Enhanced Protein Degradation Research Articles

Targeting MuRF1 to Combat Skeletal Muscle Wasting in Cardiac Cachexia: Mechanisms and Therapeutic Prospects.

Cardiac cachexia, the terminal stage of chronic heart failure, is characterized by severe systemic metabolic imbalances and significant weight loss, primarily resulting from skeletal muscle mass depletion. Despite the detrimental consequences, there is no standardized and clinically-approved intervention currently available for cardiac cachexia. In the context of cardiac cachexia, accelerated protein turnover, that is, inhibited protein synthesis and enhanced protein degradation, plays a crucial role in skeletal muscle wasting. This process is primarily mediated by various proteins encoded by atrogenes. Among them, the atrogene Trim63 (tripartite motif family 63) and its encoded protein MuRF1 have been extensively studied. This review article aims to elucidate the pathogenic mechanisms underlying skeletal muscle wasting in cardiac cachexia, describe the biochemical characteristics of MuRF1, and provide an overview of the investigation into MuRF1-targeting inhibitors. The ultimate goal is to offer novel strategies for the clinical treatment for skeletal muscle wasting associated with cardiac cachexia.

An Essential Role for Calnexin in ER-Phagy and the Unfolded Protein Response.

ER-phagy is a specialized form of autophagy, defined by the lysosomal degradation of ER subdomains. ER-phagy has been implicated in relieving the ER from misfolded proteins during ER stress upon activation of the unfolded protein response (UPR). Here, we identified an essential role for the ER chaperone calnexin in regulating ER-phagy and the UPR in neurons. We showed that chemical induction of ER stress triggers ER-phagy in the somata and axons of primary cultured motoneurons. Under basal conditions, the depletion of calnexin leads to an enhanced ER-phagy in axons. However, upon ER stress induction, ER-phagy did not further increase in calnexin-deficient motoneurons. In addition to increased ER-phagy under basal conditions, we also detected an elevated proteasomal turnover of insoluble proteins, suggesting enhanced protein degradation by default. Surprisingly, we detected a diminished UPR in calnexin-deficient early cortical neurons under ER stress conditions. In summary, our data suggest a central role for calnexin in orchestrating both ER-phagy and the UPR to maintain protein homeostasis within the ER.

Tumour-induced alterations in single-nucleus transcriptome of atrophying muscles indicate enhanced protein degradation and reduced oxidative metabolism.

Tumour-induced skeletal muscle wasting in the context of cancer cachexia is a condition with profound implications for patient survival. The loss of muscle mass is a significant clinical obstacle and is linked to reduced tolerance to chemotherapy and increased frailty. Understanding the molecular mechanisms driving muscle atrophy is crucial for the design of new therapeutics. Lewis lung carcinoma tumours were utilized to induce cachexia and muscle atrophy in mice. Single-nucleus libraries of the tibialis anterior (TA) muscle from tumour-bearing mice and their non-tumour-bearing controls were constructed using 10X Genomics applications following the manufacturer's guidelines. RNA sequencing results were analysed with Cell Ranger software and the Seurat R package. Oxygen consumption of mitochondria isolated from TA muscle was measured using an Oroboros O2k-FluoRespirometer. Mouse primary myotubes were treated with a recombinant ectodysplasin A2 (EDA-A2) protein to activate EDA-A2 receptor (EDA2R) signalling and study changes in gene expression and oxygen consumption. Tumour-bearing mice were sacrificed while exhibiting moderate cachexia. Average TA muscle weight was reduced by 11% (P=0.0207) in these mice. A total of 12335 nuclei, comprising 6422 nuclei from the control group and 5892 nuclei from atrophying muscles, were studied. The analysis of single-nucleus transcriptomes identified distinct myonuclear gene signatures and a shift towards type IIb myonuclei. Muscle atrophy-related genes, including Atrogin1, MuRF1 and Eda2r, were upregulated in these myonuclei, emphasizing their crucial roles in muscle wasting. Gene set enrichment analysis demonstrated that EDA2R activation and tumour inoculation led to similar expression patterns in muscle cells, including the stimulation of nuclear factor-kappa B, Janus kinase-signal transducer and activator of transcription and transforming growth factor-beta pathways and the suppression of myogenesis and oxidative phosphorylation. Muscle oxidative metabolism was suppressed by both tumours and EDA2R activation. This study identified tumour-induced transcriptional changes in muscle tissue at single-nucleus resolution and highlighted the negative impact of tumours on oxidative metabolism. These findings contribute to a deeper understanding of the molecular mechanisms underlying muscle wasting.

