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International Journal of Molecular... Jun 2024In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing...
The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation.
In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome-lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication.
Topics: Humans; Viral Nonstructural Proteins; Virus Replication; Autophagosomes; HeLa Cells; Phlebovirus; Autophagy; Tyrosine; Tryptophan; TOR Serine-Threonine Kinases; Mutation; Amino Acid Substitution; Severe Fever with Thrombocytopenia Syndrome; Lysosomes; Nucleoproteins
PubMed: 38928101
DOI: 10.3390/ijms25126394 -
Biomolecules May 2024The p53 protein is the master regulator of cellular integrity, primarily due to its tumor-suppressing functions. Approximately half of all human cancers carry mutations... (Review)
Review
The p53 protein is the master regulator of cellular integrity, primarily due to its tumor-suppressing functions. Approximately half of all human cancers carry mutations in the TP53 gene, which not only abrogate the tumor-suppressive functions but also confer p53 mutant proteins with oncogenic potential. The latter is achieved through so-called gain-of-function (GOF) mutations that promote cancer progression, metastasis, and therapy resistance by deregulating transcriptional networks, signaling pathways, metabolism, immune surveillance, and cellular compositions of the microenvironment. Despite recent progress in understanding the complexity of mutp53 in neoplastic development, the exact mechanisms of how mutp53 contributes to cancer development and how they escape proteasomal and lysosomal degradation remain only partially understood. In this review, we address recent findings in the field of oncogenic functions of mutp53 specifically regarding, but not limited to, its implications in metabolic pathways, the secretome of cancer cells, the cancer microenvironment, and the regulating scenarios of the aberrant proteasomal degradation. By analyzing proteasomal and lysosomal protein degradation, as well as its connection with autophagy, we propose new therapeutical approaches that aim to destabilize mutp53 proteins and deactivate its oncogenic functions, thereby providing a fundamental basis for further investigation and rational treatment approaches for TP53-mutated cancers.
Topics: Humans; Tumor Suppressor Protein p53; Neoplasms; Tumor Microenvironment; Proteolysis; Proteasome Endopeptidase Complex; Autophagy; Animals; Mutation; Lysosomes; Carcinogenesis
PubMed: 38927053
DOI: 10.3390/biom14060649 -
Biomolecules May 2024Renal interstitial fibrosis (RIF) is a classic pathophysiological process of chronic kidney disease (CKD). However, the mechanisms underlying RIF remain unclear. The...
Renal interstitial fibrosis (RIF) is a classic pathophysiological process of chronic kidney disease (CKD). However, the mechanisms underlying RIF remain unclear. The present study found that a novel circular RNA, cirInpp5b, might be involved in RIF by high-throughput sequencing. Subsequent experiments revealed that circInpp5b was reduced in UUO mouse kidney tissues and TGF-β1-treated proximal tubular cells. The overexpression of circInpp5b inhibited RIF in UUO mice and prevented extracellular matrix (ECM) deposition in TGF-β1-treated proximal tubular cells. Furthermore, overexpression of circInpp5b down-regulated the protein level of DDX1. Mechanistically, circInpp5b bound to the DDX1 protein and promoted its lysosomal degradation. Collectively, the findings of our study demonstrate that circInpp5b ameliorates RIF by binding to the DDX1 protein and promoting its lysosomal degradation.
Topics: DEAD-box RNA Helicases; Animals; Mice; Lysosomes; Fibrosis; RNA, Circular; Proteolysis; Male; Mice, Inbred C57BL; Humans; Kidney; Kidney Diseases
PubMed: 38927017
DOI: 10.3390/biom14060613 -
Journal of Functional Biomaterials Jun 2024Extracellular vesicles (EVs) can be isolated from biological fluids and cell culture medium. Their nanometric dimension, relative stability, and biocompatibility have...
