Search Results (271)

Background/Objectives: A Petit Bleu de Gascogne (PBDG) dog presented with a progressive neurological disorder characterized by hind-limb weakness, anxiety and hallucinatory episodes, lip smacking, progressive vision loss, muscle atrophy, and ataxia. Magnetic resonance imaging revealed diffuse brain atrophy. The dog was euthanized at approximately 23 months of age due to the progression of neurological signs. A study was undertaken to identify the molecular genetic basis of the disorder in this dog. Methods: Microscopic analyses were performed to characterize the disease pathology and whole-genome sequencing was performed to identify the molecular genetic basis of the disorder. Results: The proband exhibited pronounced accumulations of autofluorescent intracellular inclusions in the brain, retina, and heart with ultrastructural appearances similar to those of lysosomal storage bodies that accumulate in the neuronal ceroid lipofuscinosis (NCLs), a group of progressive neurodegenerative disorders. Whole-genome sequence analysis of DNA from the proband identified homozygous missense variants in AP3B1 and TRAPPC9 that encode proteins involved in sorting and transport of proteins through the Golgi apparatus to lysosomes. Screening of unaffected PBDGs for these variants identified dogs that were homozygous for either variant, but no other dogs that were homozygous for both. Conclusions: These findings raise the possibility that the disease involves the combined influence of the two variants, and that the proteins encoded by these genes interact within the Golgi apparatus to mediate protein sorting and transport to lysosomes. An alteration in this interaction could underlie the NCL-like lysosomal storage disorder observed in the proband. Full article

FAM20C, previously known as Golgi casein kinase (GCK), is a serine/threonine kinase localized to the Golgi apparatus and classified within the acidophilic kinase family. Its phosphorylation motif is characterized by a glutamic acid residue at the +2 position relative to the target site. Before its molecular identity was established, analysis of a limited number of phosphosites in secreted proteins showed that around 70% matched the GCK consensus sequence, suggesting that GCK is the principal kinase for secreted proteins. Following the identification of GCK as FAM20C, the generation of FAM20C knockout cell lines and phosphoproteomic data confirmed its role: approximately 80% of serine/threonine phosphosites in the secretome of two different human cell lines were shown to depend on FAM20C. In this study, comparative analysis of in vitro phosphorylation datasets from a broad panel of recombinant Ser/Thr kinases confirmed that the FAM20C consensus sequence is distinct from those of other acidophilic kinases. Examination of experimentally identified human phosphosites within the secretory pathway revealed strong conservation of the FAM20C consensus, firmly establishing this enzyme as the master Ser kinase of the entire pathway. From this dataset, we defined the putative FAM20C substratome, comprising 443 phosphosites across 256 proteins, ~77% of which had not been previously linked to FAM20C. This represents the most extensive FAM20C substratome to date and a valuable resource for functional studies. Notably, enrichment analysis highlights strong links between FAM20C and major extracellular pathways, including collagen fibril organization, complement activation, and blood coagulation, underscoring an underappreciated role for this kinase in regulating hemostasis and innate immunity. Full article