Dietary Protease Supplementation Improved Growth Performance and Nutrients Digestion via Modulating Intestine Barrier, Immunological Response, and Microbiota Composition in Weaned Piglets.

Despite mounting evidence for dietary protease benefits, the mechanisms beyond enhanced protein degradation are poorly understood. This study aims to thoroughly investigate the impact of protease addition on the growth performance, intestinal function, and microbial composition of weaned piglets. Ninety 28-day-old weaned pigs were randomly assigned to the following three experimental diets based on their initial body weight for a 28-day experiment: (1) control (CC), a basic diet with composite enzymes without protease; (2) negative control (NC), a diet with no enzymes; and (3) dietary protease (PR), a control diet with protease. The results show that dietary proteases significantly enhanced growth performance and boosted antioxidant capacity, increasing the total antioxidant capacity (T-AOC) levels (p < 0.05) while reducing malonaldehyde levels (p < 0.05). Additionally, protease addition reduced serum levels of inflammatory markers TNF-α, IL-1β, and IL-6 (p < 0.05), suppressed mRNA expression of pro-inflammatory factors in the jejunum (p < 0.01), and inhibited MAPK and NF-κB signaling pathways. Moreover, protease-supplemented diets improved intestinal morphology and barrier integrity, including zonula occludens protein 1(ZO-1), Occludin, and Claudin-1 (p < 0.05). Microbiota compositions were also significantly altered by protease addition with increased abundance of beneficial bacteria (Lachnospiraceae_AC2044_group and Prevotellaceae_UCG-001) (p < 0.05) and reduced harmful Terrisporobacter (p < 0.05). Further correlation analysis revealed a positive link between beneficial bacteria and growth performance and a negative association with inflammatory factors and intestinal permeability. In summary, dietary protease addition enhanced growth performance in weaned piglets, beneficial effects which were associated with improved intestinal barrier integrity, immunological response, and microbiota composition.

Spray drying and ultrasonication processing of camel whey protein concentrate: Characterization and impact on bioactive properties

The production of whey protein concentrates (WPCs) from camel milk whey represents an effective approach to valorize this processing by-product. These concentrates harbor active ingredients with significant bioactive properties. Camel WPCs were spray-dried (SD) at inlet temperature of 170, 185 and 200°C, or Ultrasonicated (US) for 5, 10 and 15 min, then freeze-dried to obtain fine powder. The impact of both treatments on protein degradation was studied by sodium dodecyl sulfate-PAGE and reverse-phase ultraperformance liquid chromatography (RP-UPLC) techniques. Significantly enhanced protein degradation was observed after US treatment when compared with SD. Both SD and US treatments slightly enhanced the WPCs samples' antioxidant activities. The US exposure for 15 min exhibited highest 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity (12.12 mmol TE/g). Moreover, US treatment for 10 min exhibited the highest in vitro anti-diabetic properties (α-amylase and α-glucosidase inhibition), and dipeptidyl-peptidase-IV inhibitory activity among all samples. In addition, the ultrasonication for 10 min and SD at 170°C showed the lowest IC50 values for in vitro anti-hypercholesterolemic activities in terms of pancreatic lipase and cholesteryl esterase inhibition. Conclusively, these green techniques can be adapted in the preservation and processing of camel milk whey into active ingredients with high bioactive properties.

Structural Basis of Conformational Dynamics in the PROTAC-Induced Protein Degradation.