Extracellular vesicles (EVs) can be isolated from biological fluids and cell culture medium. Their nanometric dimension, relative stability, and biocompatibility have raised considerable interest for their therapeutic use as delivery vehicles of macromolecules, namely nucleic acids and proteins. Deficiency in lysosomal enzymes and associated proteins is at the basis of a group of genetic diseases known as lysosomal storage disorders (LSDs), characterized by the accumulation of undigested substrates into lysosomes. Among them, GM2 gangliosidoses are due to a deficiency in the activity of lysosomal enzyme β-hexosaminidase, leading to the accumulation of the GM2 ganglioside and severe neurological symptoms. Current therapeutic approaches, including enzyme replacement therapy (ERT), have proven unable to significantly treat these conditions. Here, we provide evidence that the lysosomal β-hexosaminidase enzyme is associated with EVs released by HEK cells and that the EV-associated activity can be increased by overexpressing the α-subunit of β-hexosaminidase. The delivery of EVs to β-hexosaminidase-deficient fibroblasts results in a partial cross-correction of the enzymatic defect. Overall findings indicate that EVs could be a source of β-hexosaminidase that is potentially exploitable for developing therapeutic approaches for currently untreatable LSDs.
PubMed: 38921527
DOI: 10.3390/jfb15060153 -
Cells Jun 2024Venetoclax and obinutuzumab are becoming frontline therapies for chronic lymphocytic leukemia (CLL) patients. Unfortunately, drug resistance still occurs, and the...
Lysosome-Disrupting Agents in Combination with Venetoclax Increase Apoptotic Response in Primary Chronic Lymphocytic Leukemia (CLL) Cells Mediated by Lysosomal Cathepsin D Release and Inhibition of Autophagy.
Venetoclax and obinutuzumab are becoming frontline therapies for chronic lymphocytic leukemia (CLL) patients. Unfortunately, drug resistance still occurs, and the combination could be immunosuppressive. Lysosomes have previously been identified as a target for obinutuzumab cytotoxicity in CLL cells, but the mechanism remains unclear. In addition, studies have shown that lysosomotropic agents can cause synergistic cell death in vitro when combined with the BTK inhibitor, ibrutinib, in primary CLL cells. This indicates that targeting lysosomes could be a treatment strategy for CLL. In this study, we have shown that obinutuzumab induces lysosome membrane permeabilization (LMP) and cathepsin D release in CLL cells. Inhibition of cathepsins reduced obinutuzumab-induced cell death in CLL cells. We further determined that the lysosomotropic agent siramesine in combination with venetoclax increased cell death in primary CLL cells through an increase in reactive oxygen species (ROS) and cathepsin release. Siramesine treatment also induced synergistic cytotoxicity when combined with venetoclax. Microenvironmental factors IL4 and CD40L or incubation with HS-5 stromal cells failed to significantly protect CLL cells from siramesine- and venetoclax-induced apoptosis. We also found that siramesine treatment inhibited autophagy through reduced autolysosomes. Finally, the autophagy inhibitor chloroquine failed to further increase siramesine-induced cell death. Taken together, lysosome-targeting drugs could be an effective strategy in combination with venetoclax to overcome drug resistance in CLL.
Topics: Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Bridged Bicyclo Compounds, Heterocyclic; Sulfonamides; Lysosomes; Apoptosis; Autophagy; Cathepsin D; Reactive Oxygen Species; Drug Synergism; Cell Line, Tumor
PubMed: 38920669
DOI: 10.3390/cells13121041 -
Nature Communications Jun 2024Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the...
Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts.
Topics: Mucopolysaccharidosis III; Humans; Lysosomes; Acetyltransferases; Cryoelectron Microscopy; Catalytic Domain; Mutation; Heparitin Sulfate; Acetyl Coenzyme A; Models, Molecular; Glucosamine; Acetylation; Intracellular Membranes
PubMed: 38918376
DOI: 10.1038/s41467-024-49614-1 -
BioRxiv : the Preprint Server For... Jun 2024Injury can cause differentiated cells to undergo massive reprogramming to become proliferative to repair tissue via a cellular program called paligenosis. Gastric...
