Search Results (45)

RNA viruses, such as SARS-CoV-2 and influenza, pose a persistent threat to global public health. Their high mutation rates undermine the effectiveness of conventional direct-acting antivirals (DAAs) and facilitate drug resistance. As obligate intracellular parasites, RNA viruses rely extensively on host cellular machinery and metabolic pathways throughout their life cycle. This dependency has prompted a strategic shift in antiviral research—from targeting the mutable virus to targeting relatively conserved host dependency factors (HDFs). In this review, we systematically analyze how RNA viruses exploit HDFs at each stage of infection: utilizing host receptors for entry; remodeling endomembrane systems to establish replication organelles; hijacking transcriptional, translational, and metabolic systems for genome replication and protein synthesis; and co-opting trafficking and budding machinery for assembly and egress. By comparing strategies across diverse RNA viruses, we highlight the broad-spectrum potential of HDF-targeting approaches, which offer a higher genetic barrier to resistance, providing a rational framework for developing host-targeting antiviral therapies. Full article

Protein S-palmitoylation is a reversible lipid modification that regulates various aspects of protein function, including membrane association, subcellular localization, trafficking, stability, and activity. The depalmitoylase ABHD17A removes palmitate from multiple substrates, but its cellular positioning and the role of its own palmitoylation in regulating its function remain unclear. This study identifies a palmitoylation code within the conserved N-terminal cysteine cluster of ABHD17A, which governs its intracellular distribution and plasma membrane (PM) targeting. N-terminal palmitoylation is essential for PM localization. Through the use of code-restricted mutants, we found that modifications in the middle region (C14, C15) are critical for PM targeting and catalytic activity, while modifications at the front (C10, C11) and rear (C18) influence endosomal routing and delivery to the PM. Alanine scanning revealed that adjacent hydrophobic residues, particularly L9 and F13, are crucial for initial engagement with endomembranes. Sequence analysis and mutagenesis identified two tyrosine-based YXXØ motifs within the alpha/beta hydrolase fold; disruption of the proximal motif (L115A) decreased surface abundance and redirected ABHD17A to autophagosomes, indicating a need for YXXØ-dependent endosomal sorting, likely at the trans-Golgi network. Biochemical assays demonstrated a continuum of acylation states influenced by the palmitoylation code. This requirement for the middle region was conserved in ABHD17B and ABHD17C. Overall, our findings suggest a stepwise mechanism for ABHD17A delivery to the PM, enabling its depalmitoylase activity on membrane-bound substrates. Full article

Galectin-3 (Gal-3) is a β-galactoside-binding lectin that mediates diverse signaling events in multiple cell types, including immune cells. It is also a prognostic indicator for multiple clinically important disorders, including cardiovascular disease. Gal-3 binds to cell surface glycans to form lattices that modulate surface receptor signaling and internalization. However, the tissue-specific regulation of Gal-3 surface expression remains poorly understood. Here, we review evidence for the involvement of Gal-3 in cell surface signaling, intranuclear events, and intracellular trafficking. Our focus will be on the O-GlcNAc modification as a regulator of Gal-3 biosynthesis, non-canonical secretion, and recycling. We argue that the nutrient-driven cytoplasmic hexosamine biosynthetic pathway (HBP) and endomembrane transport mechanisms generate unique pools of nucleotide sugars. The differing levels of nucleotide sugars in the cytosol, endoplasmic reticulum (ER), and Golgi apparatus generate differential thresholds for the responsiveness of O-GlcNAc cycling, N- and O-linked glycan synthesis/branching, and glycolipid synthesis. By regulating Gal-3 synthesis and non-canonical secretion, O-GlcNAc cycling may serve as a nexus constraining Gal-3 cell surface expression and lattice formation. This homeostatic feedback mechanism would be critical under conditions where extensive glycan synthesis and branching in the endomembrane system and on the cell surface are maintained by elevated hexosamine synthesis. Thus, O-GlcNAc cycling and Gal-3 synergize to regulate Gal-3 secretion and influence cellular signaling. In humans, Gal-3 serves as an early-stage prognostic indicator for heart disease, kidney disease, viral infection, autoimmune disease, and neurodegenerative disorders. Since O-GlcNAc cycling has also been linked to these pathologic states, exploring the interconnections between O-GlcNAc cycling and Gal-3 expression and synthesis is likely to emerge as an exciting area of research. Full article