Pronounced conformational dynamics is unveiled upon analyzing multiple crystal structures of the same proteins recruited to the same E3 ligases by PROTACs, and yet, is largely permissive for targeted protein degradation due to the intrinsic mobility of E3 assemblies creating a large ubiquitylation zone. Mathematical modelling of ternary dynamics on ubiquitylation probability confirms the experimental finding that ternary complex rigidification need not correlate with enhanced protein degradation. Salt bridges are found to prevail in the PROTAC-induced ternary complexes, and may contribute to a positive cooperativity and prolonged half-life. The analysis highlights the importance of presenting lysines close to the active site of the E2 enzyme while constraining ternary dynamics in PROTAC design to achieve high degradation efficiency.

Effect of cadmium and lead on the morphology and protein profile of Calligonum comosum seeds

BackgroundSoils contaminated with heavy metals (HMs) pose a risk to human health via the food chain, as many edible plants absorb these metals. In turn, some of these plants could be used as phytoremediators for such soils. Calligonum comosum is an indigenous medicinal shrub that grows naturally in wide swaths of sandy soil in Saudi Arabia and has many advantages that render it a promising candidate for the treatment of HM-contaminated areas. But the impact of HM on this plant remains unknown, especially in the early stages of its development.ObjectiveThis investigation aimed to study the effects of lead (Pb) and cadmium (Cd) on the germination of C. comosum seeds under laboratory conditions, and assess the seeds’ response to these metals.MethodsThe C. comosum seed germination was monitored in Petri dishes containing Pb and Cd at increasing concentrations (25, 50, 75, and 100 µM) for up to 3 weeks. SDS-PAGE was used to examine the protein profile of germinated seeds and the western blot was used to assess the influence of HMs on the activities of the catalase enzyme and the beta subunit of ATP synthase (AtpB).ResultsThe germination rate and speed of C. comosum seeds were delayed by increasing concentrations of either Pb or Cd, but this effect was dose-dependent. SDS-PAGE analysis results revealed that exposure to both metals led to altered protein profiles as indicated by the resulting band intensities and disappearance of some proteins compared with the untreated controls. Further, the western blot analysis detected greater activity of catalase enzyme as well as AtpB in the Cd- and Pb-treated seeds.ConclusionC. comosum seeds treated with Cd or Pb enhance protein degradation and denaturation beside oxidative stress, leading to reduced seed viability. These results suggest oxidoreduction proteins and those involved in ATP synthesis are enhanced in C. comosum seeds in response to Cd and Pb stressors, which is a probable mechanism by which seeds may tolerate heavy metal stress.

Degradation and mechanism analysis of protein macromolecules by functional bacteria in tobacco leaves.

Tobacco, a crop of significant economic importance, was greatly influenced in leaf quality by protein content. However, current processing parameters fail to adequately meet the requirements for protein degradation. Microorganisms possess potential advantages for degrading proteins and enhancing the quality of tobacco leaves, and hold substantial potential in the process of curing. To effectively reduce the protein content in tobacco leaves, thereby improving the quality and safety of the tobacco leaves. In this study, tobacco leaf were used as experimental material. From these, the BSP1 strain capable of effectively degrading proteins was isolated and identified as Bacillus subtilis by 16S rDNA analysis. Furthermore, the mechanisms were analyzed by integrating microbiome, transcriptome, and metabolome. Before curing, BSP1 was applied to the surface of tobacco leaves. The results indicated that BSP1 effectively improves the activity of key enzymes and the content of related substances, thereby enhancing protein degradation. Additionally, protein degradation was achieved by regulating the diversity of the microbial community on the surface of the tobacco leaves and the ubiquitin-proteasome pathway. This study provided new strategies for extracting and utilizing functional strains from tobacco leaves, opening new avenues for enhancing the quality of tobacco leaves.

Strategies targeting endoplasmic reticulum stress to improve Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disorder with motor symptoms, which is caused by the progressive death of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Accumulating evidence shows that endoplasmic reticulum (ER) stress occurring in the SNpc DA neurons is an early event in the development of PD. ER stress triggers the activation of unfolded protein response (UPR) to reduce stress and restore ER function. However, excessive and continuous ER stress and UPR exacerbate the risk of DA neuron death through crosstalk with other PD events. Thus, ER stress is considered a promising therapeutic target for the treatment of PD. Various strategies targeting ER stress through the modulation of UPR signaling, the increase of ER's protein folding ability, and the enhancement of protein degradation are developed to alleviate neuronal death in PD models. In this review, we summarize the pathological role of ER stress in PD and update the strategies targeting ER stress to improve ER protein homeostasis and PD-related events.

