Expression Of Membrane Proteins Research Articles

Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway

Objective: To investigate the mechanism of proanthocyanidin (PA) in regulating the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs), and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB (TFEB) and on the autophagy-lysosome pathway. Methods: PDLSCs were divided into control group and PA group, which were subjected to RNA sequencing analysis (RNA Seq) to detect differentially expressed genes. The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR (RT-qPCR) analysis, alkaline phosphatase (ALP) staining and transmission electron microscope (TEM), respectively. Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs. Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes. The total protein expression of transcription factor EB (TFEB) as well as that in cytoplasm and nucleus were detected by Western blotting. Confocal laser scanning microscope (CLSM) was used to observe the nuclear translocation of TFEB. The PDLSCs were treated with small interfering RNA (siRNA) technology to knock down the expression levels of TFEB gene with or without PA treatment. Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3 (LC3B), as well as osteogenic-related proteins runt-related transcription factor 2 (RUNX2), ALP, and osteocalcin in PDLSCs. Results: Compared with the control group, the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment (P<0.05). The mRNA expression levels of osteogenic-related genes RUNX2 (2.32±0.15) and collagen type Ⅰ alpha 1 (COL1α1) (1.80±0.18), as well as the autophagy related genes LC3B (1.87±0.08) and Beclin1 (1.63±0.08) were significantly increased in the PA group, compared with the control group (1.01±0.16, 1.00±0.10, 1.00±0.07, 1.00±0.06, respectively, all P<0.01). Compared with the control group, the PA group had higher ALP activity, and more autophagosomes and autophagolysosomes observed by TEM. PA promoted the migration of PDLSCs (P<0.05) and the increased number of lysosomes and the expression of lysosomal associated membrane protein 1 (LAMP1). In the PA group, the relative expression level of total TFEB protein (1.49±0.07) and the nuclear/cytoplasmic expression of TFEB protein (1.52±0.12) were significantly higher than the control group (1.00±0.11, 1.00±0.13, respectively) (t=6.43, P<0.01; t=5.07, P<0.01). The relative nuclear/cytoplasmic fluorescence intensity of TFEB in the PA group (0.79±0.09) was increased compared with the control group (0.11±0.08) (t=8.32, P<0.01). Knocking down TFEB significantly reduced the expression of TFEB (1.00±0.15 vs 0.64±0.04), LAMP1 (1.00±0.10 vs 0.69±0.09), Beclin1 (1.00±0.05 vs 0.60±0.05), and LC3B Ⅱ/Ⅰ (1.00±0.06 vs 0.73±0.07) in PDLSCs (P<0.05, P<0.05, P<0.01, P<0.01). When TFEB gene was knocked down, the expression levels of Beclin1 (1.05±0.11), LC3B Ⅱ/Ⅰ (1.02±0.09), RUNX2 (1.04±0.10), ALP (1.04±0.16), and osteocalcin (1.03±0.15) proteins were significantly decreased in the PA group compared with the pre-knockdown period (1.28±0.03, 1.44±0.11, 1.38±0.11, 1.62±0.11, 1.65±0.17, respectively) (P<0.05, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). Conclusions: PA promotes the osteogenic differentiation of PDLSCs through inducing the expression and nuclear translocation of TFEB and activating the autophagy-lysosome pathway.

REMEMProt: a resource of membrane-enriched proteome profiles, their disease associations, and biomarker status.

The differential expression of plasma membrane proteins is integrally analyzed for their diagnosis, prognosis, and therapeutic applications in diverse clinical manifestations. Necessarily, distinct membrane protein enrichment methods and mass spectrometry platforms are employed for their global and relative quantitation. First of its kind to explore, we compiled membrane-associated proteomes in human and mouse systems into a database named, Resource of Experimental Membrane-Enriched Mass spectrometry-derived Proteome (REMEMProt). It currently hosts 14,626 proteins (9,507 proteins in Homo sapiens; 5,119 proteins in Mus musculus) with information on their membrane-protein enrichment methods, experimental/physiological context of detection in cells or tissues, transmembrane domain analysis, and their current attribution as biomarkers. Based on these annotations and the transmembrane domain analysis in proteins or their binary/complex protein-protein interactors, REMEMProt facilitates the assessment of the plasma membrane localization potential of proteins through batch query. A cross-study enrichment analysis platform is enabled in REMEMProt for comparative analysis of proteomes using novel/modified membrane enrichment methods and evaluation of methods for targeted enrichment of membrane proteins. REMEMProt data are made freely accessible to explore and download at https://rememprot.ciods.in/.

