Search Results (36)

Retromer is an evolutionarily conserved protein complex first identified in budding yeast. It was originally described for its essential role in endosome-to-Golgi retrieval of lysosomal hydrolase receptors. Retromer is now known to mediate trafficking of many endosomal cargoes. The mammalian retromer is constituted by a core heterotrimer encoded by the vacuolar protein sorting (VPS) gene products VPS26, VPS35, and VPS29. To mediate cargo recognition and endosomal sorting into various pathways, this trimer can cooperate with phosphoinositide-binding sorting nexin family members. Defective retromer functioning has been associated with alterations in cellular homeostasis, leading to disease. To gain insights into how it may mediate these broad processes, a proteomic strategy in polarized Madin-Darby canine kidney cells was devised to identify retromer-interacting proteins. Subsequent validation of one of the candidates, i.e., cytokeratin 19, led to the unexpected finding that retromer localizes to the pericentriolar region in dividing cells and subsequently translocates to the midbody during cytokinesis. Retromer was found interacting with CK19, and its antisense depletion led to delocalization from CK19. Subcellular fractionation and live cell monitoring of depleted cells provided evidence of a role by retromer in post-metaphase progression and in epithelial cell migration, thereby connecting retromer with key processes of cellular dynamics. Full article

Background/Objectives: The retromer protein complex is involved in various physiological processes, especially endosomal trafficking, and its dysregulation has been linked to Alzheimer’s disease and Parkinson’s disease, as well as VPS35 knockout (KO), causing early embryonic lethality. We aimed to investigate the cellular consequences of VPS35 deficiency. Methods: To investigate the effects of VPS35 loss, we used CRISPR/Cas9 to generate VPS35 KO human embryonic kidney 293 (HEK293) cells. We analyzed changes in retromer component expression, cell proliferation, apoptosis, and mitochondrial dynamics using Western blotting, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and confocal microscopy. Results: VPS35 KO led to a significant reduction in cell proliferation and decreased expression of VPS29 and VPS26, both essential for retromer complex assembly. Consequently, retromer formation was impaired. Compared to control cells, KO cells exhibited elevated levels of cleaved caspase-3, poly(ADP-ribose) polymerase, cytochrome C, and p21, while the expression of Ki-67, CDK4, and cyclin D was reduced. Additionally, VPS35 deletion also promoted mitochondrial fragmentation, associated with increased expression of mitochondrial fission-related proteins. Finally, the rescue experiment using the human VPS35 gene confirmed that the recovery of VPS35 not only led to the recovery of the essential elements constituting the retromer but also the recovery of molecules related to the cell cycle, restoring cell death to a normal level. Conclusions: These findings suggest that VPS35 plays a critical role in cell growth and survival by modulating apoptosis, mitochondrial dynamics, and cell cycle progression. Full article

Background and Objectives: VPS26A, a core component of the retromer complex, is pivotal to endosomal trafficking and membrane protein recycling. However, its expression profile, prognostic significance, and clinical relevance in liver hepatocellular carcinoma (LIHC) remain unexplored. This study investigates the prognostic potential of VPS26A by extensively analyzing publicly available LIHC-related databases. Materials and Methods: Multiple databases, including TIMER, UALCAN, HPA, GSCA, KM Plotter, OSlihc, MethSurv, miRNet, OncomiR, LinkedOmics, GeneMANIA, and STRING, were used to evaluate VPS26A expression patterns, prognostic implications, correlations with tumor-infiltrating immune cells (TIICs), epigenetic modifications, drug sensitivity, co-expression networks, and protein–protein interactions in LIHC. Results: VPS26A was significantly overexpressed at both the mRNA and protein levels in LIHC tissues compared to that in normal tissues. This upregulation was strongly associated with a poor prognosis. Furthermore, VPS26A expression was both positively and negatively correlated with various TIICs. Epigenetic analysis indicated that VPS26A is regulated by promoter and regional DNA methylation. Additionally, VPS26A influences the sensitivity of LIHC cells to a broad range of anticancer agents. Functional enrichment and co-expression analyses revealed that VPS26A serves as a central regulator of the LIHC transcriptomic landscape, with positively correlated gene sets linked to poor prognosis. Additionally, VPS26A contributes to the molecular architecture governing vesicular trafficking, with potential relevance to diseases involving defects in endosomal transport and autophagy. Notably, miRNAs targeting VPS26A-associated gene networks have emerged as potential prognostic biomarkers for LIHC. VPS26A was found to be integrated into a highly interconnected signaling network comprising proteins in cancer progression, immune regulation, and cellular metabolism. Conclusions: Overall, VPS26A may serve as a potential prognostic biomarker and therapeutic target in LIHC. This study provides novel insights into the molecular mechanisms underlying LIHC progression, and highlights the multifaceted role of VPS26A in tumor biology. Full article