Injury can cause differentiated cells to undergo massive reprogramming to become proliferative to repair tissue via a cellular program called paligenosis. Gastric digestive-enzyme-secreting chief cells use paligenosis to reprogram into progenitor-like Spasmolytic-Polypeptide Expressing Metaplasia (SPEM) cells. Stage 1 of paligenosis is to downscale mature cell architecture via a process involving lysosomes. Here, we noticed that sulfated glycoproteins (which are metaplasia and cancer markers in mice and humans) were not digested during paligenosis but excreted into the gland lumen. Various genetic and pharmacological approaches showed that endoplasmic reticulum membranes and secretory granule cargo were also excreted and that the process proceeded in parallel with, but was independent lysosomal activity. 3-dimensional light and electron-microscopy demonstrated that excretion occurred via unique, complex, multi-chambered invaginations of the apical plasma membrane. As this lysosome-independent cell cleansing process does not seem to have been priorly described, we termed it "cathartocytosis". Cathartocytosis allows a cell to rapidly eject excess material (likely in times of extreme stress such as are induced by paligenosis) without waiting for autophagic and lysosomal digestion. We speculate the ejection of sulfated glycoproteins (likely mucins) would aid in downscaling and might also help bind and flush pathogens (like which causes SPEM) away from tissue.
PubMed: 38915707
DOI: 10.1101/2024.06.11.598489 -
BioRxiv : the Preprint Server For... Jun 2024Underlying drivers of late-onset Alzheimer's disease (LOAD) pathology remain unknown. However, multiple biologically diverse risk factors share a common pathological...
UNLABELLED
Underlying drivers of late-onset Alzheimer's disease (LOAD) pathology remain unknown. However, multiple biologically diverse risk factors share a common pathological progression. To identify convergent molecular abnormalities that drive LOAD pathogenesis we compared two common midlife risk factors for LOAD, heavy alcohol use and obesity. This revealed that disrupted lipophagy is an underlying cause of LOAD pathogenesis. Both exposures reduced lysosomal flux, with a loss of neuronal lysosomal acid lipase (LAL). This resulted in neuronal lysosomal lipid (NLL) accumulation, which opposed Aβ localization to lysosomes. Neuronal LAL loss both preceded (with aging) and promoted (targeted knockdown) Aβ pathology and cognitive deficits in AD mice. The addition of recombinant LAL and neuronal LAL overexpression prevented amyloid increases and improved cognition. In WT mice, neuronal LAL declined with aging and correlated negatively with entorhinal Aβ. In healthy human brain, LAL also declined with age, suggesting this contributes to the age-related vulnerability for AD. In human LOAD LAL was further reduced, correlated negatively with Aβ , and occurred with polymerase pausing at the LAL gene. Together, this finds that the loss of neuronal LAL promotes NLL accumulation to impede degradation of Aβ in neuronal lysosomes to drive AD amyloid pathology.
SUMMARY
Cellular and molecular drivers of late-onset Alzheimer's disease (LOAD) are unknown, though several risk factors account for the majority of disease incidence . Though diverse in their biological natures, each of these risk exposures converge on a shared pathological progression with the accumulation of amyloid early in the disease. Human genetic and transcriptomic studies suggest a role for altered lipid metabolism , though the mechanism has been unknown. Here, using two common midlife risk exposures for LOAD, we found that dysfunctional lipophagy caused by the loss of lysosomal acid lipase (LAL) promotes early LOAD pathogenesis. Both midlife obesity and heavy alcohol reduced neuronal LAL, causing an increase in neuronal lysosomal lipid, and a subsequent accumulation of Aβ in the extra-lysosomal cytosol. This loss of LAL preceded and promoted Aβ pathology and cognitive deficits in AD mice. The addition of recombinant LAL and neuronal LAL overexpression prevented increases in amyloid and improved cognition. In human brain, LAL declined with age in healthy subjects, similar to rodents, showing robust losses in LOAD subjects with polymerase pausing. Together, this implicates neuronal LAL loss in LOAD pathogenesis and presents LAL as a promising diagnostic, preventative, and/or therapeutic target for AD.