Porin Deficiency or Plasmid Copy Number Increase Mediated Carbapenem-Resistant Escherichia coli Resistance Evolution

Aims: This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Methods: Ancestral strain ECNX52 was constructed by introducing the bla NDM-5-carrying IncX3 plasmid into E. coli C600, and was subjected to laboratory evolution under different concentrations of meropenem pressure. Minimal inhibitory concentrations and conjugation frequency were determined. Fitness of these strains was assessed. Whole genome sequencing and transcriptional changes were performed. Ancestral host or plasmids were recombined with evolved hosts or plasmids to verify plasmid or host factors in resistance evolution. Role of the repA mutation on plasmid copy number was determined. Results: Two out of the four clones (EM2N1 and EM2N3) exhibited four-fold increase in MIC when exposed to a continuous pressure of 2 μg/mL MEM (1/32 MIC), by down regulating expression of outer membrane protein ompF. Besides, all four clones displayed four-fold increase in MIC and higher conjugation frequency when subjected to a continuous pressure of 4 μg/mL MEM (1/16 MIC), attributing to increasing plasmid copy number generated by repA D140Y (GAT→TAT) mutation. Conclusion: Bacterial hosts and conjugative plasmids can undergo resistance evolution under certain concentrations of antimicrobial pressure by reducing the expression of outer membrane proteins or increasing plasmid copy numbers.

NECTIN4 Amplification Is Frequent in Solid Tumors and Predicts Enfortumab Vedotin Response in Metastatic Urothelial Cancer.

The anti-NECTIN4 antibody-drug conjugate enfortumab vedotin (EV) is approved for patients with metastatic urothelial cancer (mUC). However, durable benefit is only achieved in a small, yet uncharacterized patient subset. NECTIN4 is located on chromosome 1q23.3, and 1q23.3 gains represent frequent copy number variations (CNVs) in urothelial cancer. Here, we aimed to evaluate NECTIN4 amplifications as a genomic biomarker to predict EV response in patients with mUC. We established a NECTIN4-specific fluorescence in situ hybridization (FISH) assay to assess the predictive value of NECTIN4 CNVs in a multicenter EV-treated mUC patient cohort (mUC-EV, n = 108). CNVs were correlated with membranous NECTIN4 protein expression, EV treatment responses, and outcomes. We also assessed the prognostic value of NECTIN4 CNVs measured in metastatic biopsies of non-EV-treated mUC (mUC-non-EV, n = 103). Furthermore, we queried The Cancer Genome Atlas (TCGA) data sets (10,712 patients across 32 cancer types) for NECTIN4 CNVs. NECTIN4 amplifications are frequent genomic events in muscle-invasive bladder cancer (TCGA bladder cancer data set: approximately 17%) and mUC (approximately 26% in our mUC cohorts). In mUC-EV, NECTIN4 amplification represents a stable genomic alteration during metastatic progression and associates with enhanced membranous NECTIN4 protein expression. Ninety-six percent (27 of 28) of patients with NECTIN4 amplifications demonstrated objective responses to EV compared with 32% (24 of 74) in the nonamplified subgroup (P < .001). In multivariable Cox analysis adjusted for age, sex, and Bellmunt risk factors, NECTIN4 amplifications led to a 92% risk reduction for death (hazard ratio, 0.08 [95% CI, 0.02 to 0.34]; P < .001). In the mUC-non-EV, NECTIN4 amplifications were not associated with outcomes. TCGA Pan-Cancer analysis demonstrated that NECTIN4 amplifications occur frequently in other cancers, for example, in 5%-10% of breast and lung cancers. NECTIN4 amplifications are genomic predictors of EV responses and long-term survival in patients with mUC.

Exposure to imipenem at sub-minimum inhibitory concentration leads to altered expression of major outer membrane proteins in Acinetobacter baumannii.