Factor VIII (FVIII) interacts with Endoplasmic Reticulum (ER) chaperones Calnexin (CANX) and Calreticulin (CALR) and with ER-Golgi Intermediate Compartment (ERGIC) transporters, Lectin, mannose-binding 1 (LMAN1) and Multiple Coagulation Deficiency 2 (MCFD2). We previously reported that the Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) also influence FVIII secretion. Here, we further investigated the intracellular dynamics of FVIII using single and double CRISPR/Cas9 Knockout (KO) models of the abovementioned chaperones as well as the GABARAP proteins in HEK293 cells expressing FVIII. Cellular pathways were manipulated by Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and subjected to glucose starvation. The effect of each KO on FVIII secretion and organelle distribution was assessed by a two-stage chromogenic assay and immunofluorescence (IF) microscopy, prior and upon cell treatments. Using these approaches, we first observed distinct effects of each studied protein on FVIII trafficking. Notably, intracellular localization patterns revealed clustering of FVIII phenotypes in GABARAP^KO, CANX^KO, and CALR^KO cells together under both basal and treated conditions, an observation that was also reflected in their respective double KO combinations. Besides, a clear involvement of additional components of the endomembrane system was evident, specifically at the trans-Golgi space, as marked by FVIII colocalization with the Ras-like proteins in brain (Rab8 and Rab7) and with the Vesicle-Associated Membrane Protein (VAMP8), along with the observed impact of the selected cell treatments on FVIII phenotypes. These outcomes enhance our understanding of the molecular mechanisms regulating FVIII and pave the way for new perspectives, which could be further projected into FVIII replacement, cell and gene therapies. Full article

Members of the phloem protein 16 (PP16) gene family are induced by elicitors in rice and the corresponding proteins from cucurbits, which display RNA binding and intercellular transport activities, are accumulated in phloem sap. These proteins facilitate the movement of protein complexes through the phloem translocation flow and may be involved in the response to water deficit, among other functions. However, there is scant information regarding their function in other plants, including the identification of paralog genes in non-vascular plants and chlorophytes. In the present work, an evolutionary and structural analysis of the PP16 family in green plants (Viridiplantae) was carried out. Data mining in different databases indicated that PP16 likely originated from a larger gene present in an ancestral lineage that gave rise to chlorophytes and multicellular plants. This gene encodes a protein related to synaptotagmin, which is involved in vesicular transport in animal systems, although other members of this family play a role in lipid turnover in endomembranes and organelles. These proteins contain a membrane-binding C2 domain shared with PP16 proteins in vascular plants. In silico analysis of the predicted structure of the PP16 protein family identified several β-sheets, one α-helix, and intrinsically disordered regions. PP16 may have been originally involved in vesicular trafficking and/or membrane maintenance but specialized in long-distance signaling during the emergence of the plant vascular system. Full article

The endomembrane system in plant cells enables the cell to manage and coordinate a variety of membranous compartments so that they and their contents arrive at the right location. The secretory pathway is an essential part of this complex network and has its gateway at the Endoplasmic Reticulum. Therefore, alterations at the ER can affect how protein trafficking takes place and how cargo leaves this organelle. With this work, we assessed how abnormalities at the Endoplasmic Reticulum would interfere with protein sorting and trafficking. We used an Arabidopsis mutant—leb-2 GFP-h, presenting abnormal ER morphology, and evaluated the expression of aspartic proteinases and genes related to vacuolar transport along with the localization of a specific vacuolar sorting signal—plant-specific insert (PSI). Our results show that alterations in the leb-2 GFP-h mutant did not disrupt the transport of PSI–mCherry to the vacuole but influenced the expression of endogenous aspartic proteinases. Furthermore, the study of key endomembrane genes expression revealed an upregulation of the SNARE proteins AtVAMP722 and AtVAMP723. The leb-2 mutant seems not to interfere with vacuolar routes but may be implicated in secretion events. Full article

