control. Consequently, the ChP markers and Endosomal Sorting Complexes Required for Transport-dependent and -independent EV biogenesis genes were significantly upregulated (2–3-fold) in the VWBR, producing four-fold-higher EVs per mL media than the static control. The EVs retained similar size and zeta potential after lyophilization and re-hydration. The cells exposed to amyloid beta 42 oligomers and treated with the curcumin-loaded re-hydrated EVs showed high viability and the reduced inflammatory response determined by TNF-α and IL-6 expression. Conclusions: This study demonstrates a scalable bioreactor system to promote ChP organoid differentiation and generation of EV-based cell-free therapeutics to treat neural inflammation in various neurological disorders. Full article

The endosomal sorting complexes required for transport (ESCRT) comprise a fundamental cellular machinery with remarkable versatility in membrane remodeling. It is multifunctional in the multivesicular body (MVB) biogenesis, exosome formation and secretion, virus budding, cytokinesis, plasma membrane repair, neuron pruning, and autophagy. ESCRT’s involvement in cellular mechanisms extends beyond basic membrane trafficking. By directly interacting with autophagy-related (ATG) proteins and facilitating autophagosome-lysosome fusion, ESCRT ensures cellular homeostasis. Dysregulation in ESCRT function has been implicated in cancer, neurodegenerative disorders, and infectious diseases, underscoring its critical role in numerous pathologies. Hepatitis B virus (HBV) is an enveloped virus that exploits ESCRT and autophagy pathways for viral replication, assembly, and secretion. This review synthesizes recent mechanistic insights into ESCRT’s multifaceted roles, particularly focusing on its interactions with autophagy formation and the HBV lifecycle. Full article

The ESCRT (endosomal sorting complex required for transport) machinery is essential for various cellular processes, yet its role in head and neck squamous cell carcinoma (HNSCC) is poorly understood. We utilized The Cancer Genome Atlas (TCGA) datasets to analyze the expression of ESCRT genes. Bulk RNA-sequencing data and HNSCC tissue microarrays (TMAs) were used to evaluate VPS25 expression and its clinical significance. Single-cell RNA sequencing of tumor tissues and VPS25 knockdown experiments in CAL27 cells were used to investigate its biological functions. Immunohistochemistry, spatial transcriptomics, and immunotherapy datasets highlighted the involvement of VPS25 in immune suppression and its potential as a predictive biomarker. The results demonstrated significant VPS25 overexpression in HNSCC tissues, which correlated with poor clinical outcomes. It promoted tumor cell proliferation and migration while reducing immune cell infiltration in the tumor microenvironment (TME). Additionally, by upregulating PVR expression in tumor cells, VPS25 activated the immunosuppressive PVR-TIGIT signaling axis, thereby facilitating immune evasion. Furthermore, VPS25 emerged as a potential biomarker for predicting immunotherapy response. These findings highlight VPS25 as a pivotal regulator of tumor progression and immune evasion in HNSCC and a promising target for therapeutic strategies. Full article

We previously isolated a cDNA clone for galactosylceramide expression factor 1, which is the rat homologue of hepatocyte-growth-factor-regulated tyrosine kinase substrate (HGS) and induces galactosylceramide expression and morphological changes in COS-7 cells, and reported that overexpression of HGS induced morphological changes in canine kidney epithelial MDCK cells. HGS is a component of the endosomal sorting complexes required for transport machinery that mediates endosomal multivesicle body formation. In this study, the overexpression of HGS induced epithelial–mesenchymal transition and caused transformation in MDCK cells, whereas the overexpression of a coiled-coil domain of HGS inhibited induction of epithelial–mesenchymal transition by HGF stimulation. The overexpression of HGS in mouse melanoma B16 cells and human colorectal cancer COLO205 cells promoted cancer characteristic anchorage-independent cell growth ability and tumor growth, whereas the overexpression of the coiled-coil domain of HGS in these cells suppressed them. The oligopeptide OP12-462 constituting the coiled-coil domain suppressed the anchorage-independent cell growth ability and tumor growth of COLO205 cells. The coiled-coil domain of HGS and OP12-462 are novel tumor growth inhibitors that do not directly destroy cancer cells but rather inhibit only the anchorage-independent cell growth ability of cancer cells. Full article