Acinetobacter baumannii is a nosocomial pathogen known to be multidrug-resistant (MDR), especially to drugs of the carbapenem class. Several factors contribute to resistance, including efflux pumps, β-lactamases, alteration of target sites, and permeability defects. In addition, outer membrane proteins (OMPs), like porins are involved in the passage of antibiotics, and their alteration could lead to resistance development. This study aimed to explore the possible involvement of porins and OMPs in developing carbapenem resistance due to differential expression. The antibiotic-susceptible and MDR isolates of A. baumannii were first studied for differences in their transcriptional levels of OMP expression and OMP profiles. The antibiotic-susceptible isolates were further treated with imipenem, and it was found that the omp genes were differentially expressed. Six of the nine genes studied were upregulated at 1h of exposure to imipenem. Their expression gradually decreased with time, further confirmed by their OMP profile and two-dimensional gel electrophoresis. This study could identify OMPs that were differentially expressed on exposure to imipenem. Hence, this study provides insights into the role of specific OMPs in antibiotic resistance in A. baumannii.

Rhodopsin mislocalization drives ciliary dysregulation in a novel autosomal dominant retinitis pigmentosa knock-in mouse model.

Rhodopsin mislocalization encompasses various blind conditions. Rhodopsin mislocalization is the primary factor leading to rod photoreceptor dysfunction and degeneration in autosomal dominant retinitis pigmentosa (adRP) caused by class I mutations. In this study, we report a new knock-in mouse model that harbors a class I Q344X mutation in the endogenous rhodopsin gene, which causes rod photoreceptor degeneration in an autosomal dominant pattern. In RhoQ344X/+ mice, mRNA transcripts from the wild-type (Rho) and RhoQ344X mutant rhodopsin alleles are expressed at equal levels. However, the amount of RHOQ344X mutant protein is 2.7 times lower than that of wild-type rhodopsin, a finding consistent with the rapid degradation of the mutant protein. Immunofluorescence microscopy indicates that RHOQ344X is mislocalized to the inner segment and outer nuclear layers of rod photoreceptors in both RhoQ344X/+ and RhoQ344X/Q344X mice, confirming the essential role of the C-terminal VxPx motif in promoting OS delivery of rhodopsin. The mislocalization of RHOQ344X is associated with the concurrent mislocalization of wild-type rhodopsin in RhoQ344X/+ mice. To understand the global changes in proteostasis, we conducted quantitative proteomics analysis and found attenuated expression of rod-specific OS membrane proteins accompanying reduced expression of ciliopathy causative gene products, including constituents of BBSome and axonemal dynein subunit. Those studies unveil a novel negative feedback regulation involving ciliopathy-associated proteins. In this process, a defect in the trafficking signal leads to a reduced quantity of the trafficking apparatus, culminating in a widespread reduction in the transport of ciliary proteins.

Engineering water exchange is a safe and effective method for magnetic resonance imaging in diverse cell types

Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

EMP2 Serves as a Functional Biomarker for Chemotherapy-Resistant Triple-Negative Breast Cancer.

Breast cancer (BC) remains among the most commonly diagnosed cancers in women worldwide. Triple-negative BC (TNBC) is a subset of BC characterized by aggressive behavior, a high risk of distant recurrence, and poor overall survival rates. Chemotherapy is the backbone for treatment in patients with TNBC, but outcomes remain poor compared to other BC subtypes, in part due to the lack of recognized functional targets. In this study, the expression of the tetraspan protein epithelial membrane protein 2 (EMP2) was explored as a predictor of TNBC response to standard chemotherapy. We demonstrate that EMP2 functions as a prognostic biomarker for patients treated with taxane-based chemotherapy, with high expression at both transcriptomic and protein levels following treatment correlating with poor overall survival. Moreover, we show that targeting EMP2 in combination with docetaxel reduces tumor load in syngeneic and xenograft models of TNBC. These results provide support for the prognostic and therapeutic potential of this tetraspan protein, suggesting that anti-EMP2 therapy may be beneficial for the treatment of select chemotherapy-resistant TNBC tumors.

Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles.

Abstract 4682: Membrane protein expression and activity following inhibition of B-cell receptor-initiated signaling by WBMP-4, a therapeutic antibody for leukemia/lymphoma

