Changes In Protein Levels Research Articles

Ubiquitin signalling in Drosophila innate immune responses.

Cells respond to invading pathogens and danger signals from the environment by adapting gene expression to meet the need for protective effector molecules. While this innate immune response is required for the cell and the organism to recover, excess immune activation may lead to loss of homeostasis, thereby promoting chronic inflammation and cancer progression. The molecular basis of innate immune defence is comprised of factors promoting survival and proliferation, such as cytokines, antimicrobial peptides and anti-apoptotic proteins. As the molecular mechanisms regulating innate immune responses are conserved through evolution, the fruit fly Drosophila melanogaster serves as a convenient, affordable and ethical model organism to enhance understanding of immune signalling. Fly immunity against bacterial infection is built up by both cellular and humoral responses, where the latter is regulated by the Imd and Toll pathways activating NF-κB transcription factors Relish, Dorsal and Dif, as well as JNK activation and JAK/STAT signalling. As in mammals, the Drosophila innate immune signalling pathways are characterised by ubiquitination of signalling molecules followed by ubiquitin receptors binding to the ubiquitin chains, as well as by rapid changes in protein levels by ubiquitin-mediated targeted proteasomal and lysosomal degradation. In this review, we summarise the molecular signalling pathways regulating immune responses to pathogen infection in Drosophila, with a focus on ubiquitin-dependent control of innate immunity and inflammatory signalling.

Altered microheterogeneity at several N-glycosylation sites in OPSCC in constant protein expression conditions.

Protein glycosylation responds sensitively to disease states. It is implicated in every hallmark of cancer and has recently started to be considered as a hallmark itself. Changes in N-glycosylation microheterogeneity are more dramatic than those of protein expression due to the non-template nature of protein glycosylation. This enables their potential use in serum-based diagnostics. Here, we perform glycopeptidomics on serum from patients with oropharyngeal squamous cell carcinoma (OPSCC), compared to controls and comparing between cancers based on etiology (human papilloma virus- positive or negative). Using MS2, we then targeted glycoforms, significantly different between the groups, to identify their glycopeptide compositions. Simultaneously we investigate the same serum proteins, comparing whether N-glycosylation changes reflect protein-level changes. Significant glycoforms were identified from proteins such as alpha-1-antitrypsin (SERPINA1), haptoglobin, and different immunoglobulins. SERPINA1 had glycovariance at 2 N-glycosylation sites, that were up to 35 times more abundant in even early-stage OPSCCs, despite minimal differences between SERPINA1 protein levels between groups. Some identified glycoforms' fold changes (FCs) were in line with serum protein level FCs, others were less abundant in early-stage cancers but with great variance in higher-stage cancers, such as on immunoglobulin heavy constant gamma 2, despite no change in protein levels. Such findings indicate that glycovariant analysis might be more beneficial than proteomic analysis, which is yet to be fruitful in the search for biomarkers. Highly sensitive glycopeptide changes could potentially be used in the future for cancer screening. Additionally, characterizing the glycopeptide changes in OPSCC is valuable in the search for potential therapeutic targets.

Cellular and molecular alterations in a human hepatocellular in vitro model of nonalcoholic fatty liver disease development and stratification

The rapidly increasing incidence of nonalcoholic fatty liver disease (NAFLD) is a growing health crisis worldwide. If not detected early, NAFLD progression can lead to irreversible pathological states, including liver fibrosis and cirrhosis. Using in vitro models to understand the molecular pathogenesis has been extremely beneficial; however, most studies have utilized only short-term exposures, highlighting a limitation in current research to model extended fat-induced liver injury. We treated Hep3B cells continuously with a low dose of oleic and palmitic free fatty acids (FFAs) for 7 or 28 days. Transcriptomic analysis identified dysregulated molecular pathways and differential expression of 984 and 917 genes after FFA treatment for 7 and 28 days respectively. DNA methylation analysis of altered DNA methylated regions (DMRs) found 7 DMRs in common. Pathway analysis of differentially expressed genes (DEGs) revealed transcriptomic changes primarily involved in lipid metabolism, small molecule biochemistry, and molecular transport. Western blot analysis revealed changes in PDK4 and CPT1A protein levels, indicative of mitochondrial stress. In line with this, there was mitochondrial morphological change demonstrating breakdown of the mitochondrial network. This in vitro model of human NAFL mimics results observed in human patients and may be used as a pre-clinical model for drug intervention.