Despite significant advances in understanding the genetics of Parkinson’s disease (PD) and Parkinsonism, the diagnostic yield remains low. Pathogenic variants of GBA1, which encodes the lysosomal enzyme β-glucocerebrosidase and causes recessive Gaucher dis-ease, are recognized as the most important genetic risk factor for PD in heterozygous carriers. This study focuses on the functional genomics of rare genetic variations in other lysosomal hydrolytic enzymes genes in patient-derived fibroblasts. We examined 49 early-onset PD patients using whole exome sequencing and in silico panel analysis based on a curated PD gene list. Two patients were found to carry the p.Asp313Tyr variant in the X-linked GLA gene (encoding GALA, typically associated with Fabry disease), and one patient carried the p.Arg419Gln variant in GLB1 (encoding β-Gal, linked to the recessive GM1 gangliosidosis and mucopolysaccharidosis type IVB). The in silico study of both variants supports a potentially damaging impact on the encoded protein function and structural destabilization. Additional candidate variants were found related to lysosomes, Golgi apparatus and neurodegeneration, suggesting a multifactorial contribution to the disease. However, none of these variants met diagnostic standards. Functional assays showed a significant decrease in GALA expression and partial retention of the enzyme in the trans-Golgi network in fibroblasts with GLA:p.Asp313Tyr, while altered Golgi morphology was observed in fibroblasts with GLB1:p.Arg419Gln. Moreover, all patients exhibited abnormalities in lysosomal morphology, altered lysosomal pH, and impaired autophagic flux. Our findings suggest that rare, heterozygous variants in lysosomal-related genes, even when individually insufficient for monogenic disease, can converge to impair lysosomal homeostasis and autophagic flux in EOPD. The underlying genetic and cellular heterogeneity among patients emphasizes the importance of combining genetic and functional approaches to better understand the mechanisms behind the EOPD, which could enhance both diagnosis and future treatments. Full article

DROSHA protein is widely known for its essential role in the microRNA (miRNA/miR) biogenesis pathway where, together with its co-factor DGCR8, it forms the “Microprocessor” complex and catalyzes the primary miRNA (pri-miRNA) processing in the nucleus. Nevertheless, DROSHA also seems to participate in several miRNA-independent cellular mechanisms, such as transcriptional regulation, RNA processing and genome integrity maintenance. Hence, the present study aims to further investigate novel miRNA-independent activities of DROSHA protein, with potentially regulatory roles in the oncogenesis of human cancer cells. Our results reveal a new, strong profile of microprocessor-independent DROSHA localization at the Golgi apparatus in several human cancer cell lines of different tissue origin, with hepatic carcinoma, thyroid cancer, urothelial bladder cancer, colon carcinoma and melanoma being the cellular model systems herein examined. Notably, oncogenic activity, malignancy grade and metastatic capacity are shown to be strongly associated with DROSHA’s compartmentalization at Golgi, a phenotype that does not seem to rely on p53 protein’s functionality. Taken together, through employment of advanced confocal laser scanning microscopy (CLSM) and molecular modeling, we herein unveil the ability of DROSHA, but not AGO2 and DICER, to reside at Golgi, where DROSHA can physically interact with the GM130 Golgi-specific component, thus indicating DROSHA’s engagement in non-canonical and miRNA-independent—but also Golgi apparatus-dependent—novel mechanisms that can be tightly coupled with malignancy dynamics and beneficially utilized as potential biomarkers and therapeutic targets for human cancer. Full article

The vast majority of viruses causing human and animal diseases are enveloped—their virions contain an outer lipid bilayer originating from a host cell. Small molecule antivirals targeting the lipid bilayer cover the broadest spectrum of viruses. In this context, we consider the chemical nature and mechanisms of action of membrane-targeting antivirals. They can affect virions by (1) physically modulating membrane properties to inhibit fusion of the viral envelope with the cell membrane, (2) physically affecting envelope lipids and proteins leading to membrane damage, pore formation and lysis, (3) causing photochemical damage of unsaturated membrane lipids resulting in integrity loss and fusion arrest. Other membrane-active compounds can target host cell membranes involved in virion’s maturation, coating, and egress (endoplasmic reticulum, Golgi apparatus, and outer membrane) affecting these last stages of viral reproduction. Both virion- and host-targeting membrane-active molecules are promising concepts for broad-spectrum antivirals. A panel of approved antivirals would be a superior weapon to respond to and control emerging disease outbreaks caused by new viral strains and variants. 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