Abstract The B-cell Receptor (BCR), comprising membrane Ig (mIg) and CD79a/b, controls normal signaling in B-cells, but dysregulation of this signaling is the dominant driver of leukemia/lymphoma. The class-specific mIg subunit of the BCR is a rational drug target for B-cell malignancies. It plays a critical role in the functioning of these cells, and allows for restricted targeting of only B-cells expressing an Ig class, offering a more tumor-specific approach than current pan-B-cell treatments. We developed a first-in-class antibody, WBMP-4, to the mIgM-BCR, which inhibits cell growth and induces apoptosis via widespread down-regulation of tyrosine kinase activity. Given the potent inhibition of intracellular signaling induced upon WBMP-4 binding to the mIgM-BCR, we expect that translation of membrane proteins will be impacted. Here we investigate the expression and phosphorylation profile of cell surface proteins following treatment of malignant B-cells with WBMP-4. Using western blot and flow cytometry, we measure levels of CD79a/b, CD19, CD20, CD21, CD22, CD32a, CD81, and CXCR4 at 6, 24, and 48 hours after low (5ug/mL) and high (20ug/mL) dose WBMP-4 treatment, as compared to control, untreated Burkitt lymphoma (CA46) cells. We analyze phosphorylation levels of those surface proteins that serve as kinases in this system (CD79a/b, CD19, CXCR4, etc.). Using these same methods, we also examine the expression and phosphorylation of key B-cell transcription factors (e.g. NF-kB, c-MYC, STAT5, PAX5, EBF1, FOXO, and BCL-2). Preliminary analyses show a complete absence of CD79a protein (western blot), and decreased phospho-kinase activity of CD79b (Pamgene phosphokinase array) following treatment of Burkitt lymphoma cells with WBMP-4, as compared to untreated cells. While the effect of an antibody binding to mIg may be most pronounced for CD79a/b relative to other proteins, as mIg and CD79a/b form a complex, we expect that the absence of CD79a/b is a result of WBMP-4-induced mIgM inhibition modulating downstream signaling and transcription factor activity. By examining cell surface markers, we may gain insight into how WBMP-4 impacts a cell’s interaction with the tumor microenvironment, and this information is valuable for diagnostic and patient monitoring purposes, and to determine drug combination strategies. This work will be considered in the context of our ongoing research on WBMP-4’s inhibition of intracellular signaling pathways, and the resulting biologic effects, and will contribute to the continued development of WBMP-4 by providing an understanding of the effects achieved (cell growth inhibition, cell differentiation, apoptosis) at different dosages and if these effects are maintained over time. As a novel, efficacious, and low toxicity treatment strategy for B-cell leukemia and lymphoma, WBMP-4 has significant clinical potential. Citation Format: Rachel S. Welt, David Kostyal, Virginia Raymond, Jose M. Lobo, Sydney Welt. Membrane protein expression and activity following inhibition of B-cell receptor-initiated signaling by WBMP-4, a therapeutic antibody for leukemia/lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4682.

Pharmacological suppression of HHLA2 glycosylation restores anti-tumor immunity in colorectal cancer

Immunotherapy aimed at inhibiting the negative co-stimulatory molecule programmed cell death-ligand 1 (PD-L1) has limited effectiveness, with clinical response rates remaining below 10%–15%. Therefore, new immune checkpoints need to be explored. Our study focused on human endogenous retrovirus H long terminal repeat–associating protein 2 (HHLA2), a highly glycosylated member of the B7 family that is widely expressed in colorectal cancer. HHLA2 expression negatively correlates with the prognosis of colorectal cancer. Glycosylation of HHLA2, which is regulated by the glycosyltransferase STT3 oligosaccharyltransferase complex catalytic subunit A (STT3A), is crucial for protein stability and expression in cell membranes. Additionally, the binding of HHLA2 to the receptors killer cell immunoglobulin-like receptor, three immunoglobulin domains and long cytoplasmic tail 3 (KIR3DL3) and transmembrane and immunoglobulin (Ig) domain containing 2 (TMIGD2) is dependent on N-glycosylation. Moreover, N-glycosylation of HHLA2 promotes immune evasion in colorectal cancer by suppressing the immune response of NK cells. Notably, the STT3A inhibitor NGI-1 enhances the anti-tumor immune response of NK cells. Our findings provide new insights and a molecular basis for targeting HHLA2 in immunotherapy for colorectal cancer.