Mir324 knockout regulates the structure of dendritic spines and impairs hippocampal long-term potentiation

MicroRNAs are an emerging class of synaptic regulators. These small noncoding RNAs post-transcriptionally regulate gene expression, thereby altering neuronal pathways and shaping cell-to-cell communication. Their ability to rapidly alter gene expression and target multiple pathways makes them interesting candidates in the study of synaptic plasticity. Here, we demonstrate that the proconvulsive microRNA miR-324-5p regulates excitatory synapse structure and function in the hippocampus of mice. Both Mir324 knockout (KO) and miR-324-5p antagomir treatment significantly reduce dendritic spine density in the hippocampal CA1 subregion, and Mir324 KO, but not miR-324-5p antagomir treatment, shift dendritic spine morphology, reducing the proportion of thin, “unstable” spines. Western blot and quantitative Real-Time PCR revealed changes in protein and mRNA levels for potassium channels, cytoskeletal components, and synaptic markers, including MAP2 and Kv4.2, which are important for long-term potentiation (LTP). In line with these findings, slice electrophysiology revealed that LTP is severely impaired in Mir324 KO mice, while neurotransmitter release probability remains unchanged. Overall, this study demonstrates that miR-324-5p regulates dendritic spine density, morphology, and plasticity in the hippocampus, potentially via multiple cytoskeletal and synaptic modulators.

RCN1 deficiency inhibits oral squamous cell carcinoma progression and THP-1 macrophage M2 polarization

Reticulocalbin 1 (RCN1), a calcium-binding protein located in the endoplasmic reticulum (ER) lumen, contains six conserved regions. Its main functions include maintaining intracellular homeostasis and regulating cell proliferation and apoptosis, and it plays an important role in the development of various tumours. However, the exact function of RCN1 in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the aim of this study was to investigate the effects of RCN1 on the biological behaviour of OSCC and the regulation of tumour-associated macrophage (TAM) polarization. The expression of RCN1 in OSCC and normal oral mucosa was evaluated through bioinformatics analysis and immunohistochemical staining. The growth, migration, and invasion of OSCC cells were observed after knockdown of RCN1 using CCK-8 and Transwell assays. Apoptosis was detected by flow cytometry. The effect of tumour cell-derived RCN1 on the polarization of THP-1 macrophages was investigated by establishing a coculture model of THP-1 macrophages and OSCC cells. Additionally, changes in the expression levels of relevant proteins were detected using Western blotting. The upregulation of RCN1 in tumour tissues compared to normal oral mucosal tissues is associated with a poor prognosis and can be utilized as a prognostic indicator for OSCC. Knockdown of RCN1 inhibited the proliferation, migration, and invasion of OSCC cells. Additionally, knockdown of RCN1 in Cal-27 and SCC-25 cells resulted in inhibition of the M2 polarization of THP-1 macrophages. RCN1 knockdown inhibits OSCC progression and M2 macrophage polarization. Targeting RCN1 may be a promising approach for OSCC treatment.

Citrullination profile analysis reveals peptidylarginine deaminase 3 as an HSV-1 target to dampen the activity of candidate antiviral restriction factors.

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that remains latent in neuronal cell bodies but reactivates throughout an individual's life, causing severe adverse reactions, such as herpes simplex encephalitis (HSE). Recently, it has also been implicated in the etiology of Alzheimer's disease (AD). The absence of an effective vaccine and the emergence of numerous drug-resistant variants have called for the development of new antiviral agents that can tackle HSV-1 infection. Host-targeting antivirals (HTAs) have recently emerged as promising antiviral compounds that act on host-cell factors essential for viral replication. Here we show that a new class of HTAs targeting peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes catalyzing protein citrullination, exhibits a marked inhibitory activity against HSV-1. Furthermore, we show that HSV-1 infection leads to enhanced protein citrullination through transcriptional activation of three PAD isoforms: PAD2, PAD3, and PAD4. Interestingly, PAD3-depletion by specific drugs or siRNAs dramatically inhibits HSV-1 replication. Finally, an analysis of the citrullinome reveals significant changes in the deimination levels of both cellular and viral proteins, with the interferon (IFN)-inducible proteins IFIT1 and IFIT2 being among the most heavily deiminated ones. As genetic depletion of IFIT1 and IFIT2 strongly enhances HSV-1 growth, we propose that viral-induced citrullination of IFIT1 and 2 is a highly efficient HSV-1 evasion mechanism from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection and demonstrate that PAD inhibitors efficiently suppress HSV-1 infection in vitro, which may provide the rationale for their repurposing as HSV-1 antiviral drugs.

LncRNA H19 Influences Cellular Activities via the miR-454-3p/BHLHE40 Axis in Anaplastic Thyroid Carcinoma.

Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy threatening patients' life quality. Our previous study has demonstrated that inhibition of long non-coding RNA H19 (lncRNA h19; H19) blocks ATC growth and metastasis. The current study aimed to further explore the potential mechanism of H19 in ATC. Expression of H19, miR-454-3p, and BHLHE40 mRNA was measured using RT-qPCR in tissue samples and cell lines. The dual-luciferase reporter assay and Pearson correlation analysis were used to explore the interaction among H19, miR-454-3p, and BHLHE40. The biological process of proliferation, migration, and invasion was determined using loss- or gain-function CCK-8 and Transwell assays. Western blot assay was used to evaluate the changes in protein levels. H19 was elevated in ATC tissues and cell lines. Based on online prediction database results, miR-454-3p might be a target of H19, and BHLHE40 might be a direct target of miR-454-3p. miR-454-3p expression was decreased in ATC and had a negative interaction with H19. BHLHE40 mRNA expression was increased and has a negative correlation with miR-454-3p and a positive correlation with H19. Downregulation of miR-454-3p and upregulation of BHLHE40 could reverse the decreased cellular activities caused by si-H19. Moreover, the silence of H19 modulates BHLHE40 to affect the PI3K/AKT protein levels and apoptotic-related protein levels. The current study provided a potential detailed mechanism of H19 in ATC, and lncRNA H19-miR-454-3p-BHLHE40 interaction may be a new experimental basis for prognosis and targeted therapy for ATC patients.

Myocardial Angiotensin-Converting Enzyme 2 Protein Expression in Ischemic Heart Failure.

The angiotensin-converting enzyme 2 (ACE2)-angiotensin-(1-7)-Mas receptor axis plays a significant role in regulating myocardial remodeling and the development of heart failure (HF), with ACE2 being the primary focus. However, contemporary understanding of the membrane-bound form of the human ACE2 protein remains insufficient. The purpose of this study was to determine the expression of ACE2 protein in different cells of the left ventricular myocardium in non-diseased hearts and at various stages of ischemic HF. A total of 103 myocardial tissue samples from the left ventricle underwent quantitative and semi-quantitative immunohistochemical analysis. Upon assessing ACE2 immunostaining in all myocardial cells through unselective digital image analysis, there was no change in the stage A HF group. Nevertheless, the expression of ACE2 membrane protein in cardiomyocytes showed a tendency to increase, while non-cardiomyocyte ACE2 expression decreased significantly (p < 0.001). In the stage B HF group, the intensity of ACE2 immunostaining continued to increase with rising cardiomyocyte ACE2 expression (p < 0.001). Non-cardiomyocyte expression, in contrast, remained similar to that observed in the stage A HF group. In the stages C/D HF group, ACE2 expression reached its highest level in cardiomyocytes (p < 0.001), while ACE2 expression in non-cardiomyocytes was the lowest (p < 0.001). These changes in ACE2 protein levels are associated with left ventricular remodeling in ischemic HF.

Cell-type proteomic and metabolomic resolution of early and late grain filling stages of wheat endosperm.

The nutritional value of wheat grains, particularly their protein and metabolite composition, is a result of the grain-filling process, especially in the endosperm. Here, we employ laser microdissection (LMD) combined with shotgun proteomics and metabolomics to generate a cell type-specific proteome and metabolome inventory of developing wheat endosperm at the early (15 DAA) and late (26 DAA) grain-filling stages. We identified 1803 proteins and 41 metabolites from four different cell types (aleurone (AL), sub-aleurone (SA), starchy endosperm (SE) and endosperm transfer cells (ETCs). Differentially expressed proteins were detected, 67 in the AL, 31 in the SA, 27 in the SE and 50 in the ETCs between these two-time points. Cell-type accumulation of specific SUT and GLUT transporters, sucrose converting and starch biosynthesis enzymes correlate well with the respective sugar metabolites, suggesting sugar upload and starch accumulation via nucellar projection and ETC at 15 DAA in contrast to the later stage at 26 DAA. Changes in various protein levels between AL, SA and ETC support this metabolic switch from 15 to 26 DAA. The distinct spatial and temporal abundances of proteins and metabolites revealed a contrasting activity of nitrogen assimilation pathways, e.g. for GOGAT, GDH and glutamic acid, in the different cell types from 15 to 26 DAA, which can be correlated with specific protein accumulation in the endosperm. The integration of cell-type specific proteome and metabolome data revealed a complex metabolic interplay of the different cell types and a functional switch during grain development and grain-filling processes.

Angiotensin‐converting enzyme‐1, ACE‐1, and ACE‐2 are dysregulated in Alzheimer’s disease and related to ACE1 genotype

