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Investigative Ophthalmology & Visual... Jul 2024Glucocorticoid-induced glaucoma (GIG) is a prevalent complication associated with glucocorticoids (GCs), resulting in irreversible blindness. GIG is characterized by the...
PURPOSE
Glucocorticoid-induced glaucoma (GIG) is a prevalent complication associated with glucocorticoids (GCs), resulting in irreversible blindness. GIG is characterized by the abnormal deposition of extracellular matrix (ECM) in the trabecular meshwork (TM), elevation of intraocular pressure (IOP), and loss of retinal ganglion cells (RGCs). The objective of this study is to investigate the effects of nicotinamide riboside (NR) on TM in GIG.
METHODS
Primary human TM cells (pHTMs) and C57BL/6J mice responsive to GCs were utilized to establish in vitro and in vivo GIG models, respectively. The study assessed the expression of ECM-related proteins in TM and the functions of pHTMs to reflect the effects of NR. Mitochondrial morphology and function were also examined in the GIG cell model. GIG progression was monitored through IOP, RGCs, and mitochondrial morphology. Intracellular nicotinamide adenine dinucleotide (NAD+) levels of pHTMs were enzymatically assayed.
RESULTS
NR significantly prevented the expression of ECM-related proteins and alleviated dysfunction in pHTMs after dexamethasone treatment. Importantly, NR protected damaged ATP synthesis, preventing overexpression of mitochondrial reactive oxygen species (ROS), and also protect against decreased mitochondrial membrane potential induced by GCs in vitro. In the GIG mouse model, NR partially prevented the elevation of IOP and the loss of RGCs. Furthermore, NR effectively suppressed the excessive expression of ECM-associated proteins and mitigated mitochondrial damage in vivo.
CONCLUSIONS
Based on the results, NR effectively enhances intracellular levels of NAD+, thereby mitigating abnormal ECM deposition and TM dysfunction in GIG by attenuating mitochondrial damage induced by GCs. Thus, NR has promising potential as a therapeutic candidate for GIG treatment.
Topics: Animals; Niacinamide; Pyridinium Compounds; Glucocorticoids; Mice, Inbred C57BL; Mitochondria; Mice; Glaucoma; Extracellular Matrix; Intraocular Pressure; Humans; Disease Models, Animal; Trabecular Meshwork; Cells, Cultured; Retinal Ganglion Cells; Reactive Oxygen Species; Dexamethasone; Male
PubMed: 38949632
DOI: 10.1167/iovs.65.8.1 -
Molecular Plant-microbe Interactions :... Jul 2024Hemibiotrophic fungi in the genus employ a biotrophic phase invading host epidermal cells followed by a necrotrophic phase spreading through neighboring mesophyll and...
Hemibiotrophic fungi in the genus employ a biotrophic phase invading host epidermal cells followed by a necrotrophic phase spreading through neighboring mesophyll and epidermal cells. We used serial block face scanning electron microscopy (SBF-SEM) to compare subcellular changes that occur in (alfalfa) cotyledons during infection by (compatible on ) and (incompatible on ). Three-dimensional reconstruction of serial images revealed that alfalfa epidermal cells infected with undergo massive cytological changes during the first 60 hours following inoculation to accommodate extensive intracellular hyphal growth. Conversely, inoculation with the incompatible species resulted in no successful penetration events and frequent formation of papilla-like structures and cytoplasmic aggregates beneath attempted fungal penetration sites. Further analysis of the incompatible interaction using focused ion beam-scanning electron microcopy (FIB-SEM) revealed formation of large multivesicular body-like structures that appeared spherical and were not visible in compatible interactions. These structures often fused with the host plasma membrane, giving rise to paramural bodies that appeared to be releasing extracellular vesicles (EVs). Isolation of EVs from the apoplastic space of alfalfa leaves at 60h post inoculation showed significantly more vesicles secreted from alfalfa infected with incompatible fungus compared to compatible fungus, which in turn was more than produced by non-infected plants. Thus, the increased frequency of paramural bodies during incompatible interactions correlated with an increase in EV quantity in apoplastic wash fluids. Together, these results suggest that EVs and paramural bodies contribute to immunity during pathogen attack in alfalfa.
PubMed: 38949504
DOI: 10.1094/MPMI-04-24-0045-R -
Molecular Imaging and Radionuclide... Jun 2024We present the case of a patient with newly diagnosed high-risk prostate cancer. The patient underwent nephrectomy for renal cell carcinoma (RCC) in 2009. The...