DDDR-08. DISCOVERY OF ER STRESS-INDUCING COMPOUNDS FOR GBM: A PROMISING APPROACH TOWARDS THERAPEUTIC DEVELOPMENT

Abstract The unfolded protein response (UPR) is a pro-survival mechanism triggered by cellular stresses encountered by tumor cells, such as hypoxia, acidosis, reactive oxygen species and even radiation and chemotherapy. In response to increased unfolded or mis-folded proteins in the endoplasmic reticulum (ER), the UPR initiates specific cell signaling pathways aiming to alleviate ER stress and restore ER proteostasis which include increasing the expression of chaperones to aid in protein folding, enhancing protein degradation and reducing global protein synthesis. However, if homeostasis cannot be restored, the UPR switches from survival to cell death. The UPR is a relatively novel therapeutic target for glioblastoma (GBM), and specifically GBM stem-like cells (GSCs) which maintain tumor heterogeneity, drive GBM tumor growth and therapy resistance. Here we tested a series of curcumin derivatives against three GSC lines and identified a trimethoxy bis-chalcone which promotes robust GSC death with an average IC50 of just below 300 nM, a 100-fold increase in activity compared to curcumin. Furthermore, this bis-chalcone was non-toxic to normal human mesenchymal stem cells. Western blot analysis indicated that the bis-chalcone induced robust activation of UPR pro-death protein C/EBP Homologous Protein (CHOP) while simultaneously decreasing the expression of pro-survival chaperone GRP78/Bip. Lastly our bis-chalcone induced caspase 7 expression and PARP cleavage indicating apoptosis. Most ER stress inducing agents promote both the survival, upregulation of Grp78, and cell death arms of the UPR. Here we identified a compound that only promotes UPR cell death signaling and is highly toxic to radiation and temozolomide resistant GSCs while demonstrating no toxicity to non-tumor cells. This is a promising lead compound for the development of UPR targeted GSC/GBM therapeutics.

Harnessing artificial intelligence for enhanced Parkinson’s disease management: Pathways, treatment, and prospects

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the accumulation of misfolded proteins and impaired protein degradation mechanisms. Dysregulation of protein degradation processes, including autophagy and the ubiquitin-proteasome system, has been implicated in the pathogenesis of PD. Recently, artificial intelligence (AI) has emerged as a powerful tool to enhance our understanding of protein degradation in PD. This abstract provides an overview of the advancements in studying protein degradation in PD with the aid of AI. The integration of AI techniques, such as machine learning and data mining, has enabled the identification and characterization of protein degradation pathways involved in PD. By analyzing large-scale protein-protein interaction networks, AI algorithms have revealed key interactions and pathways underlying protein degradation dysfunction in PD. Furthermore, AI models can predict the efficiency of protein degradation processes and identify potential targets for enhancing protein degradation in PD, aiding in the development of novel therapeutic interventions.AI-based approaches have also been instrumental in drug discovery and target identification, as they can screen vast databases of compounds to identify potential drugs or small molecules that modulate protein degradation pathways relevant to PD. Additionally, deep learning algorithms have facilitated the analysis of protein structures, predicting protein stability and folding patterns that impact protein degradation. Moreover, AI has played a crucial role in the identification of protein biomarkers associated with protein degradation dysfunction in PD. These biomarkers can aid in early diagnosis and monitoring of the disease, enabling timely intervention and personalized treatment strategies. The advancements presented in this abstract highlight the transformative potential of AI in elucidating the intricate mechanisms of protein degradation in PD. Collaborations between AI researchers, biologists, and clinicians are essential to translate these findings into effective diagnostic tools and therapeutic interventions for PD patients.

Metabolic and microbial analyses of the surface and inner part of wet-aged and dry-aged beef.