Tsg101, a component of the endosomal sorting complex required for transport (ESCRT), is responsible for recognition of events requiring the machinery, as signaled by cargo tagging with ubiquitin (Ub), and for recruitment of downstream acting subunits to the site. Although much is known about the latter function, little is known about its role in the earlier event. The N-terminal domain of Tsg101 is a structural homologue of Ub conjugases (E2 enzymes) and the protein associates with Ub ligases (E3 enzymes) that regulate several cellular processes including virus budding. A pocket in the domain recognizes a motif, PT/SAP, that permits its recruitment. PT/SAP disruption makes budding dependent on Nedd4L E3 ligases. Using HIV-1 encoding a PT/SAP mutation that makes budding Nedd4L-dependent, we identified as critical for rescue the residues in the catalytic (HECT) domain of the E3 enzyme that lie in proximity to sites in Tsg101 that bind Ub non-covalently. Mutation of these residues impaired rescue by Nedd4L but the same mutations had no apparent effect in the context of a Nedd4 isomer, Nedd4-2s, whose N-terminal (C2) domain is naturally truncated, precluding C2-HECT auto-inhibition. Surprisingly, like small molecules that disrupt Tsg101 Ub-binding, small molecules that interfered with Nedd4 substrate recognition arrested budding at an early stage, supporting the conclusion that Tsg101–Ub–Nedd4 interaction promotes enzyme activation and regulates Nedd4 signaling for viral egress. Tsg101 regulation of E3 ligases may underlie its broad ability to function as an effector in various cellular activities, including viral particle assembly and budding. Full article

Newcastle disease virus (NDV) is a highly pathogenic avian infectious disease agent and also a promising oncolytic virus with broad application prospects. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery has been increasingly recognized for its crucial role in the life cycles of enveloped viruses, influencing processes such as viral entry, replication, and budding. In this study, we employed an RNA interference screening approach to identify key ESCRT components that regulate NDV replication in tumor cells. qPCR, immunofluorescence, and Western blot assays demonstrated that knockdown of HRS, CHMP4A, CHMP4B, and CHMP4C significantly impaired NDV replication in HeLa cells, with HRS exhibiting the most pronounced inhibitory effect. Additionally, HRS knockout significantly inhibited viral budding and suppressed NDV-induced cell death in HeLa cells. Notably, NDV infection was shown to significantly upregulate HRS gene and protein expression in a time-dependent manner. In conclusion, this study systematically identifies critical ESCRT components involved in NDV replication within tumor cells, with a particular focus on the role of HRS in promoting NDV’s replication by promoting viral budding, offering new insights for the development of NDV-based oncolytic therapies. Full article

The endosomal sorting complex required for transport (ESCRT) machinery is composed of an articulated architecture of proteins that assemble at multiple cellular sites. The ESCRT machinery is involved in pathways that are pivotal for the physiology of the cell, including vesicle transport, cell division, and membrane repair. The subunits of the ESCRT I complex are mainly responsible for anchoring the machinery to the action site. The ESCRT II subunits function to bridge and recruit the ESCRT III subunits. The latter are responsible for finalizing operations that, independently of the action site, involve the repair and fusion of membrane edges. In this review, we report on the data related to the activity of the ESCRT machinery at two sites: the nuclear membrane and the midbody and the bridge linking cells in the final stages of cytokinesis. In these contexts, the machinery plays a significant role for the protection of genome integrity by contributing to the control of the abscission checkpoint and to nuclear envelope reorganization and correlated resilience. Consistently, several studies show how the dysfunction of the ESCRT machinery causes genome damage and is a codriver of pathologies, such as laminopathies and cancer. Full article