AbstractBackgroundThe renin‐angiotensin system (RAS) is dysregulated in Alzheimer’s disease (AD): angiotensin‐converting enzyme (ACE‐1) activity, a rate‐limiting enzyme in the disease‐associated classical RAS (cRAS) pathway, is elevated, whereas ACE‐2 activity, a central mediator of the counter‐regulatory protective pathway (rRAS) (that generates Ang‐(1‐7) from Ang‐II) is defective. Previous meta‐analyses, more recently confirmed in GWAS studies, indicate that ACE1 variants, including an insertion (I)/deletion (D) (indel) polymorphism at intron 16, are risk variants in AD. In this study, we have determined if ACE‐1 and ACE‐2 are dysregulated in AD in relation to ACE1 variant (rs4343) (a proxy marker for the more commonly studied indel polymorphism).MethodWe studied brain tissue from the frontal and parietal cortex from 147 dementia cases (AD (n = 94) and vascular dementia n = 20 and mixed AD/VaD (n = 33)) and 104 age‐matched controls from the South West Dementia Brain Bank, University of Bristol. ACE‐1 and ACE‐2 protein levels were measured by ELISA, and enzyme activities were measured using fluorogenic peptide substrate assays. ACE1 (rs4343) polymorphism was genotyped by PCR.ResultACE‐1 protein level was increased (p&lt;0.0001) and ACE‐2 protein level reduced (p = 0.0103) in AD cases compared to controls in the parietal cortex. In contrast, no significant changes in protein level were observed in the frontal cortex. ACE‐1 enzyme activity was elevated (p&lt;0.0001) as was ACE‐2 enzyme activity in both frontal and parietal cortices (p&lt;0.0001 and p = 0.001 respectively) in AD. Individuals with I/I compared with D/D and I/D genotypes in AD had unchanged ACE‐1 level (p = 0.079), lower ACE‐1 enzyme activity (p = 0.029), higher ACE‐2 level (p = 0.011) and unaltered ACE‐2 enzyme activity (p = 0.085) in the frontal cortex.ConclusionThese data confirm our previous findings showing that RAS is dysregulated in AD. Interestingly, we report regional differences in RAS signalling in AD and a reciprocal relationship between ACE‐2 protein level (reduced) and enzyme activity (increased) in AD that warrants further investigation. RAS appears to be most dysregulated in individuals with an I/I genotype in AD.

Arctigenin Suppresses the Proliferation and Metastasis, and Induces Apoptosis and Cycle Arrest of Osteosarcoma Cells by inhibiting HMOX1 Expression.

Osteosarcoma is the most common malignant bone tumor, with highly proliferative and metastatic properties. Previous studies have reported that arctigenin (Arc), a bioactive lignin compound, showed excellent anti-tumor activities in a variety of human cancers. However, its role in osteosarcoma has not been studied. We aimed to investigate the anti-tumor effects of Arc on osteosarcoma cell proliferation, migration, invasion, apoptosis, and cell cycle. Effects of Arc on osteosarcoma cell proliferation were detected by MTT and colony formation assay. Flow cytometry analysis was performed to assess the cell apoptosis and cycle arrest. Transwell assay was used to evaluate the capability of migration and invasion. qRT-PCR and Western blot were employed to determine the changes in mRNA and protein levels. Arc could significantly suppress the proliferation, colony formation, and induce cell apoptosis and S phase cycle arrest of MG63 and U-2 OS cells in a dose-dependent manner. In addition, we also observed an inhibitory effect of Arc treatment on osteosarcoma cell invasion, migration, and epithelial-mesenchymal transition (EMT). HMOX1, encoding enzyme heme oxygenase-1, was predicted to be a candidate target of Arc using STITCH. Arc treatment significantly reduced the mRNA and protein levels of HMOX1. Furthermore, overexpression of HMOX1 could partly reverse the inhibitory effects of Arc on osteosarcoma cell malignant phenotypes. Our results suggest that Arc inhibits the proliferation, metastasis and promotes cell apoptosis and cycle arrest of osteosarcoma cells by downregulating HMOX1 expression.

Characteristics of AD‐brain tau aggregate internalisation by human astrocytes and the responses induced

AbstractBackgroundHighly phosphorylated tau aggregates are deposited in tauopathy brain as disease progresses, driven by a prion‐like spread of tau seeds. Evidence suggests that astrocytes may influence tau spread. However, the efficiency of uptake of disease‐associated tau species is not well defined, nor are the effects of tau uptake on astrocyte reactivity and function. We investigate human astrocyte ability to internalise and clear tau aggregates derived from multiple Alzheimer’s disease (AD) cases, and their impact on astrocyte function and reactivity. We further explore alterations in endogenous tau expression, APP processing and cytoskeletal proteins following tau uptake.MethodControl human iPSC‐derived astrocytes were cultured for 70 days. Aggregated (sarkosyl‐insoluble) tau was isolated from postmortem AD brain. Post‐translational modifications (PTM) of isolated AD‐brain tau aggregates were analysed by mass spectrometry. Tau (0.1 ng/µL) and extracts from control human brain were spiked into astrocyte cultures. Internalisation of aggregated tau and the localised response of S100B and GFAP were quantified by ICC. Changes to tau, APP, functional and reactivity related genes were measured by qPCR and/or proteins by SDS‐PAGE.ResultAggregated tau from AD brain is readily internalised by human astrocytes, and sequesters GFAP and S100B. Differences in the rate of uptake, clearance and seeding of tau correlates with cytoskeletal protein accumulation and specific changes in astrocyte mRNA and protein levels. Work is in progress to relate these changes to characteristics of tau aggregates.ConclusionOur data shows that the capacity of AD brain aggregates to be internalised, cleared and induce seeding of endogenous tau varies by case. Investigations are underway to pinpoint the tau characteristics that underlie these differences. We also show that seeding ability is associated with changes in cytoskeletal elements and reactivity markers at mRNA and protein level. This work agrees with growing evidence that tau variability in AD is linked with specific cell type vulnerabilities that contribute to tau spread and disease progression.