In eukaryotic cells, vesicle-mediated transport interconnects the endomembrane system. These vesicles are formed by coat proteins via deformation of donor membranes. Here, we constructed a set of fluorescent protein-based markers for major coat protein complexes in the yeast model system, and examined their subcellular localization patterns. Our markers covered COPII, COPI, AP-1, AP-2, AP-3, and retromer complexes. Our live cell imaging demonstrates that COPII puncta were primarily associated with the endoplasmic reticulum (ER), and occasionally with early Golgi. COPI was present on both early Golgi and late Golgi/early endosomes. AP-1 puncta were present on late Golgi/early endosomes. AP-2 was present on plasma membrane (PM)-associated puncta, and around the bud neck. AP-3 puncta were present on late Golgi/early endosomes and on the surface of vacuoles. Retromer was present on the surface of vacuoles, late endosomes, and other perivacuolar puncta. Notably, more than half of AP-1 puncta and AP-3 puncta were not associated with the donor compartments where they are thought to be generated, implying that these were coated transport vesicles. This work provides a convenient tool set for the investigation of vesicular transport in yeast and live cell imaging evidence for the presence of certain coated transport vesicles. Full article

In eukaryotes, the retromer complex is critical for the transport of cargo proteins from endosomes to the trans-Golgi network (TGN). Despite its importance, there is a lack of research on the retromer-mediated transport of cargo proteins regulating the growth, development, and pathogenicity of filamentous fungi. In the present study, transcriptome analysis showed that the expression levels of the retromer complex (CcVPS35, CcVPS29 and CcVPS26) were significantly elevated during the early stages of Corynespora cassiicola invasion. Gene knockout and complementation analyses further highlighted the critical role of the retromer complex in C. cassiicola infection. Subcellular localization analysis showed that the retromer complex was mainly localized to the vacuolar membrane and partially to endosomes and the TGN. Further research found that the retromer core subunit CcVps35 can interact with the cargo protein CcSnc1. Subcellular localization showed that CcSnc1 is mainly located at the hyphal tip and partially in endosomes and the Golgi apparatus. Deletion of CcVPS35 resulted in the missorting of CcSnc1 into the vacuolar degradation pathway, indicating that the retromer can sort CcSnc1 from endosomes and transport it to the TGN. Additionally, gene knockout and complementation analyses demonstrated that CcSnc1 is critical for the growth, development, and pathogenicity of C. cassiicola. In summary, the vesicular transport pathway involving the retromer complex regulates the sorting and transport of the cargo protein CcSnc1, which is important for the growth, development and pathogenicity of C. cassiicola. Full article

The tight regulation of protein composition within the plasma membranes of plant cells is crucial for the proper development of plants and for their ability to respond to a changing environment. Upon being endocytosed, integral membrane proteins can be secreted, sorted into multivesicular bodies/late endosomes, and degraded in the lytic vacuole, or recycled back to the plasma membrane to continue functioning. The evolutionarily conserved retromer complex has attracted the interest of plant cell biologists for over a decade as it has emerged as a key regulator of the trafficking of endocytosed integral plasma membrane proteins. Recently, a related recycling complex that shares a subunit with retromer was described in metazoan species. Named “retriever”, homologs to the proteins that comprise this new recycling complex and its accessory proteins are found within plant lineages. Initial experiments indicate that there is conservation of function between metazoan and plant retriever proteins, suggesting that it is prudent to re-evaluate the available plant retromer data with the added potential of a plant retriever complex. Full article

In the last two decades, alpha-synuclein (alpha-syn) assumed a prominent role as a major component and seeding structure of Lewy bodies (LBs). This concept is driving ongoing research on the pathophysiology of Parkinson’s disease (PD). In line with this, alpha-syn is considered to be the guilty protein in the disease process, and it may be targeted through precision medicine to modify disease progression. Therefore, designing specific tools to block the aggregation and spreading of alpha-syn represents a major effort in the development of disease-modifying therapies in PD. The present article analyzes concrete evidence about the significance of alpha-syn within LBs. In this effort, some dogmas are challenged. This concerns the question of whether alpha-syn is more abundant compared with other proteins within LBs. Again, the occurrence of alpha-syn compared with non-protein constituents is scrutinized. Finally, the prominent role of alpha-syn in seeding LBs as the guilty structure causing PD is questioned. These revisited concepts may be helpful in the process of validating which proteins, organelles, and pathways are likely to be involved in the damage to meso-striatal dopamine neurons and other brain regions involved in PD. Full article