PubMed: 38915509
DOI: 10.1101/2024.06.09.596693 -
Frontiers in Aging Neuroscience 2024Parkinson's disease (PD), the second most prevalent neurodegenerative condition, has a multifaceted etiology. Cathepsin-cysteine proteases situated within lysosomes...
BACKGROUND
Parkinson's disease (PD), the second most prevalent neurodegenerative condition, has a multifaceted etiology. Cathepsin-cysteine proteases situated within lysosomes participate in a range of physiological and pathological processes, including the degradation of harmful proteins. Prior research has pointed towards a potential link between cathepsins and PD; however, the precise causal relationship between the cathepsin family and PD remains unclear.
METHODS
This study employed univariate and multivariate Mendelian randomization (MR) analyses to explore the causal relationship between the nine cathepsins and Parkinson's disease (PD) risk. For the primary analysis, genome-wide association study (GWAS) summary statistics for the plasma levels of the nine cathepsins and PD was obtained from the INTERVAL study and the International Parkinson's Disease Genomics Consortium. GWAS for PD replication analysis were obtained from the FinnGen consortium, and a meta-analysis was performed for the primary and replication analyses to evaluate the association between genetically predicted cathepsin plasma levels and PD risk. After identifying significant MR estimates, genetic co-localization analyses were conducted to determine whether shared or distinct causal variants influenced both cathepsins and PD.
RESULTS
Elevated cathepsin B levels were associated with a decreased risk of PD in univariate MR analysis (odds ratio [OR] = 0.890, 95% confidence interval [CI]: 0.831-0.954, pFDR = 0.009). However, there was no indication that PD affected cathepsin B levels (OR = 0.965, 95% CI: 0.858-1.087, = 0.852). In addition, after adjusting for the remaining cathepsins, cathepsin B levels independently and significantly contributed to the reduced risk of PD in multivariate MR analysis (OR = 0.887, 95% CI: 0.823-0.957, = 0.002). The results of the replication MR analysis with the FinnGen GWAS for PD (OR = 0.921, 95% CI: 0.860-0.987, = 0.020) and meta-analysis (OR = 0.905, 95% CI: 0.862-0.951, < 0.001) were consistent with those of the primary analysis. Colocalization analysis did not provide any evidence of a shared causal variant between cathepsins and PD (PP.H4.abf = 0.005).
CONCLUSION
This genetic investigation supports the hypothesis that cathepsin B exerts a protective effect against PD. The quantification of cathepsin B levels could potentially serve as a predictive biomarker for susceptibility to PD, providing new insights into the pathomechanisms of the disease and possible interventions.
PubMed: 38903897
DOI: 10.3389/fnagi.2024.1380483 -
BioRxiv : the Preprint Server For... Apr 2024Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, ) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to...
Dynein cytoplasmic 1 light intermediate chain 1 (LIC1, ) is a core subunit of the dynein motor complex. The LIC1 subunit also interacts with various cargo adaptors to regulate Rab-mediated endosomal recycling and lysosomal degradation. Defects in this gene are predicted to alter dynein motor function, Rab binding capabilities, and cytoplasmic cargo trafficking. Here, we have identified a zebrafish mutant, harboring a premature stop codon at the exon 12/13 splice acceptor site, that displays increased angiogenesis. , LIC1-deficient human endothelial cells display increases in cell surface levels of the pro-angiogenic receptor VEGFR2, SRC phosphorylation, and Rab11-mediated endosomal recycling. , endothelial-specific expression of constitutively active leads to excessive angiogenesis, similar to the mutants. Increased angiogenesis is also evident in zebrafish harboring mutations in , the adaptor proteins that promote Rab docking to Lic1 to mediate lysosomal targeting. These findings suggest that LIC1 and the Rab-adaptor proteins RILPL1 and 2 restrict angiogenesis by promoting degradation of VEGFR2-containing recycling endosomes. Disruption of LIC1- and RILPL1/2-mediated lysosomal targeting increases Rab11-mediated recycling endosome activity, promoting excessive SRC signaling and angiogenesis.
PubMed: 38903077
DOI: 10.1101/2024.04.01.587559