Lipid-lowering effect of drug pair Scutellariae Radix-Coptidis Rhizoma based on lipomics

This study investigated the mechanism of action of Scutellariae Radix-Coptidis Rhizoma(SR-CR) in intervening in non-alcoholic fatty liver disease(NAFLD) in rats based on lipidomics. Thirty-six SD rats were divided into a control group, a model group, SR-CR groups of different doses, and a simvastatin group, with six rats in each group. Rats in the control group were fed on a normal diet, while those in the remaining groups were fed on a high-lipid diet. After four weeks of feeding, drug treatment was carried out and rats were sacrificed after 12 weeks. Serum liver function and lipid indexes were detected using kits, and the pathomorphology of liver tissues was evaluated by hematoxylin-eosin(HE) staining and oil red O staining. Changes in lipid levels in rats were detected using the LC-MS technique. Differential lipid metabolites were screened by multivariate statistical analysis, and lipid metabolic pathways were plotted. The changes in lipid-related protein levels were further verified by Western blot. The results showed that compared with the control group, the model group showed increased levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c)(P&lt;0.01), and decreased levels of γ-glutamyl transferase(γ-GT) and high-density lipoprotein cholesterol(HDL-c)(P&lt;0.01), which were significantly recovered by the intervention of SR-CR. HE staining and oil red O staining showed that different doses of SR-CR could reverse the steatosis in the rat liver in a dose-dependent manner. After lipidomics analysis, there were significant differences in lipid metabolism between the model group and the control group, with 54 lipids significantly altered, mainly including glycerolipids, phosphatidylcholine, and sphingolipids. After administration, 44 differential lipids tended to normal levels, which indicated that SR-CR groups of different doses significantly improved the lipid metabolism level in NAFLD rats. Western blot showed that SR-CR significantly decreased TG-synthesis enzyme 1(DGAT1), recombinant lipin 1(LPIN1), fatty acid synthase(FASN), acetyl-CoA carboxylase 1(ACC1), and increased the phosphorylation level of ACC1. These changes significantly decreased the synthesis of TG and increased the rate of its decomposition, which enhanced the level of lipid metabolism in the body and finally achieved the lipid-lowering effect. SR-CR can improve NAFLD by inhibiting the synthesis of fatty acids and TG.

Analysis of temperature effects on the protein accumulation of the FT-FD module using newly generated Arabidopsis transgenic plants.

Arabidopsis flowering is dependent on interactions between a component of the florigens FLOWERING LOCUS T (FT) and the basic leucine zipper (bZIP) transcription factor FD. These proteins form a complex that activates the genes required for flowering competence and integrates environmental cues, such as photoperiod and temperature. However, it remains largely unknown how FT and FD are regulated at the protein level. To address this, we created FT transgenic plants that express the N-terminal FLAG-tagged FT fusion protein under the control of its own promoter in ft mutant backgrounds. FT transgenic plants complemented the delayed flowering of the ft mutant and exhibited similar FT expression patterns to wild-type Col-0 plants in response to changes in photoperiod and temperature. Similarly, we generated FD transgenic plants in fd mutant backgrounds that express the N-terminal MYC-tagged FD fusion protein under the FD promoter, rescuing the late flowering phenotypes in the fd mutant. Using these transgenic plants, we investigated how temperature regulates the expression of FT and FD proteins. Temperature-dependent changes in FT and FD protein levels are primarily regulated at the transcript level, but protein-level temperature effects have also been observed to some extent. In addition, our examination of the expression patterns of FT and FD in different tissues revealed that similar to the spatial expression pattern of FT, FD mRNA was expressed in both the leaf and shoot apex, but FD protein was only detected in the apex, suggesting a regulatory mechanism that restricts FD protein expression in the leaf during the vegetative growth phase. These transgenic plants provided a valuable platform for investigating the role of the FT-FD module in flowering time regulation.

The Protective Role of L-carnitine on Psychosocial Stress-induced Changes in Gene Expression and Protein Levels of Matrix Metalloproteinases, Serum Corticosterone in a Rat Model.