Development of a novel prostate Cancer-Stroma Sphere (CSS) model for In Vitro tumor microenvironment studies

Tumor employs non-cancerous cells to gain beneficial features that promote growth and survival of cancer cells. Despite intensive research in the area of tumor microenvironment, there is still a lack of reliable and reproducible in vitro model for tumor and tumor-microenvironment cell interaction studies. Herein we report the successful development of a heterogeneous cancer-stroma sphere (CSS) model composed of prostate adenocarcinoma PC3 cells and immortalized mesenchymal stem cells (MSC). The CSS model demonstrated a structured spatial layout of the cells, with stromal cells concentrated at the center of the spheres and tumor cells located on the periphery. Significant increase in the levels of VEGFA, IL-10, and IL1a has been detected in the conditioned media of CSS as compared to PC3 spheres. Single cell RNA sequencing data revealed that VEGFA was secreted by MSC cells within heterogeneous spheroids. Enhanced expression of extracellular membrane (ECM) proteins was also shown for CSS-derived MSCs. Furthermore, we demonstrated that the multicellular architecture altered cancer cell response to chemotherapeutic agents: the inhibition of sphere formation by topotecan was 74.92 ± 4.56 % for PC3 spheres and 45.95 ± 7.84 % for CSS spheres (p < 0.01), docetaxel showed 37,51± 20,88 % and 15,67± 14,08 % inhibition, respectively (p < 0.05). Thus, CSS present an effective in vitro model for examining the extracellular matrix composition and cell-to-cell interactions within the tumor, as well as for evaluating the antitumor activity of drugs.

HPV16 E7 modulates the cell surface expression of MET and CD109 via the AP2 complex

Multiple cellular pathways are affected by HPV E6 and E7 oncoproteins, including endocytic and cellular trafficking. HPV-16 E7 can target the adaptor protein (AP) complex, which contains proteins important during endocytosis transport. To further investigate the role of HPV E7 during this process, we analysed the expression of cell surface proteins in NIKS cells expressing HPV-16 E7. We show that different cell surface proteins are regulated by HPV-16 E7 via interaction with AP2. We observed that the expression of MET and CD109 membrane protein seems to be upregulated in cells expressing E7. Moreover, the interaction of MET and CD109 with AP2 proteins is disrupted by HPV-16 E7. In addition, in the absence of HPV-16 E7, there is a downregulation of the cell membrane expression of MET and CD109 in HPV-positive cell lines. These results expand our knowledge of the functions of E7 and open new potential cellular pathways affected by this oncoprotein.

GLA Mutations Suppress Autophagy and Stimulate Lysosome Generation in Fabry Disease.

Fabry disease (FD) is an X-linked recessive inheritance lysosomal storage disorder caused by pathogenic mutations in the GLA gene leading to a deficiency of the enzyme alpha-galactosidase A (α-Gal A). Multiple organ systems are implicated in FD, most notably the kidney, heart, and central nervous system. In our previous study, we identified four GLA mutations from four independent Fabry disease families with kidney disease or neuropathic pain: c.119C>A (p.P40H), c.280T>C (C94R), c.680G>C (p.R227P) and c.801+1G>A (p.L268fsX3). To reveal the molecular mechanism underlying the predisposition to Fabry disease caused by GLA mutations, we analyzed the effects of these four GLA mutations on the protein structure of α-galactosidase A using bioinformatics methods. The results showed that these mutations have a significant impact on the internal dynamics and structures of GLA, and all these altered amino acids are close to the enzyme activity center and lead to significantly reduced enzyme activity. Furthermore, these mutations led to the accumulation of autophagosomes and impairment of autophagy in the cells, which may in turn negatively regulate autophagy by slightly increasing the phosphorylation of mTOR. Moreover, the overexpression of these GLA mutants promoted the expression of lysosome-associated membrane protein 2 (LAMP2), resulting in an increased number of lysosomes. Our study reveals the pathogenesis of these four GLA mutations in FD and provides a scientific foundation for accurate diagnosis and precise medical intervention for FD.

Mechanism of Induction of P-gp Activity During MET Induced by DEX in Lung Cancer Cell Line

Lung cancer metastasis often leads to a poor prognosis for patients. Mesenchymal-epithelial transition (MET) is one key process associated with metastasis. MET has also been linked to multidrug drug resistance (MDR). MDR arises from the overactivity of drug efflux transporters such as P-glycoprotein (P-gp) which operate at the cell plasma membrane, under the regulatory control of the scaffold proteins ezrin (Ezr), radixin (Rdx), and moesin (Msn), collectively known as ERM proteins. The current study was intended to clarify the functional changing of P-gp and the underlying mechanisms in the context of dexamethasone (DEX)-induced MET in lung cancer cells. We found that the mRNA and membrane protein expression of Ezr and P-gp was increased in response to DEX treatment. Moreover, the DEX-treated group exhibited an increase in Rho123 efflux, and it was reversed by treatment with the P-gp inhibitor verapamil or Ezr siRNA. The decrease in cell viability with paclitaxel (PTX) treatment was mitigated by pretreatment with DEX. The increased expression and activation of P-gp during the progression of lung cancer MET was regulated by Ezr. The regulatory mechanism of P-gp expression and activity may differ depending on the cell status.