Monomer-to-excimer transition has become a valuable technique in fluorescence imaging because of its ability to enhance imaging contrast. However, from a practical perspective, the accuracy of excimer formation at target sites warrants further exploration. Enzyme-triggered peptide self-assembly provides a promising solution to this limitation. As a proof-of-concept, in this study, we developed a furin-triggered peptide self-assembling fluorescent probe RF-Cou by coupling a coumarin dye 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid (Cou) with a furin-responsive peptide scaffold for precision live-cell imaging. Upon entering furin-overexpressing 4T1 tumor cells, RF-Cou underwent enzymatic cleavage, releasing an amphiphilic peptide motif and self-assembling into nanoparticles largely concentrated in the Golgi apparatus to confine the diffusion of Cou. During this process, the Cou excimers were formed and induced a red shift in the fluorescence emission, validating the feasibility of RF-Cou in efficient excimer imaging of furin-overexpressing tumor cells. We expect that our findings will highlight the potential of stimuli-responsive small molecular peptide probes to advance excimer-based imaging platforms, particularly for enzyme-specific cell imaging and therapeutic monitoring. Full article

The proteolytic processing of the SARS-CoV-2 spike glycoprotein by host cell membrane-associated proteases is a key step in both the entry of the invading virus into the cell and the release of the newly generated viral particles from the infected cell. Because of the critical importance of this step for the viral infectivity cycle, it has been a target of extensive efforts aimed at identifying highly specific protease inhibitors as potential antiviral agents. An alternative strategy to disrupt the pre-fusioviden processing of the SARS-CoV-2 S glycoprotein aims to protect the substrate rather than directly inhibit the proteases. In this work, we focused on furin, a serine protease located primarily in the Golgi apparatus, but also present on the cell membrane. Its cleavage site within the S glycoprotein is located within the stalk region of the latter and comprises an arginine-rich segment (SPRRARS), which fits the definition of the Cardin–Weintraub glycosaminoglycan recognition motif. Native mass spectrometry (MS) measurements confirmed the binding of a hexadecameric peptide representing the loop region at the S1/S2 interface and incorporating the furin cleavage site (FCS) to heparin fragments of various lengths, as well as unfractionated heparin (UFH), although at the physiological ionic strength, only UFH remains tightly bound to the FCS. The direct LC/MS monitoring of FCS digestion with furin revealed a significant impact of both heparin fragments and UFH on the proteolysis kinetics, although only the latter had IC50 values that could be considered physiologically relevant (0.6 ± 0.1 mg/mL). The results of this work highlight the importance of the long-range and relatively non-specific electrostatic interactions in modulating physiological and pathological processes and emphasize the multi-faceted role played by heparin in managing coronavirus infections. Full article

Hydroxyproline O-arabinosyltransferase (HPAT), a critical enzyme in plant glycosylation pathways, catalyzes the transfer of arabinose to the hydroxyl group of hydroxyproline residues. This enzyme contains a canonical GT95 glycosyltransferase, a structural hallmark of this carbohydrate-active enzyme family. HPAT mediates arabinosylation of diverse cellular targets, including cell wall extension and small signaling peptides. Emerging evidence has shown that HPAT orthologs regulate plant development and symbiotic interactions through post-translational modification of CLV1/LRR Extracellular (CLE) peptides. Although the molecular functions of HPAT genes have been characterized in model plants such as Arabidopsis thaliana and Lotus japonicus, their roles remain unexplored in Zea mays L. In this study, we used ZmHPAT2 homozygous mutants to explore the function of the maize HPAT gene. Sequence analysis identified a N-terminal signal peptide targeting the Golgi apparatus and promoter elements responsive to AM fungal colonization. Phenotypic analysis revealed its negative regulatory role: zmhpat2 promotes vegetative growth (increased plant height and accelerated flowering) and enhances AM symbiosis (increased colonization rate). Mechanistic studies demonstrated that ZmHPAT2 possesses dual regulatory functions—the activation of auxin signaling and repression of ZmMYB1-mediated arbuscular degradation pathways. In addition, overexpression of ZmHPAT2 in Lotus japonicus inhibits growth (reduced plant height) and impairs symbiotic interactions. Our findings establish ZmHPAT2 as a critical node to regulate auxin and symbiotic signaling, providing novel insights into plant glycosylation-mediated development. This work not only advances our understanding of maize growth regulation but also identifies potential targets for crop improvement through arabinosylation pathway manipulation. Full article