We present the case of a patient with newly diagnosed high-risk prostate cancer. The patient underwent nephrectomy for renal cell carcinoma (RCC) in 2009. The prostate-specific membrane antigen (PSMA) scan revealed a primary tumor with seminal vessel involvement, PSMA-positive regional lymph nodes, several nodular lung lesions with mild PSMA uptake, PSMA-positive mediastinal lymph nodes, and a PSMA-positive mass in the pancreatic head. Ultrasound-guided biopsy was performed for the pancreatic lesions revealing metastasis from a RCC. Simultaneous treatment for prostate cancer and metastatic RCC was initiated. To separate metastatic sites for both primaries, we attempted to use fluorodeoxyglucose positron emission tomography/computed tomography, which was moderately positive for the pancreatic mass but not for the other locations. RCC is a Ga PSMA-positive tumor; the synchronous combination of RCC with prostate cancer can be confusing and requires more complex clinical interpretation.
PubMed: 38949477
DOI: 10.4274/mirt.galenos.2023.90582 -
Paths of least resistance: Unconventional effector secretion by fungal and oomycete plant pathogens.Molecular Plant-microbe Interactions :... Jul 2024Effector secretion by different routes mediates the molecular interplay between host plant and pathogen, but mechanistic details in eukaryotes are sparse. This may limit...
Effector secretion by different routes mediates the molecular interplay between host plant and pathogen, but mechanistic details in eukaryotes are sparse. This may limit the discovery of new effectors that could be utilized for improving host plant disease resistance. In fungi and oomycetes, apoplastic effectors are secreted via the conventional ER-Golgi pathway while cytoplasmic effectors are packaged into vesicles that bypass Golgi in an unconventional protein secretion (UPS) pathway. In , the Golgi bypass UPS pathway incorporates components of the exocyst complex and a t-SNARE, presumably to fuse Golgi bypass vesicles to the fungal plasma membrane. Upstream, cytoplasmic effector mRNA translation in requires the efficient decoding of AA-ending codons. This involves the modification of wobble uridines in the anticodon loop of cognate tRNAs and fine-tunes cytoplasmic effector translation and secretion rates to maintain biotrophic interfacial complex integrity and permit host infection. Thus, plant-fungal interface integrity is intimately tied to effector codon usage, a surprising constraint on pathogenicity. Here, we discuss these findings within the context of fungal and oomycete effector discovery, delivery, and function in host cells. We show how cracking the codon code for unconventional cytoplasmic effector secretion in has revealed AA-ending codon usage bias in cytoplasmic effector mRNAs across kingdoms, including within the RxLR-dEER motif-encoding sequence of a bona fide cytoplasmic effector, suggesting its subjection to translational speed control. By focusing on recent developments in understanding unconventional effector secretion, we draw attention to this important but understudied area of host-pathogen interactions.
PubMed: 38949402
DOI: 10.1094/MPMI-12-23-0212-CR -
The Journal of Clinical Investigation Jul 2024Ubiquitination plays an essential role in protein stability, subcellular localization, and interactions. Crosstalk between different types of ubiquitination results in...
Ubiquitination plays an essential role in protein stability, subcellular localization, and interactions. Crosstalk between different types of ubiquitination results in distinct biological outcomes for proteins. However, the role of ubiquitination-related crosstalk in lymph node (LN) metastasis and the key regulatory factors controlling this process have not been determined. Using high-throughput sequencing, we found that ubiquitin-conjugating enzyme E2 C (UBE2C) was overexpressed in bladder cancer (BCa) and was strongly associated with an unfavorable prognosis. Overexpression of UBE2C increased BCa lymphangiogenesis and promoted LN metastasis both in vitro and in vivo. Mechanistically, UBE2C mediated sodium-coupled neutral amino acid transporter 2 (SNAT2) monoubiquitination at lysine 59 to inhibit K63-linked polyubiquitination at lysine 33 of SNAT2. Crosstalk between monoubiquitination and K63-linked polyubiquitination increased SNAT2 membrane protein levels by suppressing epsin 1-mediated (EPN1-mediated) endocytosis. SNAT2 facilitated glutamine uptake and metabolism to promote VEGFC secretion, ultimately leading to lymphangiogenesis and LN metastasis in patients with BCa. Importantly, inhibition of UBE2C significantly attenuated BCa lymphangiogenesis in a patient-derived xenograft model. Our results reveal the mechanism by which UBE2C mediates crosstalk between the monoubiquitination and K63-linked polyubiquitination of SNAT2 to promote BCa metastasis and identify UBE2C as a promising target for treating LN-metastatic BCa.