Psychosocial stress (STS) is a common stress in modern societies. Chronic STS is associated with the impairment of a broad range of cognitive functions in humans and with prolonged abnormalities in memory. Matrix metalloproteinases (MMPs), a protein family of zinccontaining endopeptidases, are essential in neuro-inflammation and involved in neurodegenerative diseases. L-Car possessed neuroprotective, antioxidant, and anti-inflammatory properties and was shown to modulate MMPs. The current study aimed to examine the protective effect of L-Carnitine (L-CAR) on STSinduced changes in serum corticosterone levels, MMP-2, -9, and -12 protein and mRNA expression in the hippocampus as a possible mechanism for L-CAR protective effect on STS-induced memory impairment. The chronic STS and L-CAR (300 mg/kg/day, i.p) were simultaneously administered for 6 weeks to adult male Wistar rats. Serum corticosterone and protein levels of MMP-2, -9 and -12 were evaluated using ELISA. Real-Time PCR techniques were used to determine the mRNA levels of MMP-2, -9 and -12 in the hippocampus. The findings showed that serum corticosterone levels and MMP-2 and -9 protein levels were significantly increased (p<0.05) in the STS group compared to the control. Similarly, RT-PCR findings showed that the mRNA of those proteinases significantly increased (p<0.05) following the intruder method. On the other hand, the administration of L-CAR restored the alterations in corticosterone levels and MMPs gene and protein expression induced by chronic STS. STS induced elevations in corticosterone and MMP-2 and -9 levels in the hippocampus. L-CAR, on the other hand, exhibited protective effects against the STS-induced changes in MMP-2 and -9.

Novel Pi3kδ Inhibitor Roginolisib Synergizes with the Bcl-2 Inhibitor Venetoclax in Hematological Malignancies

Roginolisib (IOA-244) is a first in class allosteric modulator and non-ATP competitive, selective PI3Kδ inhibitor currently in a Phase 1 clinical study in lymphoma and solid tumors. Initial structural and biochemical studies identified unique chemical structure, excellent selectivity, and excellent PK properties for roginolisib as compared to earlier PI3Kδ inhibitors, while the clinical trial has demonstrated a favorable safety profile. Consistent with prior PI3Kδ inhibitors, roginolisib inhibits the in vitro growth of lymphoma cells. In contrast to other PI3Kδ inhibitors, roginolisib activity is correlated with the expression levels of PIK3CD, suggesting specific and on target cancer cell-intrinsic effects (PMID: 37066023). To identify rational combination strategies, initial pharmacological screening of 474 compounds in combination with roginolisib was carried out using a MTT assay in the lymphoma cell lines HH (cutaneous T cell lymphoma, CTCL) and SP-53 (mantle cell lymphoma, MCL). Initial screening identified that roginolisib synergized with the bcl-2 inhibitor venetoclax in the HH cell line with a combination index value (CI) of 0.003 by the Chou-Talalay method. To further validate this finding, the combination of roginolisib with venetoclax or with another bcl-2 inhibitor S55746 was tested in a broad range of lymphoma cell lines including GRANTA519, JVM2, SP49 (MCL); FARAGE, TMD8 (diffuse large B cell lymphoma); MEC1 (chronic lymphocytic leukemia); MJ (cutaneous T cell lymphoma); and YT (NK lymphoma). The combination showed synergism in all the cell lines (CI 0.81 - 0.05) except SP49 (CI- 1.3). Similarly, the combination of roginolisib with S55746 showed synergism in most of the cell lines (CI- 0.46 - 0.089), additive effect in SP49 (CI- 1) and antagonistic effect in GRANTA519 (CI- 1.3) and FARAGE (CI- 2.17). To assess the tumor-intrinsic effect of roginolisib in patient derived CLL cells, PBMCs isolated from CLL patients responding to prior BTK inhibitor (i) (n=4) or progressed on BTKi (n=6) were tested to check the effect of roginolisib on BCR signalling. Cells were treated with increasing doses of roginolisib from 0.625 μM to 5 μM in the presence of anti-IgM. Other BCRi such as idelalisib, duvelisib and acalabrutinib were used at 5 μM concentrations as positive controls. Roginolisib showed a dose dependent suppression of the activity of BCR downstream kinases such as AKT and ERK. The suppression of phosphorylation of AKT and ERK were comparable with other BCRi tested. Additionally, the apoptosis analysis by annexin/7AAD showed comparable activity of roginolisib with idelalisib, duvelisib or acalabrutinib at 5 μM concentration. To investigate the possible synergy of roginolisib with venetoclax in CLL patient samples who are BTKi responders (n=6) or BTKi progressors (n=8), apoptosis analysis was carried out in patient PBMCs. Viability of CLL cells following 48h of ex vivo treatment with increasing concentrations of roginolisib (0.625 to 5 μM) and venetoclax (1 to 8 nM) alone or in combination was tested. Both the BTKi responders and progressors showed strong synergy with the combination, with slightly better cell killing activity in BTKi progressors ( p=0.007) than responders ( p=0.019). To understand the mechanism by which roginolisib increases the sensitivity to venetoclax, changes in the expression levels of various Bcl-2 family proteins that have been implicated in venetoclax resistance such as Mcl-1, Bcl-xL, Bcl-2, A1 and BIM were measured by immunoblotting. Changes in expression were evaluated in PBMCs from both the BTKi responders (n=8) and progressors (n=7) after 24h treatment with 0.625 and 5 μM concentrations of roginolisib. A significant dose dependent downregulation of Mcl-1 was observed compared to DMSO treated cells in both BTKi responders ( p=0.0033) and progressors ( p= 0.0025). In addition, a significant increase in proapoptotic BIM EL was also observed at both the 0.625 μM ( p=0.0054) and 5 μM ( p=0.0194) concentrations compared to DMSO. In summary, the novel PI3Kδ selective inhibitor roginolisib shows tumor cell killing on various hematologic malignancies including CLL cells from patients with disease progression on BTK inhibitors. In addition, roginolisib synergized with venetoclax in lymphoma cell lines and CLL patient samples. Our data support extending this combination strategy to clinical trials in hematological malignancies.