Autophagy is a catabolic pathway capable of degrading cellular components ranging from individual molecules to organelles. Autophagy helps cells cope with stress by removing superfluous or hazardous material. In a previous work, we demonstrated that transcriptional upregulation of two autophagy-related genes, ATG5 and ATG7, in Arabidopsis thaliana positively affected agronomically important traits: biomass, seed yield, tolerance to pathogens and oxidative stress. Although the occurrence of these traits correlated with enhanced autophagic activity, it is possible that autophagy-independent roles of ATG5 and ATG7 also contributed to the phenotypes. In this study, we employed affinity purification and LC-MS/MS to identify the interactome of wild-type ATG5 and its autophagy-inactive substitution mutant, ATG5^K128R Here we present the first interactome of plant ATG5, encompassing not only known autophagy regulators but also stress-response factors, components of the ubiquitin-proteasome system, proteins involved in endomembrane trafficking, and potential partners of the nuclear fraction of ATG5. Furthermore, we discovered post-translational modifications, such as phosphorylation and acetylation present on ATG5 complex components that are likely to play regulatory functions. These results strongly indicate that plant ATG5 complex proteins have roles beyond autophagy itself, opening avenues for further investigations on the complex roles of autophagy in plant growth and stress responses. Full article

Coenzyme Q (CoQ) is an essential lipid with many cellular functions, such as electron transport for cellular respiration, antioxidant protection, redox homeostasis, and ferroptosis suppression. Deficiencies in CoQ due to aging, genetic disease, or medication can be ameliorated by high-dose supplementation. As such, an understanding of the uptake and transport of CoQ may inform methods of clinical use and identify how to better treat deficiency. Here, we review what is known about the cellular uptake and intracellular distribution of CoQ from yeast, mammalian cell culture, and rodent models, as well as its absorption at the organism level. We discuss the use of these model organisms to probe the mechanisms of uptake and distribution. The literature indicates that CoQ uptake and distribution are multifaceted processes likely to have redundancies in its transport, utilizing the endomembrane system and newly identified proteins that function as lipid transporters. Impairment of the trafficking of either endogenous or exogenous CoQ exerts profound effects on metabolism and stress response. This review also highlights significant gaps in our knowledge of how CoQ is distributed within the cell and suggests future directions of research to better understand this process. Full article

BnSIP1-1 is the first identified SIP1 (6b Interacting Protein1) subfamily gene of the trihelix transcription factor family from Brassica napus (B. napus). We previously used a reverse genetic method to reveal its abiotic stress response function in endowing plants resistance to drought and salinity, as well as ABA (Abscisic acid). However, the molecular mechanisms of BnSIP1-1 are unclear. In this study, the global transcriptome files of BnSIP1-1-overexpressing transgenic and wildtype B. napus seedlings under ABA treatment were constructed using RNA-seq. A total of 1823 and 5512 DEGs (Differentially Expressed Genes) were identified in OE vs. WT and OE_ABA vs. WT_ABA comparison groups, which included 751 and 2567 up-regulated DEGs, and 1072 and 2945 down-regulated DEGs, separately. The impact of overexpressed BnSIP1-1 on plants was amplified by ABA, indicating BnSIP1-1 was an ABA-conditioned responsive gene. More interestingly, we found the reasons for BnSIP1-1 increasing plants’ insensitivity to ABA were not by regulating ABA synthesis and catabolism, but by manipulating ABA transportation, ABA signal perception and transduction, inositol phosphate metabolism, as well as endomembrane trafficking, indirectly suggesting this gene may play roles upstream of the core ABA response pathway. Our results provided new insights into improving the knowledge about the function of BnSIP1-1 and the ABA signaling mechanism in B. napus. Full article

Viruses infect all cellular life forms and cause various diseases and significant economic losses worldwide. The majority of viruses are positive-sense RNA viruses. A common feature of infection by diverse RNA viruses is to induce the formation of altered membrane structures in infected host cells. Indeed, upon entry into host cells, plant-infecting RNA viruses target preferred organelles of the cellular endomembrane system and remodel organellar membranes to form organelle-like structures for virus genome replication, termed as the viral replication organelle (VRO) or the viral replication complex (VRC). Different viruses may recruit different host factors for membrane modifications. These membrane-enclosed virus-induced replication factories provide an optimum, protective microenvironment to concentrate viral and host components for robust viral replication. Although different viruses prefer specific organelles to build VROs, at least some of them have the ability to exploit alternative organellar membranes for replication. Besides being responsible for viral replication, VROs of some viruses can be mobile to reach plasmodesmata (PD) via the endomembrane system, as well as the cytoskeleton machinery. Viral movement protein (MP) and/or MP-associated viral movement complexes also exploit the endomembrane-cytoskeleton network for trafficking to PD where progeny viruses pass through the cell-wall barrier to enter neighboring cells. Full article