The effects of aging and microbial growth on the metabolome of aged beef were investigated in this study. The metabolome of beef is influenced by the aging method applied. This includes the aging-related changes in metabolism and the presence of microorganisms on the beef during aging that may affect the beef and its quality. The inner part and the trimmed surface of dry-aged (the surface of dry-aged beef is also called the "crust" due to its drying during aging) and wet-aged beef were analyzed by 1 H nuclear magnetic resonance (NMR) spectroscopy over aging periods up to 28 days at intervals of 7 days, and the former also by microbiological analysis. The metabolome detected by 1 H NMR spectroscopy demonstrated changes over the aging time of beef and differed depending on the sampling location (surface or inner part of beef). The influence of the microbiota on changes in the metabolome can be negligible due to the low microbial growth on the surface of dry-aged beef (<3logCFU/g). Therefore, the aging-related metabolism postmortem of the analyzed dry-aged beef might be the main factor for metabolic changes. The significantly (p<0.05) higher amino acids and inosine concentrations and lower inosine 5'-monophosphate concentrations suggested enhanced protein degradation and energy metabolism in the wet-aged beef compared to the dry-aged beef, probably due to the combined influence of the aging and the microbiota on the wet-aged beef and, thus, its metabolic changes.

The α-arrestin SUP-13/ARRD-15 promotes isoform turnover of actin-interacting protein 1 in Caenorhabditis elegans striated muscle.

Precise arrangement of actin, myosin, and other regulatory components in a sarcomeric pattern is critical for producing contractile forces in striated muscles. Actin-interacting protein 1 (AIP1), also known as WD-repeat protein 1 (WDR1), is one of essential factors that regulate sarcomeric assembly of actin filaments. In the nematode Caenorhabditis elegans, mutation in unc-78, encoding one of the two AIP1 isoforms, causes severe disorganization of sarcomeric actin filaments and near paralysis, but mutation in sup-13 suppresses the unc-78-mutant phenotypes to restore nearly normal sarcomeric actin organization and worm motility. Here, we identified that sup-13 is a nonsense allele of arrd-15 encoding an α-arrestin. The sup-13/arrd-15 mutation suppressed the phenotypes of unc-78 null mutant but required aipl-1 that encodes a second AIP1 isoform. aipl-1 was normally expressed highly in embryos and downregulated in mature muscle. However, in the sup-13/arrd-15 mutant, the AIPL-1 protein was maintained at high levels in adult muscle to compensate for the absence of the UNC-78 protein. The sup-13/arrd-15 mutation caused accumulation of ubiquitinated AIPL-1 protein, suggesting that a normal function of sup-13/arrd-15 is to enhance degradation of ubiquitinated AIPL-1, thereby promoting transition of AIP1 isoforms from AIPL-1 to UNC-78 in developing muscle. These results suggest that α-arrestin is a novel factor to promote isoform turnover by enhancing protein degradation.

TRIM55 inhibits colorectal cancer development via enhancing protein degradation of c-Myc.

Colorectal cancer (CRC) is one of the most common and lethal malignancies which including colon and rectum cancer. Tripartite motif containing 55 (TRIM55) is an E3 ubiquitin ligase belonging to the TRIM family. Although the aberrant TRIM55 expression has been implicated in several tumors, its functional role, and molecular mechanisms in CRC remain unknown. Immunohistochemical studies, qRT-PCR, and Western blot were performed to analyze the expression of TRIM55 in CRC patients and cell lines. TRIM55 expression and its relevance to clinical traits and prognosis were further explored in the TCGA database, and in our 87 clinical samples. Subsequently, we performed a series of functional assays to explore the effect of TRIM55 on CRC progression. Finally, the molecular mechanism of TRIM55 was investigated by immunoprecipitation and ubiquitination analyses. Here, we demonstrated that TRIM55 was markedly downregulated in CRC cell lines and tumors from CRC patients. Moreover, overexpression of TRIM55 could suppress CRC cell growth in vitro and inhibit CRC xenograft tumor development in vivo. Additionally, TRIM55 overexpression dampened CRC cell migration and invasion. Further bioinformatics analysis indicated that TRIM55 suppressed cyclin D1 and c-Myc expression. Mechanistically, co-immunoprecipitation assay revealed that TRIM55 directly interacted with c-Myc and down-regulated its protein expression level via protein ubiquitination. Intriguingly, c-Myc overexpression partially antagonized the function of TRIM55 overexpression. Taken together, our findings suggest that TRIM55 inhibits CRC tumor development via, at least in part, enhancing protein degradation of c-Myc. Targeting TRIM55 could provide a new therapeutic approach for CRC patients.