The endoplasmic reticulum–Golgi apparatus system is an important organelle regulating male reproduction. USO1 vesicle transport factor (USO1), as an important molecule in this system, is a general vesicular transport factor and regulates various biological processes in vivo. However, the potential role of USO1 in mammalian testis development and spermatogenesis has not been investigated. We documented the presence of USO1 in mouse germ cells and its functional roles by generating Uso1-knockout germ cell lines. Uso1 depletion suppressed cell proliferation and growth while stimulating apoptosis in GC1 and GC2 cells. In addition, the Uso1 knockout blocked cell cycle progression and weakened DNA damage repair. Mechanistically, USO1 is associated with male reproduction by regulating the expression of genes involved in spermatogenesis, and we found evidence that USO1 is closely linked to centriolar satellites (CSs), which may play an important biological role. Overall, our findings reveal a vital role for USO1 in male fertility and offer a significant understanding of the functions of golgin proteins in reproductive biology. Full article

TM9SF2 belongs to a family of highly conserved nonaspanin proteins, and has been frequently identified as one of the important host factors for a plethora of lethal pathogens and toxins in previous genome-wide screening studies. We reported herein a novel molecular mechanism of TM9SF2 in mediating the cytotoxicity of ricin, a type II ribosome-inactivating protein. We first showed that TM9SF2 displays a non-redundant requirement for ricin-induced cytotoxicity within the nonaspanin family. Then we found that genetic interference of TM9SF2 substantially affects/remodels intracellular cholesterol trafficking, which results in abnormal cholesterol accumulation in Golgi compartments and causes severe Golgi fragmentation. The disruption of Golgi integrity and network impedes the retrograde transport of ricin and thus attenuates ricin-induced cytotoxicity. We further verified this mechanism by pharmacological manipulation of cholesterol metabolism (e.g., by using A939572 and avasimibe, etc.), which well restores the integrity of the Golgi apparatus and reverses the ricin-resistant phenotype induced by TM9SF2 knockdown. Our finding provides new mechanistic insights into the pathology and toxicology of ricin and could potentially be applied to other ribosome-inactivating toxins. Full article

Human papillomavirus (HPV) 16 is transported in a retrograde fashion from the cell surface to the Golgi apparatus. Prior to mitosis, the virus loses association with the Golgi and, following nuclear envelope breakdown, is found associated with the condensed mitotic chromatin. The intervening steps have not been well defined. It was previously demonstrated that the virus is transported to the mitotic chromosomes in vesicles. Here, we describe the role of the endoplasmic reticulum (ER) in the post-Golgi trafficking and the importance of the ER-generated coat protein complex II (COPII) anterograde trafficking pathway in HPV infection. HPV pseudovirus (PsV) colocalized with COPII components and silencing of this pathway inhibited HPV infection. Additionally, the inner COPII coat protein, Sec24b, could be biochemically isolated in association with HPV capsid proteins. This study provides insight into the mechanism of post-Golgi HPV trafficking. Full article

Cellular stressors have been demonstrated to exert a substantial influence on the functionality of organelles, thereby impacting cellular homeostasis and contributing to the development of disease pathogenesis. This review aims to examine the impact of diverse stressors, including environmental, chemical, biological, and physical factors, on critical organelles such as the cell membrane, mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, and membrane-less organelles. The intricate molecular mechanisms underlying cellular stress responses, encompassing oxidative stress, protein misfolding, and metabolic reprogramming, have the capacity to elicit adaptive responses or culminate in pathological conditions. The interplay between these stressors and organelle dysfunction has been implicated in a myriad of diseases, including neurodegenerative disorders, cancer, metabolic disorders, and immune-related pathologies. A comprehensive understanding of the mechanisms by which organelles respond to stress can offer valuable insights into the development of therapeutic strategies aimed at mitigating cellular damage. Full article