The hypothalamus regulates fundamental aspects of physiological homeostasis and behavior, including stress response, reproduction, growth, sleep, and feeding, several of which are affected in patients with Prader–Willi (PWS) and Schaaf–Yang syndrome (SYS). PWS is caused by paternal deletion, maternal uniparental disomy, or imprinting defects that lead to loss of expression of a maternally imprinted region of chromosome 15 encompassing non-coding RNAs and five protein-coding genes; SYS patients have a mutation in one of them, MAGEL2. Throughout life, PWS and SYS patients suffer from musculoskeletal deficiencies, intellectual disabilities, and hormonal abnormalities, which lead to compulsive behaviors like hyperphagia and temper outbursts. Management of PWS and SYS is mostly symptomatic and cures for these debilitating disorders do not exist, highlighting a clear, unmet medical need. Research over several decades into the molecular and cellular roles of PWS genes has uncovered that several impinge on the neuroendocrine system. In this review, we will discuss the expression and molecular functions of PWS genes, connecting them with hormonal imbalances in patients and animal models. Besides the observed hormonal imbalances, we will describe the recent findings about how the loss of individual genes, particularly MAGEL2, affects the molecular mechanisms of hormone secretion. These results suggest that MAGEL2 evolved as a mammalian-specific regulator of hypothalamic neuroendocrine function. Full article

Fungi are an important group of microorganisms that play crucial roles in a variety of ecological and biotechnological processes. Fungi depend on intracellular protein trafficking, which involves moving proteins from their site of synthesis to the final destination within or outside the cell. The soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are vital components of vesicle trafficking and membrane fusion, ultimately leading to the release of cargos to the target destination. The v-SNARE (vesicle-associated SNARE) Snc1 is responsible for anterograde and retrograde vesicle trafficking between the plasma membrane (PM) and Golgi. It allows for the fusion of exocytic vesicles to the PM and the subsequent recycling of Golgi-localized proteins back to the Golgi via three distinct and parallel recycling pathways. This recycling process requires several components, including a phospholipid flippase (Drs2-Cdc50), an F-box protein (Rcy1), a sorting nexin (Snx4-Atg20), a retromer submit, and the COPI coat complex. Snc1 interacts with exocytic SNAREs (Sso1/2, Sec9) and the exocytic complex to complete the process of exocytosis. It also interacts with endocytic SNAREs (Tlg1 and Tlg2) during endocytic trafficking. Snc1 has been extensively investigated in fungi and has been found to play crucial roles in various aspects of intracellular protein trafficking. When Snc1 is overexpressed alone or in combination with some key secretory components, it results in enhanced protein production. This article will cover the role of Snc1 in the anterograde and retrograde trafficking of fungi and its interactions with other proteins for efficient cellular transportation. Full article

Entamoeba histolytica is the enteric protozoan parasite responsible for amebiasis. Trophozoites of E. histolytica ingest human cells in the intestine and other organs, which is the hallmark of its pathogenesis. Phagocytosis and trogocytosis are pivotal biological functions for its virulence and also contribute to the proliferation of nutrient uptake from the environment. We previously elucidated the role of a variety of proteins associated with phagocytosis and trogocytosis, including Rab small GTPases, Rab effectors, including retromer, phosphoinositide-binding proteins, lysosomal hydrolase receptors, protein kinases, and cytoskeletal proteins. However, a number of proteins involved in phagocytosis and trogocytosis remain to be identified, and mechanistic details of their involvement must be elucidated at the molecular level. To date, a number of studies in which a repertoire of proteins associated with phagosomes and potentially involved in phagocytosis have been conducted. In this review, we revisited all phagosome proteome studies we previously conducted in order to reiterate information on the proteome of phagosomes. We demonstrated the core set of constitutive phagosomal proteins and also the set of phagosomal proteins recruited only transiently or in condition-dependent fashions. The catalogs of phagosome proteomes resulting from such analyses can be a useful source of information for future mechanistic studies as well as for confirming or excluding a possibility of whether a protein of interest in various investigations is likely or is potentially involved in phagocytosis and phagosome biogenesis. Full article

SNX27 belongs to the sorting nexin (SNX) family of proteins that play a critical role in protein sorting and trafficking in the endocytosis pathway. This protein family is characterized by the presence of a Phox (PX) domain; however, SNX27 is unique in containing an additional PDZ domain. Recently, SNX27 has gained popularity as an important sorting protein that is associated with the retromer complex and mediates the recycling of internalized proteins from endosomes to the plasma membrane in a PDZ domain-dependent manner. Over 100 cell surface proteins have been identified as binding partners of the SNX27–retromer complex. However, the roles and underlying mechanisms governed by SNX27 in tumorigenesis remains to be poorly understood. Many of its known binding partners include several G-protein coupled receptors, such as β2-andrenergic receptor and parathyroid hormone receptor, are associated with multiple pathways implicated in oncogenic signaling and tumorigenesis. Additionally, SNX27 mediates the recycling of GLUT1 and the activation of mTORC1, both of which can regulate intracellular energy balance and promote cell survival and proliferation under conditions of nutrient deprivation. In this review, we summarize the structure and fundamental roles of SNX proteins, with a focus on SNX27, and provide the current evidence indicating towards the role of SNX27 in human cancers. We also discuss the gap in the field and future direction of SNX27 research. Insights into the emerging roles and mechanism of SNX27 in cancers will provide better development strategies to prevent and treat tumorigenesis. Full article