Topics: Ubiquitin-Conjugating Enzymes; Humans; Ubiquitination; Urinary Bladder Neoplasms; Animals; Lymphatic Metastasis; Mice; Cell Line, Tumor; Lymphangiogenesis; Female; Male; Vascular Endothelial Growth Factor C; Neoplasm Proteins; Minor Histocompatibility Antigens; Amino Acid Transport System ASC
PubMed: 38949026
DOI: 10.1172/JCI179122 -
Oncoimmunology 2024Deregulation or loss of the human leukocyte antigen class I (HLA-I) molecules on tumor cells leading to inhibition of CD8 T cell recognition is an important tumor immune...
Deregulation or loss of the human leukocyte antigen class I (HLA-I) molecules on tumor cells leading to inhibition of CD8 T cell recognition is an important tumor immune escape strategy, which could be caused by a posttranscriptional control of molecules in the HLA-I pathway mediated by RNA-binding proteins (RBPs). So far, there exists only limited information about the interaction of RBPs with HLA-I-associated molecules, but own work demonstrated a binding of the heterogeneous ribonucleoprotein C (hnRNP C) to the 3' untranslated region (UTR) of the TAP-associated glycoprotein tapasin (tpn). In this study, analysis of pan-cancer TCGA datasets revealed that hnRNP C is higher expressed in tumor specimens compared to corresponding normal tissues, which is negatively correlated to tpn expression, T cell infiltration and the overall survival of tumor patients. Functional analysis demonstrated an upregulation of tpn expression upon siRNA-mediated downregulation of hnRNP C, which is accompanied by an increased HLA-I surface expression. Thus, hnRNP C has been identified to target tpn and its inhibition could improve the HLA-I surface expression on melanoma cells suggesting its use as a possible biomarker for T-cell-based tumor immunotherapies.
Topics: Humans; Melanoma; Heterogeneous-Nuclear Ribonucleoprotein Group C; 3' Untranslated Regions; Membrane Transport Proteins; Cell Line, Tumor; Gene Expression Regulation, Neoplastic
PubMed: 38948930
DOI: 10.1080/2162402X.2024.2370928 -
BioRxiv : the Preprint Server For... Jun 2024Neurotransmiter release is triggered in microseconds by Ca -binding to the Synaptotagmin-1 C domains and by SNARE complexes that form four-helix bundles between...
UNLABELLED
Neurotransmiter release is triggered in microseconds by Ca -binding to the Synaptotagmin-1 C domains and by SNARE complexes that form four-helix bundles between synaptic vesicles and plasma membranes, but the coupling mechanism between Ca -sensing and membrane fusion is unknown. Release requires extension of SNARE helices into juxtamembrane linkers that precede transmembrane regions (linker zippering) and binding of the Synaptotagmin-1 C B domain to SNARE complexes through a 'primary interface' comprising two regions (I and II). The Synaptotagmin-1 Ca -binding loops were believed to accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but SNARE complex binding orients the Ca -binding loops away from the fusion site, hindering these putative activities. Molecular dynamics simulations now suggest that Synaptotagmin-1 C domains near the site of fusion hinder SNARE action, providing an explanation for this paradox and arguing against previous models of Sytnaptotagmin-1 action. NMR experiments reveal that binding of C B domain arginines to SNARE acidic residues at region II remains after disruption of region I. These results and fluorescence resonance energy transfer assays, together with previous data, suggest that Ca causes reorientation of the C B domain on the membrane and dissociation from the SNAREs at region I but not region II. Based on these results and molecular modeling, we propose that Synaptotagmin-1 acts as a lever that pulls the SNARE complex when Ca causes reorientation of the C B domain, facilitating linker zippering and fast membrane fusion. This hypothesis is supported by the electrophysiological data described in the accompanying paper.
SIGNIFICANCE STATEMENT
Neurotransmiter release requires SNARE complexes that fuse synaptic vesicles with the plasma membrane and the Ca -sensor synaptotagmin-1, which was thought to facilitate membrane fusion directly through its Ca -binding loops. However, binding of Synaptotagmin-1 to SNARE complexes orients these loops away from the fusion site. Using molecular dynamics simulations, we show that placing Synaptotagmin-1 at the fusion site hinders the action of SNARE complexes. Spectroscopic studies show that Ca binding to Synaptotagmin-1 can change its interactions with SNARE complexes and, together with molecular modeling, suggest that Synaptotagmin-1 acts as a lever, pulling SNARE complexes and thus facilitating their action on the membranes to induce fusion. Functional studies described in the accompanying paper support this hypothesis.