The BK polyomavirus (BKPyV) is a small DNA non-enveloped virus whose infection is asymptomatic in most of the world’s adult population. However, in cases of immunosuppression, the reactivation of the virus can cause various complications, and in particular, nephropathies in kidney transplant recipients or hemorrhagic cystitis in bone marrow transplant recipients. Recently, it was demonstrated that BKPyV virions can use extracellular vesicles to collectively traffic in and out of cells, thus exiting producing cells without cell lysis and entering target cells by diversified entry routes. By a comparison to other naked viruses, we investigated the possibility that BKPyV virions recruit the Endosomal-Sorting Complexes Required for Transport (ESCRT) machinery through late domains in order to hijack extracellular vesicles. We identified a single potential late domain in the BKPyV structural proteins, a YPX₃L motif in the VP1 protein, and used pseudovirions to study the effect of point mutations found in a BKPyV clinical isolate or known to ablate the interaction of such a domain with the ESCRT machinery. Our results suggest that this domain is not involved in BKPyV association with extracellular vesicles but is crucial for capsomere interaction and thus viral particle assembly. Full article

Entamoeba histolytica is the protozoan causative of human amoebiasis. The EhADH adhesin (687 aa) is a protein involved in tissue invasion, phagocytosis and host-cell lysis. EhADH adheres to the prey and follows its arrival to the multivesicular bodies. It is an accessory protein of the endosomal sorting complexes required for transport (ESCRT) machinery. Here, to study the role of different parts of EhADH during virulence events, we produced trophozoites overexpressing the three domains of EhADH, Bro1 (1–400 aa), Linker (246–446 aa) and Adh (444–687 aa) to evaluate their role in virulence. The TrophozBro1_1–400 slightly increased adherence and phagocytosis, but these trophozoites showed a higher ability to destroy cell monolayers, augment the permeability of cultured epithelial cells and mouse colon, and produce more damage to hamster livers. The TrophozLinker_226–446 also increased the virulence properties, but with lower effect than the TrophozBro1_1–400. In addition, this fragment participates in cholesterol transport and GTPase binding. Interestingly, the TrophozAdh_444–687 produced the highest effect on adherence and phagocytosis, but it poorly influenced the monolayers destruction; nevertheless, they augmented the colon and liver damage. To identify the protein partners of each domain, we used recombinant peptides. Pull-down assays and mass spectrometry showed that Bro1 domain interplays with EhADH, Gal/GalNAc lectin, EhCPs, ESCRT machinery components and cytoskeleton proteins. While EhADH, ubiquitin, EhRabB, EhNPC1 and EhHSP70 were associated to the Linker domain, and EhADH, EhHSP70, EhPrx and metabolic enzymes interacted to the Adh domain. The diverse protein association confirms that EhADH is a versatile molecule with multiple functions probably given by its capacity to form distinct molecular complexes. Full article

The Sit4 protein phosphatase plays a key role in orchestrating various cellular processes essential for maintaining cell viability during aging. We have previously shown that SIT4 deletion promotes vacuolar acidification, mitochondrial derepression, and oxidative stress resistance, increasing yeast chronological lifespan. In this study, we performed a proteomic analysis of isolated vacuoles and yeast genetic interaction analysis to unravel how Sit4 influences vacuolar and mitochondrial function. By employing high-resolution mass spectrometry, we show that sit4Δ vacuolar membranes were enriched in Vps27 and Hse1, two proteins that are part of the endosomal sorting complex required for transport-0. In addition, SIT4 exhibited a negative genetic interaction with VPS27, as sit4∆vps27∆ double mutants had a shortened lifespan compared to sit4∆ and vps27∆ single mutants. Our results also show that Vps27 did not increase sit4∆ lifespan by improving protein trafficking or vacuolar sorting pathways. However, Vps27 was critical for iron homeostasis and mitochondrial function in sit4∆ cells, as sit4∆vps27∆ double mutants exhibited high iron levels and impaired mitochondrial respiration. These findings show, for the first time, cross-talk between Sit4 and Vps27, providing new insights into the mechanisms governing chronological lifespan. Full article