Systemic and local immunosuppression in glioblastoma and its prognostic significance.

The effectiveness of tumor therapy, especially immunotherapy and oncolytic virotherapy, critically depends on the activity of the host immune cells. However, various local and systemic mechanisms of immunosuppression operate in cancer patients. Tumor-associated immunosuppression involves deregulation of many components of immunity, including a decrease in the number of T lymphocytes (lymphopenia), an increase in the levels or ratios of circulating and tumor-infiltrating immunosuppressive subsets [e.g., macrophages, microglia, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs)], as well as defective functions of subsets of antigen-presenting, helper and effector immune cell due to altered expression of various soluble and membrane proteins (receptors, costimulatory molecules, and cytokines). In this review, we specifically focus on data from patients with glioblastoma/glioma before standard chemoradiotherapy. We discuss glioblastoma-related immunosuppression at baseline and the prognostic significance of different subsets of circulating and tumor-infiltrating immune cells (lymphocytes, CD4+ and CD8+ T cells, Tregs, natural killer (NK) cells, neutrophils, macrophages, MDSCs, and dendritic cells), including neutrophil-to-lymphocyte ratio (NLR), focus on the immune landscape and prognostic significance of isocitrate dehydrogenase (IDH)-mutant gliomas, proneural, classical and mesenchymal molecular subtypes, and highlight the features of immune surveillance in the brain. All attempts to identify a reliable prognostic immune marker in glioblastoma tissue have led to contradictory results, which can be explained, among other things, by the unprecedented level of spatial heterogeneity of the immune infiltrate and the significant phenotypic diversity and (dys)functional states of immune subpopulations. High NLR is one of the most repeatedly confirmed independent prognostic factors for shorter overall survival in patients with glioblastoma and carcinoma, and its combination with other markers of the immune response or systemic inflammation significantly improves the accuracy of prediction; however, more prospective studies are needed to confirm the prognostic/predictive power of NLR. We call for the inclusion of dynamic assessment of NLR and other blood inflammatory markers (e.g., absolute/total lymphocyte count, platelet-to-lymphocyte ratio, lymphocyte-to-monocyte ratio, systemic immune-inflammation index, and systemic immune response index) in all neuro-oncology studies for rigorous evaluation and comparison of their individual and combinatorial prognostic/predictive significance and relative superiority.

Altered vacuole membrane protein 1 (VMP1) expression is associated with increased NLRP3 inflammasome activation and mitochondrial dysfunction.

Altered expression of vacuole membrane protein 1 (VMP1) has recently been observed in the context of multiple sclerosis and Parkinson's disease (PD). However, how changes in VMP1 expression may impact pathogenesis has not been explored. This study aimed to characterize how altered VMP1 expression affects NLRP3 inflammasome activation and mitochondrial function. VMP1 expression was depleted in a monocytic cell line using CRISPR-Cas9. The effect of VMP1 on NLRP3 inflammasome activation was examined by stimulating cells with LPS and ATP or α-synuclein fibrils. Inflammasome activation was determined by caspase-1 activation using both a FLICA assay and a biosensor as well as by the release of proinflammatory molecules measured by ELISA. RNA-sequencing was utilized to define global gene expression changes resulting from VMP1 deletion. SERCA activity and mitochondrial function were investigated using various fluorescence microscopy-based approaches including a novel method that assesses the function of individual mitochondria in a cell. Here, we report that genetic deletion of VMP1 from a monocytic cell line resulted in increased NLRP3 inflammasome activation and release of proinflammatory molecules. Examination of the VMP1-dependent changes in these cells revealed that VMP1 deficiency led to decreased SERCA activity and increased intracellular [Ca2+]. We also observed calcium overload in mitochondria in VMP1 depleted cells, which was associated with mitochondrial dysfunction and release of mitochondrial DNA into the cytoplasm and the extracellular environment. Collectively, these studies reveal VMP1 as a negative regulator of inflammatory responses, and we postulate that decreased expression of VMP1 can aggravate the inflammatory sequelae associated with neurodegenerative diseases like PD.

Occurrence of NECTIN4 amplification in solid tumors and enfortumab vedotin response in metastatic urothelial cancer.