PubMed: 38948826
DOI: 10.1101/2024.06.17.599417 -
BioRxiv : the Preprint Server For... Jun 2024Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the...
Glycosylation-deficient Chinese hamster ovary (CHO) cell lines have been instrumental in the discovery of N-glycosylation machinery. Yet, the molecular causes of the glycosylation defects in the Lec5 and Lec9 mutants have been elusive, even though for both cell lines a defect in dolichol formation from polyprenol was previously established. We recently found that dolichol synthesis from polyprenol occurs in three steps consisting of the conversion of polyprenol to polyprenal by DHRSX, the reduction of polyprenal to dolichal by SRD5A3 and the reduction of dolichal to dolichol, again by DHRSX. This led us to investigate defective dolichol synthesis in Lec5 and Lec9 cells. Both cell lines showed increased levels of polyprenol and its derivatives, concomitant with decreased levels of dolichol and derivatives, but no change in polyprenal levels, suggesting DHRSX deficiency. Accordingly, N-glycan synthesis and changes in polyisoprenoid levels were corrected by complementation with human DHRSX but not with SRD5A3. Furthermore, the typical polyprenol dehydrogenase and dolichal reductase activities of DHRSX were absent in membrane preparations derived from Lec5 and Lec9 cells, while the reduction of polyprenal to dolichal, catalyzed by SRD5A3, was unaffected. Long-read whole genome sequencing of Lec5 and Lec9 cells did not reveal mutations in the ORF of , but the genomic region containing was absent. Lastly, we established the sequence of Chinese hamster DHRSX and validated that this protein has similar kinetic properties to the human enzyme. Our work therefore identifies the basis of the dolichol synthesis defect in CHO Lec5 and Lec9 cells.
PubMed: 38948797
DOI: 10.1101/2024.06.18.599300 -
BioRxiv : the Preprint Server For... Jun 2024We take a unique approach to understanding the causes of podocyte injury in collagen IV nephropathies, a crucial step in developing targeted therapies for conditions...
RATIONALE
We take a unique approach to understanding the causes of podocyte injury in collagen IV nephropathies, a crucial step in developing targeted therapies for conditions like Alport Syndrome.
OBJECTIVES
We characterize the structural, functional, and biophysical properties of glomerular capillaries and podocytes in mice and analyze kidney cortex transcriptional profiles at various disease stages. We investigate the effects of the ER stress mitigator TUDCA on these parameters. Furthermore, we used human FSGS associated podocyte enriched genes to identify molecular pathways rescued by TUDCA thereby offering potential therapeutic targets for Alport Syndrome.
FINDINGS
We find a clear disease progression timeline in mice. Podocyte injury develops by 3 months, with glomeruli reaching maximum deformability at 4 months, associated with a 40% loss of podocytes. This is followed by progressive stiffening of glomerular capillaries, increasing proteinuria, reduced renal function, inflammatory infiltrates, and fibrosis from months 4 to 8. Bulk RNA sequencing at 2, 4, and 7 months reveals a progressive increase in expression of genes related to cytokine and chemokine signaling, matrix and cell injury, and activation of the TNF pathway, similar to observations in a NEPTUNE FSGS cohort. Podocyte-enriched genes from FSGS patients mapped to mice found that TUDCA, which mitigated glomerular and renal injury suppressed molecular pathways associated with extracellular matrix and basement membrane synthesis, podocyte stress and hypertrophy.
CONCLUSIONS
We uncover two distinct phases of nephropathy progression. The first is characterized by podocytopathy, increased glomerular capillary deformability and accelerated podocyte loss, and the second by increased capillary wall stiffening and renal inflammatory and profibrotic pathway activation. The response of podocytes to TUDCA treatment provides novel insights into downstream signaling pathways, offering potential therapeutic targets for treating Alport and related nephropathies.
PubMed: 38948788
DOI: 10.1101/2024.02.26.582201 -
BioRxiv : the Preprint Server For... Jun 2024SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death,...
SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M- K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.
PubMed: 38948778
DOI: 10.1101/2024.06.17.599107