Mammalian retromer complex contributes to multiple early endosome-associated trafficking pathways whose origins are dependent on which sorting nexin (SNX) they are complexed with. In an attempt to dissect out the contribution of individual retromer–SNX complexes, we examined the trafficking of EGFR in detail within a series of KO cell line models. We demonstrated that the depletion of retromer subunit Vps35 leads to decreased EGFR protein levels in resting cells with enhanced association of EGFR with lysosomal compartments. Compared to control cells, the addition of EGF to Vps35 KO cells resulted in a reduced rate of EGFR degradation; AKT activation and cell prolferation rates were elevated, while ERK activation remained relatively unchanged. These observations are consistent with a prolonged temporal association of EGFR within early endosomes due to the inefficiency of early endosome-associated protein trafficking pathways or organelle maturation due to retromer absence. We did not fully delineate the discrete contributions from retromer-associated SNXs to the phenotypes observed from retromer Vps35 depletion. While each of the knock-outs of SNX1/2, SNX3, or SNX27 promotes the enhanced association of EGFR with early endosomal compartments, only the decreased EGF-mediated EGFR degradation was observed in SNX1/2 dKO cells, while the enhanced AKT activation was only increased in SNX3 KO or SNX27 KO cells. Despite this, each of the knock-outs showed increased EGF-stimulated cell proliferation rates. Full article

Camellia oleifera is an edible oil tree species native to China. Anthracnose is a common disease of Ca. oleifera, which reduces the production of the trees and brings huge economic losses. We have previously identified the fungus Colletotrichum fructicola as the major pathogen of anthracnose in Ca. oleifera. The retromer complex participates in the intracellular retrograde transport of the cargos from the endosome to the trans-Golgi network in the eukaryotes. Vacuolar protein sorting 29 is a subunit of the retromer complex. Targeted CfVPS29 gene deletion revealed that CfVps29 is involved in growth, conidiation, and the response to cell wall stress. We further found that the ΔCfvps29 mutant was minimally pathogenic to Ca. oleifera leaves, as a result of its defect in appressorium formation. This study illustrated the crucial functions of CfVps29 in the development, cell wall stress response, and pathogenicity of C. fructicola and, therefore, identified it as a potential fungicide target for the control of anthracnose. Full article

The HIV-1 envelope glycoprotein (Env) is an essential structural component of the virus, serving as the receptor-binding protein and principal neutralizing determinant. Env trimers are incorporated into developing particles at the plasma membrane of infected cells. Incorporation of HIV-1 Env into particles in T cells and macrophages is regulated by the long Env cytoplasmic tail (CT) and the matrix region of Gag. The CT incorporates motifs that interact with cellular factors involved in endosomal trafficking. Env follows an unusual pathway to arrive at the site of particle assembly, first traversing the secretory pathway to the plasma membrane (PM), then undergoing endocytosis, followed by directed sorting to the site of particle assembly on the PM. Many aspects of Env trafficking remain to be defined, including the sequential events that occur following endocytosis, leading to productive recycling and particle incorporation. This review focuses on the host factors and pathways involved in Env trafficking, and discusses leading models of Env incorporation into particles. Full article

Vps35 (vacuolar protein sorting 35), a key component of retromer, plays a crucial role in selective retrieval of transmembrane proteins from endosomes to trans-Golgi networks. Dysfunctional Vps35/retromer is a risk factor for the development of neurodegenerative diseases. Vps35 is highly expressed in developing pyramidal neurons, both in the mouse neocortex and hippocampus, Although embryonic neuronal Vps35’s function in promoting neuronal terminal differentiation and survival is evident, it remains unclear whether and how neuronal Vps35 communicates with other types of brain cells, such as blood vessels (BVs), which are essential for supplying nutrients to neurons. Dysfunctional BVs contribute to the pathogenesis of various neurodegenerative disorders. Here, we provide evidence for embryonic neuronal Vps35 as critical for BV branching and maturation in the developing mouse brain. Selectively knocking out (KO) Vps35 in mouse embryonic, not postnatal, neurons results in reductions in BV branching and density, arteriole diameter, and BV-associated pericytes and microglia but an increase in BV-associated reactive astrocytes. Deletion of microglia by PLX3397 enhances these BV deficits in mutant mice. These results reveal the function of neuronal Vps35 in neurovascular coupling in the developing mouse brain and implicate BV-associated microglia as underlying this event. Full article