673 Background: The anti-NECTIN4 antibody-drug conjugate (ADC) enfortumab vedotin (EV) is approved for patients with metastatic urothelial cancer (mUC). However, durable benefit is only achieved in a small, yet uncharacterized patient subset. NECTIN4 is located on chromosome 1q23.3, and 1q23.3 gains are frequent genomic events in mUC leading to NECTIN4 amplifications. Here, we aimed to evaluate the potential of NECTIN4 amplification as a genomic biomarker to predict EV response in patients with mUC. Methods: We established a NECTIN4-specific fluorescence in-situ hybridization (FISH) assay to assess NECTIN4 copy number variations (CNVs) in a multicenter EV-treated mUC patient cohort (UC-EV, N=77), and CNV data were correlated with membranous NECTIN4 protein expression (H-score) assessed via immunohistochemistry, EV treatment responses and outcomes. Next, we conducted a pan-cancer analysis of the The Cancer Genome Atlas (TCGA) datasets, comprising 10,712 patients across 32 cancer types, to investigate the relationship between NECTIN4 CNV, mRNA expression (RNAseq) and protein levels (RPPA) across entities. Results: In TCGA cohorts, NECTIN4 amplification occurs frequently across different solid cancer types, especially in 15-20% of bladder cancers (17% in TCGA-BLCA), as well as 5-10% in breast cancer (TCGA-BRCA) and lung adenocarcinoma (TCGA-LUAD). We confirmed the amplification frequency in our UC-EV cohort (18%). NECTIN4 amplification is significantly associated with both increased NECTIN4 mRNA expression (e.g., TCGA-BLCA, BRCA, LUAD) and membranous NECTIN4 protein expression (UC-EV), and represents a stable genomic alteration during metastatic progression. In the UC-EV cohort, all patients with NECTIN4 amplification (N=14) responded to EV. In multivariable Cox adjusted for age and sex, NECTIN4 amplifications led to a 93% risk reduction for death compared to a NECTIN4non-amplified status (HR=0.07, 95%-CI 0.01–0.53; P&lt;0.001). Conclusions: Our study highlights the value of NECTIN4 amplifications to predict EV responses in patients with mUC. NECTIN4 amplifications occur frequently in different cancer types and therefore have the potential to be a novel tumor-agnostic genomic biomarker that enables tailored NECTIN4-targeted therapies in various entities.

E and M SARS-CoV-2 membrane protein expression and enrichment with plant lipid droplets.

Plants are gaining traction as a cost-effective and scalable platform for producing recombinant proteins. However, expressing integral membrane proteins in plants is challenging due to their hydrophobic nature. In our study, we used transient and stable expression systems in Nicotiana benthamiana and Camelina sativa respectively to express SARS-CoV-2 E and M integral proteins, and target them to lipid droplets (LDs). LDs offer an ideal environment for folding hydrophobic proteins and aid in their purification through flotation. We tested various protein fusions with different linkers and tags and used three dimensional structure predictions to assess their effects. E and M mostly localized in the ER in N. benthamiana leaves but E could be targeted to LDs in oil accumulating tobacco when fused with oleosin, a LD integral protein. In Camelina sativa seeds, E and M were however found associated with purified LDs. By enhancing the accumulation of E and M within LDs through oleosin, we enriched these proteins in the purified floating fraction. This strategy provides an alternative approach for efficiently producing and purifying hydrophobic pharmaceuticals and vaccines using plant systems.

High Expression of PXMP4 in Hepatocellular Carcinoma Tissues.

To analyze the high expression of peroxisome membrane protein 4 (PXMP4) in hepatocellular carcinoma (HCC) and its clinical significance. The expression of PXMP4 mRNA in HCC tissues and corresponding adjacent tissues was detected by Q-PCR, and the expression of PXMP4 protein was detected by Western blot and immunohistochemistry. The correlation of PXMP4 protein expression with clinicopathological features and prognosis of HCC was analyzed. The expression levels of PXMP4 mRNA and protein in HCC tissues were significantly higher than those in adjacent tissues (P < 0.05), and its high expression was significantly correlated with tumor differentiation, lymph node metastasis, depth of invasion and TNM stage (P < 0.05). Patients with high expression of PXMP4 had a poor prognosis (P < 0.05). The high expression of PXMP4 may promote the occurrence and development of HCC, and inhibition of PXMP4 may be one of the potential molecular targets for targeted therapy of HCC.