Regulation of proteolysis in bovine cumulus cells with possible inclusion of proton pump activators.

The aim of this study was to reveal the effects of V-ATPase proton pump activation on lysosomal acidity and protein degradation in cultured cumulus cells. Cumulus cells from bovine ovaries were cultured in the presence of 10 and 50 μM doses of V-ATPase proton pump activators PIP2, PMA and DOG for 12 and 24 h. At the end of the culture period, the level of protein degradation was evaluated through DQ-Red-BSA analysis and the lysosomes were detected through a fluorescent probe. In addition, total and phosphorylated MAPK1/3 and AKT protein levels of cumulus cells were determined through Western blotting. PIP2 and PMA were shown to increase protein degradation and lysosomal acidity in cultured bovine cumulus cells, whereas DOG did not have any significant effects on these cells. Total and phosphorylated MAPK and AKT protein levels were higher in PIP2 and PMA groups compared with the control and DOG. It was concluded that particular proton pump activators can enhance protein degradation and lysosomal acidification in cultured bovine cumulus cells without having detrimental effects on cell signalling members required for cell viability and proper functioning. Due to the cellular interactions, increasing the lysosomal activity in cumulus cells in the culture environment could also affect the removal of protein aggregates in the oocytes. This strategy could be effective for improving in vitro maturation of the oocytes by providing proteostasis.

Aged Garlic Extract Prevents Alcohol-Induced Cytotoxicity through Induction of Aldehyde Dehydrogenase 2 intheLiverofMice.

Acetaldehyde is a highly toxic primary metabolite of ethanol, and converts to nontoxic acetic acid by aldehyde dehydrogenase (ALDH). Accumulation of acetaldehyde causes significant damage to human body. Aged garlic extract (AGE) is a functional food material and possesses various health beneficial effects. This study investigates whether AGE contributes to acetaldehyde detoxification through ALDH induction and its underlying mechanism. C57BL/6J mice are orally administrated 10-1000mgkg-1 body weight (BW) of AGE for 1week before ethanol administration. AGE suppresses ethanol-caused accumulation of acetaldehyde level in the plasma through inducing mitochondrial ALDH2 but not cytosolic ALDH1A1. AGE also induces antioxidant enzymes, heme oxygenase-1, and NAD(P)H:quinone oxidoreductase 1, resulting in prevention of lipid peroxidation in the liver. In HepG2 cells, AGE prevents ethanol- and acetaldehyde-caused cytotoxicity. AGE induces mitochondrial ALDH2 through activating nuclear factor-erythroid 2-related factor 2 (Nrf2). AGE inhibits protein degradation of Nrf2 and enhances protein degradation of kelch-like ECH-associated protein 1. Furthermore, S-allyl cysteine and S-allyl mercaptocysteine as the bioactive compounds in AGE also induce ALDH2 and Nrf2. AGE prevents acetaldehyde-induced hepatotoxicity through enhancing acetaldehyde detoxification through Nrf2-dependent induction of mitochondrial ALDH2.

Enhanced protein degradation by black soldier fly larvae (Hermetia illucens L.) and its gut microbes.