Plants must adapt to cope with adverse environmental conditions that affect their growth and development. To overcome these constraints, they can alter their developmental patterns by modulating cellular processes and activating stress-responsive signals. Alongside the activation of the antioxidant (AOX) system, a high number of genes are expressed, and proteins must be distributed to the correct locations within the cell. The endomembrane system and associated vesicles thus play an important role. Several pathways have been associated with adverse environmental conditions, which is the case for the exocyst-positive organelle—EXPO. The present work, using Arabidopsis mutants with T-DNA insertions in the gene EXO70, essential for EXPO vesicles formation, was designed to characterise the anatomical (morphology and root length), biochemical (quantification of stress markers and antioxidant system components), and molecular responses (gene expression) to abiotic stresses (saline, drought, oxidative, and metal-induced toxicity). The results obtained showed that mutant plants behave differently from the wild type (WT) plants. Therefore, in the exo70 mutant, morphological changes were more noticeable in plants under stress, and the non-enzymatic component of the antioxidant system was activated, with no alterations to the enzymatic component. Furthermore, other defence strategies, such as autophagy, did not show important changes. These results confirmed the EXPO as an important structure for tolerance/adaptation to stress. Full article

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. Full article

Plant cell walls are classified as primary and secondary walls. The primary wall is necessary for plant morphogenesis and supports cell growth and expansion. Once the growth and expansion ceases, specialized cells form secondary walls in order to give strength and rigidity to the plant. Secondary cell walls are the main constituent of woody biomass. This biomass is raw material for industrial products, food, and biomaterials. Recently, there are an increasing number of studies using biomass for biofuel production and this area has gained importance. However, there are still many unknowns regarding the synthesis and structure of complex polysaccharides forming biomass. Cellulose, being one of the main components of the cell wall, is synthesized at the plasma membrane by cellulose synthase complexes and does not require transportation. On the other hand, pectin and hemicelluloses are synthesized by enzymes located in the Golgi apparatus. Therefore, they need to be transported to the plasma membrane. Even though this transport mechanism is very important, it is one of the least understood parts of the endomembrane system. Xylan is the major hemicellulose in many biomasses and is important for renewable material production. There is limited knowledge about the cellular trafficking of xylan. In this review, we cover the current information and what we know about the vesicular transport of xylan to the cell wall. Full article

Pattern recognition receptor (PRR) trafficking to the plasma membrane and endocytosis plays a crucial role in pattern triggered immunity (PTI). Dynamin-related proteins (DRPs) participate in endocytosis and recycling. In Arabidopsis, DRP1 and DRP2 are involved in plasma membrane scission during endocytosis. They are required for the PRR FLS2 endocytosis induction and PTI activation after elicitation with flg22, the MAMP recognized by FLS2. In tomato, SlDRP2A regulates the PRR LeEIX2 endocytosis and PTI activation in response to EIX, the MAMP recognized by LeEIX2. However, it is unknown if other DRPs participate in these processes. Taking advantage of bioinformatics tools, we selected SlDRP2B among the eight DRP2 tomato orthologues to study its functionality in trafficking and plant immunity. Through transient expression of SlDRP1B and its dominant-negative mutant on Nicotiana benthamiana and Nicotiana tabacum, we analyzed SlDRP1B function. We observed that SlDRP1B is physically associated with the LeEIX2 and modifies LeEIX2 trafficking, increasing its presence in endosomes. An enhancement of EIX-elicitated defense responses accompanies the role of SlDRP1B on LeEIX endocytosis. In addition, SlDRP1B overexpression enhanced flg22-elicited defense response. With these results, we conclude that SlDRP1B regulates PRR trafficking and, therefore, plant immunity, similarly to the SlDRP2A role. Full article

The Golgi Complex is the central hub in the endomembrane system and serves not only as a biosynthetic and processing center but also as a trafficking and sorting station for glycoproteins and lipids. In addition, it is an active signaling hub involved in the regulation of multiple cellular processes, including cell polarity, motility, growth, autophagy, apoptosis, inflammation, DNA repair and stress responses. As such, the dysregulation of the Golgi Complex-centered signaling cascades contributes to the onset of several pathological conditions, including cancer. This review summarizes the current knowledge on the signaling pathways regulated by the Golgi Complex and implicated in promoting cancer hallmarks and tumor progression. Full article