Plant cells utilize protein oligomerization for their functions in numerous important cellular processes. Protein-protein interactions are necessary to stabilize, optimize, and activate enzymes, as well as localize proteins to specific organelles and membranes. Glycosyltransferases—enzymes that attach sugars to polysaccharides, proteins, lipids, and RNA—across multiple plant biosynthetic processes have been demonstrated to interact with one another. The mechanisms behind these interactions are still unknown, but recent research has highlighted extensive examples of protein-protein interactions, specifically in the plant cell wall hemicellulose and pectin biosynthesis that takes place in the Golgi apparatus. In this review, we will discuss what is known so far about the interactions among Golgi-localized glycosyltransferases that are important for their functioning, trafficking, as well as structural aspects. Full article

The ultrastructural organization of the nuclei of the tegmental region in juvenile chum salmon (Oncorhynchus keta) was examined using transmission electron microscopy (TEM). The dorsal tegmental nuclei (DTN), the nucleus of fasciculus longitudinalis medialis (NFLM), and the nucleus of the oculomotor nerve (NIII) were studied. The ultrastructural examination provided detailed ultrastructural characteristics of neurons forming the tegmental nuclei and showed neuro–glial relationships in them. Neurons of three size types with a high metabolic rate, characterized by the presence of numerous mitochondria, polyribosomes, Golgi apparatus, and cytoplasmic inclusions (vacuoles, lipid droplets, and dense bodies), were distinguished. It was found that large interneurons of the NFLM formed contacts with protoplasmic astrocytes. Excitatory synaptic structures were identified in the tegmentum and their detailed characteristic are provided for the first time. Microglia-like cells were found in the NIII. The ultrastructural characteristics of neurogenic zones of the tegmentum of juvenile chum salmon were also determined for the first time. In the neurogenic zones of the tegmentum, adult-type neural stem progenitor cells (aNSPCs) corresponding to cells of types III and IVa Danio rerio. In the neurogenic zones of the tegmentum, neuroepithelial-like cells (NECs) corresponding to cells previously described from the zebrafish cerebellum were found and characterized. In the tegmentum of juvenile chum salmon, patterns of paracrine neurosecretion were observed and their ultrastructural characteristics were recorded. Patterns of apoptosis in large neurons of the tegmentum were examined by TEM. Using immunohistochemical (IHC) labeling of the brain lipid-binding protein (BLBP) and aromatase B (AroB), patterns of their expression in the tegmentum of intact animals and in the post-traumatic period after acute injury to the medulla oblongata were characterized. The response to brainstem injury in chum salmon was found to activate multiple signaling pathways, which significantly increases the BLBP and AroB expression in various regions of the tegmentum and valvula cerebelli. However, post-traumatic patterns of BLBP and AroB localizations are not the same. In addition to a general increase in BLBP expression in the tegmental parenchyma, BLBP overexpression was observed in the rostro-lateral tegmental neurogenic zone (RLTNZ), while AroB expression in the RLTNZ was completely absent. Another difference was the peripheral overexpression of AroB and the formation of dense reactive clusters in the ventro-medial zone of the tegmentum. Thus, in the post-traumatic period, various pathways were activated whose components were putative candidates for inducers of the “astrocyte-like” response in the juvenile chum salmon brain that are similar to those present in the mammalian brain. In this case, BLBP acted as a factor enhancing the differentiation of both radial glia and neurons. Estradiol from AroB+ astrocytes exerted paracrine neuroprotective effects through the potential inhibition of inflammatory processes. These results indicate a new role for neuronal aromatization as a mechanism preventing the development of neuroinflammation. Moreover, our findings support the hypothesis that BLBP is a factor enhancing neuronal and glial differentiation in the post-traumatic period in the chum salmon brain. Full article