Black soldier fly larvae (BSFL) can convert a variety of organic wastes into biomass, and its gut microbiota are involved in this process. However, the role of gut microbes in the nutrient metabolism of BSFL is unclear. In this study, germ-free BSFL (GF) and gnotobiotic BSFL (GB) were evaluated in a high-protein artificial diet model. We used 16S rDNA sequencing, ITS1 sequencing, and network analysis to study gut microbiota in BSFL that degrade proteins. The protein reduction rate of the GB BSFL group was significantly higher (increased by 73.44%) than that of the GF BSFL group. The activity of gut proteinases, such as trypsin and peptidase, in the GB group was significantly higher than the GF group. The abundances of different gut microbes, including Pseudomonas spp., Orbus spp. and Campylobacter spp., were strongly correlated with amino acid metabolic pathways. Dysgonomonas spp. were strongly correlated with protein digestion and absorption. Issatchenkia spp. had a strong correlation with pepsin activity. Campylobacter spp., Pediococcus spp. and Lactobacillus spp. were strongly correlated with trypsin activity. Lactobacillus spp. and Bacillus spp. were strongly correlated with peptidase activity. Gut microbes such as Issatchenkia spp. may promote the gut proteolytic enzyme activity of BSFL and improve the degradation rate of proteins. BSFL protein digestion and absorption involves gut microbiota that have a variety of functions. In BSFL the core gut microbiota help complete protein degradation. These results demonstrate that core gut microbes in BSFL are important in protein degradation.

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

Pyruvate prevents the onset of the cachectic features and metabolic alterations in myotubes downregulating STAT3 signaling.

Cachexia is a systemic disease associated with several pathologies, including cancer, that leads to excessive weight loss due to enhanced protein degradation. Previously, we showed that cachectic features in myotubes are provoked by a metabolic shift toward lactic fermentation. Our previous results led us to hyphotesise that increasing pyruvate concentration could impede the metabolic modifications responsible for induction of cachexia in myotubes. Here, we demonstrated that the addition of sodium pyruvate in conditioned media from CT26 colon cancer cells (CM CT26) prevents the onset of either phenotypic and metabolic cachectic features. Myotubes treated with CM CT26 containing sodium pyruvate show a phenotype similar to the healthy counterpart and display lactate production, oxygen consumption, and pyruvate dehydrogenase activity as control myotubes. The use of the Mitochondrial Pyruvate Carrier inhibitor UK5099, highlights the importance of mitochondrial pyruvate amount in the prevention of cachexia. Indeed, UK5099-treated myotubes show cachectic features as those observed in myotubes treated with CM CT26. Finally, we found that sodium pyruvate is able to decrease STAT3 phosphorylation level, a signaling pathway involved in the induction of cachexia in myotubes. Collectively, our results show that cachexia in myotubes could be prevented by the utilization of sodium pyruvate which impedes the metabolic modifications responsible for the acquisition of the cachectic features.

A heterozygous p.S143P mutation in LMNA associates with proteasome dysfunction and enhanced autophagy-mediated degradation of mutant lamins A and C.

Lamins A and C are nuclear intermediate filament proteins that form a proteinaceous meshwork called lamina beneath the inner nuclear membrane. Mutations in the LMNA gene encoding lamins A and C cause a heterogenous group of inherited degenerative diseases known as laminopathies. Previous studies have revealed altered cell signaling pathways in lamin-mutant patient cells, but little is known about the fate of mutant lamins A and C within the cells. Here, we analyzed the turnover of lamins A and C in cells derived from a dilated cardiomyopathy patient with a heterozygous p.S143P mutation in LMNA. We found that transcriptional activation and mRNA levels of LMNA are increased in the primary patient fibroblasts, but the protein levels of lamins A and C remain equal in control and patient cells because of a meticulous interplay between autophagy and the ubiquitin-proteasome system (UPS). Both endogenous and ectopic expression of p.S143P lamins A and C cause significantly reduced activity of UPS and an accumulation of K48-ubiquitin chains in the nucleus. Furthermore, K48-ubiquitinated lamins A and C are degraded by compensatory enhanced autophagy, as shown by increased autophagosome formation and binding of lamins A and C to microtubule-associated protein 1A/1B-light chain 3. Finally, chaperone 4-PBA augmented protein degradation by restoring UPS activity as well as autophagy in the patient cells. In summary, our results suggest that the p.S143P-mutant lamins A and C have overloading and deleterious effects on protein degradation machinery and pharmacological interventions with compounds enhancing protein degradation may be beneficial for cell homeostasis.