Machine learning-based classification of deubiquitinase USP26 and its cell proliferation inhibition through stabilizing KLF6 in cervical cancer

ObjectiveWe aim to accurately distinguish ubiquitin-specific proteases (USPs) from other members within the deubiquitinating enzyme families based on protein sequences. Additionally, we seek to elucidate the specific regulatory mechanisms through which USP26 modulates Krüppel-like factor 6 (KLF6) and assess the subsequent effects of this regulation on both the proliferation and migration of cervical cancer cells. MethodsAll the deubiquitinase (DUB) sequences were classified into USPs and non-USPs. Feature vectors, including 188D, n-gram, and 400D dimensions, were extracted from these sequences and subjected to binary classification via the Weka software. Next, thirty human USPs were also analyzed to identify conserved motifs and ascertained evolutionary relationships. Experimentally, more than 90 unique DUB-encoding plasmids were transfected into HeLa cell lines to assess alterations in KLF6 protein levels and to isolate a specific DUB involved in KLF6 regulation. Subsequent experiments utilized both wild-type (WT) USP26 overexpression and shRNA-mediated USP26 knockdown to examine changes in KLF6 protein levels. The half-life experiment was performed to assess the influence of USP26 on KLF6 protein stability. Immunoprecipitation was applied to confirm the USP26-KLF6 interaction, and ubiquitination assays to explore the role of USP26 in KLF6 deubiquitination. Additional cellular assays were conducted to evaluate the effects of USP26 on HeLa cell proliferation and migration. Results1. Among the extracted feature vectors of 188D, 400D, and n-gram, all 12 classifiers demonstrated excellent performance. The RandomForest classifier demonstrated superior performance in this assessment. Phylogenetic analysis of 30 human USPs revealed the presence of nine unique motifs, comprising zinc finger and ubiquitin-specific protease domains. 2. Through a systematic screening of the deubiquitinase library, USP26 was identified as the sole DUB associated with KLF6. 3. USP26 positively regulated the protein level of KLF6, as evidenced by the decrease in KLF6 protein expression upon shUSP26 knockdown in both 293T and Hela cell lines. Additionally, half-life experiments demonstrated that USP26 prolonged the stability of KLF6. 4. Immunoprecipitation experiments revealed a strong interaction between USP26 and KLF6. Notably, the functional interaction domain was mapped to amino acids 285–913 of USP26, as opposed to the 1–295 region. 5. WT USP26 was found to attenuate the ubiquitination levels of KLF6. However, the mutant USP26 abrogated its deubiquitination activity. 6. Functional biological assays demonstrated that overexpression of USP26 inhibited both proliferation and migration of HeLa cells. Conversely, knockdown of USP26 was shown to promote these oncogenic properties. Conclusions1. At the protein sequence level, members of the USP family can be effectively differentiated from non-USP proteins. Furthermore, specific functional motifs have been identified within the sequences of human USPs. 2. The deubiquitinating enzyme USP26 has been shown to target KLF6 for deubiquitination, thereby modulating its stability. Importantly, USP26 plays a pivotal role in the modulation of proliferation and migration in cervical cancer cells.

Development of a proteomic signature associated with severe disease for patients with COVID-19 using data from 5 multicenter, randomized, controlled, and prospective studies

Significant progress has been made in preventing severe COVID-19 disease through the development of vaccines. However, we still lack a validated baseline predictive biologic signature for the development of more severe disease in both outpatients and inpatients infected with SARS-CoV-2. The objective of this study was to develop and externally validate, via 5 international outpatient and inpatient trials and/or prospective cohort studies, a novel baseline proteomic signature, which predicts the development of moderate or severe (vs mild) disease in patients with COVID-19 from a proteomic analysis of 7000 + proteins. The secondary objective was exploratory, to identify (1) individual baseline protein levels and/or (2) protein level changes within the first 2 weeks of acute infection that are associated with the development of moderate/severe (vs mild) disease. For model development, samples collected from 2 randomized controlled trials were used. Plasma was isolated and the SomaLogic SomaScan platform was used to characterize protein levels for 7301 proteins of interest for all studies. We dichotomized 113 patients as having mild or moderate/severe COVID-19 disease. An elastic net approach was used to develop a predictive proteomic signature. For validation, we applied our signature to data from three independent prospective biomarker studies. We found 4110 proteins measured at baseline that significantly differed between patients with mild COVID-19 and those with moderate/severe COVID-19 after adjusting for multiple hypothesis testing. Baseline protein expression was associated with predicted disease severity with an error rate of 4.7% (AUC = 0.964). We also found that five proteins (Afamin, I-309, NKG2A, PRS57, LIPK) and patient age serve as a signature that separates patients with mild COVID-19 and patients with moderate/severe COVID-19 with an error rate of 1.77% (AUC = 0.9804). This panel was validated using data from 3 external studies with AUCs of 0.764 (Harvard University), 0.696 (University of Colorado), and 0.893 (Karolinska Institutet). In this study we developed and externally validated a baseline COVID-19 proteomic signature associated with disease severity for potential use in both outpatients and inpatients with COVID-19.