Cytokinesis, as the last stage of the cell division cycle, is a tightly controlled process amongst all eukaryotes, with defective division leading to severe cellular consequences and implicated in serious human diseases and conditions such as cancer. Both mammalian cells and the fission yeast Schizosaccharomyces pombe use binary fission to divide into two equally sized daughter cells. Similar to mammalian cells, in S. pombe, cytokinetic division is driven by the assembly of an actomyosin contractile ring (ACR) at the cell equator between the two cell tips. The ACR is composed of a complex network of membrane scaffold proteins, actin filaments, myosin motors and other cytokinesis regulators. The contraction of the ACR leads to the formation of a cleavage furrow which is severed by the endosomal sorting complex required for transport (ESCRT) proteins, leading to the final cell separation during the last stage of cytokinesis, the abscission. This review describes recent findings defining the two phases of cytokinesis in S. pombe: ACR assembly and constriction, and their coordination with septation. In summary, we provide an overview of the current understanding of the mechanisms regulating ACR-mediated cytokinesis in S. pombe and emphasize a potential role of ESCRT proteins in this process. Full article

Some charged multivesicular body protein 2B (CHMP2B) mutations are associated with autosomal-dominant neurodegenerative frontotemporal dementia and/or amyotrophic lateral sclerosis type 7 (FTDALS7). The main aim of this study is to clarify the relationship between the expression of mutated CHMP2B protein displaying FTD symptoms and defective neuronal differentiation. First, we illustrate that the expression of CHMP2B with the Asp148Tyr (D148Y) mutation, which preferentially displays FTD phenotypes, blunts neurite process elongation in rat primary cortical neurons. Similar results were observed in the N1E-115 cell line, a model that undergoes neurite elongation. Second, these effects were also accompanied by changes in neuronal differentiation marker protein expression. Third, wild-type CHMP2B protein was indeed localized in the endosomal sorting complexes required to transport (ESCRT)-like structures throughout the cytoplasm. In contrast, CHMP2B with the D148Y mutation exhibited aggregation-like structures and accumulated in the Golgi body. Fourth, among currently known Golgi stress regulators, the expression levels of Hsp47, which has protective effects on the Golgi body, were decreased in cells expressing CHMP2B with the D148Y mutation. Fifth, Arf4, another Golgi stress-signaling molecule, was increased in mutant-expressing cells. Finally, when transfecting Hsp47 or knocking down Arf4 with small interfering (si)RNA, cellular phenotypes in mutant-expressing cells were recovered. These results suggest that CHMP2B with the D148Y mutation, acting through Golgi stress signaling, is negatively involved in the regulation of neuronal cell morphological differentiation, providing evidence that a molecule controlling Golgi stress may be one of the potential FTD therapeutic targets at the molecular and cellular levels. Full article

Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus that causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The release of infectious HCV particles from infected hepatocytes is a crucial step in viral dissemination and disease progression. While the exact mechanisms of HCV particle release remain poorly understood, emerging evidence suggests that HCV utilizes intracellular membrane trafficking and secretory pathways. These pathways include the Golgi secretory pathway and the endosomal trafficking pathways, such as the recycling endosome pathway and the endosomal sorting complex required for transport (ESCRT)-dependent multivesicular bodies (MVBs) pathway. This review provides an overview of recent advances in understanding the release of infectious HCV particles, with a particular focus on the involvement of the host cell factors that participate in HCV particle release. By summarizing the current knowledge in this area, this review aims to contribute to a better understanding of endosomal pathways involved in the extracellular release of HCV particles and the development of novel antiviral strategies. Full article