Proteomic profiling of human menisci from mild joint degeneration and end-stage osteoarthritis versus healthy controls

ObjectiveTo gain new insight into the molecular changes of the meniscus by comparing the proteome profiles of healthy controls with mild degeneration and end-stage osteoarthritis (OA). MethodWe obtained tissue plugs from lateral and medial menisci of 37 individuals (central part of the posterior horn) classified as healthy (n ​= ​12), mild signs of joint damage (n ​= ​13) and end-stage OA (n ​= ​12). The protein profile was analysed by nano-liquid chromatography-mass spectrometry using data-independent acquisition and quantified by Spectronaut. Linear-mixed effects modelling was applied to extract the between-group comparisons. ResultsA similar protein profile was observed for the mild group as compared to healthy controls while the most different group was end-stage OA mainly for the medial compartment. When a pattern of gradual change in protein levels from healthy to end-stage OA was required, a 42-proteins panel was identified, suggesting a potential role in OA development. The levels of QSOX1 were lower and G6PD higher in the mild group following the proposed protein abundance pattern. Qualitative protein changes suggest lower levels of CYTL1 as a potential biomarker of early joint degradation. ConclusionFor future targeted proteomic approaches, we propose a candidate panel of 42 proteins based on gradually altered meniscal posterior horn protein abundance patterns associated with joint degradation.

The effect of proprotein convertase subtilisin/kexin type 9 inhibition on the plasma proteome: a SPIRE sub-study

Abstract Background The plasma proteome is an intermediate phenotype for disease and is a tool to inform on pharmacological mechanisms of effect. For selective low-density lipoprotein cholesterol (LDL-C) lowering by proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition, multiple studies have demonstrated that its potent LDL-C lowering effect coincides with marked reductions in both cellular and arterial plaque inflammation, despite unaltered high-sensitivity C-reactive protein (hs-CRP) levels. Purpose In the present study, we set out to investigate potential anti-inflammatory effects of PCSK9 inhibition using plasma proteomics. Methods In 190 patients participating in the SPIRE-1/2 trials, 184 targeted proteins were measured at baseline and after 12 months of bococizumab treatment using the Cardiovascular II and III panels (Sweden). Of the included patients, 96 patients received placebo and 94 received 150mg of bococizumab, once every two weeks. Considering the development of auto-antibodies in some patients, patients with LDL-C reduction below 15% were excluded. The primary outcome was the fold change in protein levels after 12 months compared to baseline. The Benjamini-Hochberg method was used to adjust for multiple comparisons with a false discovery rate (FDR) of 5%. The findings were compared to the LoDoCo2 sub-study which used the same proteomic panels. Results In the placebo group, there was no significant change in any of the proteins. In the bococizumab group (n=77, mean age 62.2±9.7 years and 70 [91%] male), LDL-C levels were reduced from 114±41 mg/dL to 47±34 mg/dL (mean reduction 66±29 mg/dL) on top of statin therapy while there was a neutral effect on hs-CRP (median change 0.0 mg/L IQR [-0.8,1.1]). PCSK9 was the only protein with change upon treatment with bococizumab (24.4±11.0 vs. 15.0±10.7; PFDR=0.033; Figure 1A). In comparison, treatment of colchicine in the LoDoCo2 sub-study was associated with a significant change in 60 proteins (Figure 1B). Conclusion Here, we show that PCSK9 inhibition with bococizumab resulted in a highly selective reduction of PCSK9 plasma levels without robustly affecting other circulating proteins in the measured panels, implying PCSK9 inhibition lowers ASCVD risk via pure lipid lowering pathways. Our study reinforces the neutral systemic inflammatory effect of PCSK9 inhibition on top of statin therapy. The local effects on cellular and vascular inflammation by PCSK9 inhibition observed in earlier studies most likely reflect a direct consequence of a reduced cholesterol content in inflammatory cells and plaques, subsequently leading to a local anti-inflammatory effect. These findings support the combination of targeted anti-inflammatory therapies on top of potent LDL-C lowering in order to further reduce the residual inflammatory risk.