Impaired Protein Synthesis Research Articles

Occurrence of the Ability to Suppress the Growth of Related Strains in Rhizobia

Abstract—Screening of 1019 strains of rhizobia Rhizobium leguminosarum and Neorhizobium galegae from the “Symbiont” collection of symbiotic rhizospheric microorganisms of the Institute of Biochemistry and Genetics, Ufa Research Center, Russian Academy of Sciences, which have been isolated from root nodules of 20 leguminous species, for the ability to suppress the growth of related strains of rhizobia was carried out. Inhibition of the growth of competing bacteria by rhizobia was shown to be rather common. On average, when grown on rich media, ~10% of rhizobial strains released antibacterial substances into the medium. At the same time, when a live culture of rhizobia was applied to the lawn of bacteria, the percentage of suppression was higher. The percentage of strains synthesizing antibacterial substances correlated also, to some extent, with the level of genetic diversity of the population of legume microsymbionts. A weak signal of impaired protein synthesis was found in the study of the mechanism of action of metabolites of 20 strains of rhizobia R. leguminosarum bv. viciae from root nodules of Lathyrus palustris and Lathyrus vernus. In other cases, the nature of the antibacterial action of bacterial metabolites could not be determined using the DualRep2 system.

L-Isoleucine reverses hyperammonemia-induced myotube mitochondrial dysfunction and post-mitotic senescence

Perturbations in the metabolism of ammonia, a cytotoxic endogenous metabolite, occur in a number of chronic diseases, with consequent hyperammonemia. Increased skeletal muscle ammonia uptake causes metabolic, molecular, and phenotype alterations including cataplerosis of (loss of tricarboxylic acid cycle (TCA) cycle intermediate) α-ketoglutarate (αKG), mitochondrial oxidative dysfunction, and senescence-associated molecular phenotype (SAMP). L-Isoleucine (Ile) is an essential, branched-chain amino acid (BCAA) that simultaneously provides acetyl-CoA as an oxidative substrate and succinyl-CoA for anaplerosis (providing TCA cycle intermediates). Our multiomics analyses in myotubes and skeletal muscle from hyperammonemic mice and human patients with cirrhosis showed perturbations in BCAA transporters and catabolism. We, therefore, determined if Ile reverses hyperammonemia-induced impaired mitochondrial oxidative function and SAMP. Studies were performed in differentiated murine C2C12 myotubes that were early passage, late passage (senescent), or those depleted of LAT1/SLC7A5 and human induced pluripotent stem cell-derived myotubes (hiPSCM). Ile reverses hyperammonemia-induced reduction in the maximum respiratory capacity, complex I, II, and III functions in early passage murine myotubes and hiPSCM. Consistently, low ATP content and impaired global protein synthesis (high energy requiring cellular process) during hyperammonemia are reversed by Ile in murine myotubes and hiPSCM. Lower abundance of critical regulators of protein synthesis in mTORC1 signaling, and increased phosphorylation of eukaryotic initiation factor 2α are also reversed by Ile. Genetic depletion studies showed that Ile responses are independent of the amino acid transporter LAT1/SLC7A5. Our studies show that Ile reverses the hyperammonemia-induced impaired mitochondrial oxidative function, cataplerosis, and SAMP in a LAT1/SLC7A5 transporter-independent manner.

Data-driven transcriptomics analysis identifies PCSK9 as a novel key regulator in liver aging.

The liver, as a crucial metabolic organ, undergoes significant pathological changes during the aging process, which can have a profound impact on overall health. To gain a comprehensive understanding of these alterations, we employed data-driven approaches, along with biochemical methods, histology, and immunohistochemistry techniques, to systematically investigate the effects of aging on the liver. Our study utilized a well-established rat aging model provided by the National Institute of Aging. Systems biology approaches were used to analyze genome-wide transcriptomics data from liver samples obtained from young (4-5months old) and aging (20-21months old) Fischer 344 rats. Our findings revealed pathological changes occurring in various essential biological processes in aging livers. These included mitochondrial dysfunction, increased oxidative/nitrative stress, decreased NAD + content, impaired amino acid and protein synthesis, heightened inflammation, disrupted lipid metabolism, enhanced apoptosis, senescence, and fibrosis. These results were validated using independent datasets from both human and rat aging studies. Furthermore, by employing co-expression network analysis, we identified novel driver genes responsible for liver aging, confirmed our findings in human aging subjects, and pointed out the cellular localization of the driver genes using single-cell RNA-sequencing human data. Our study led to the discovery and validation of a liver-specific gene, proprotein convertase subtilisin/kexin type 9 (PCSK9), as a potential therapeutic target for mitigating the pathological processes associated with aging in the liver. This finding envisions new possibilities for developing interventions aimed to improve liver health during the aging process.

Phenylalanine-tRNA aminoacylation is compromised by ALS/FTD-associated C9orf72 C4G2 repeat RNA

The expanded hexanucleotide GGGGCC repeat mutation in the C9orf72 gene is the main genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Under one disease mechanism, sense and antisense transcripts of the repeat are predicted to bind various RNA-binding proteins, compromise their function and cause cytotoxicity. Here we identify phenylalanine-tRNA synthetase (FARS) subunit alpha (FARSA) as the main interactor of the CCCCGG antisense repeat RNA in cytosol. The aminoacylation of tRNAPhe by FARS is inhibited by antisense RNA, leading to decreased levels of charged tRNAPhe. Remarkably, this is associated with global reduction of phenylalanine incorporation in the proteome and decrease in expression of phenylalanine-rich proteins in cellular models and patient tissues. In conclusion, this study reveals functional inhibition of FARSA in the presence of antisense RNA repeats. Compromised aminoacylation of tRNA could lead to impairments in protein synthesis and further contribute to C9orf72 mutation-associated pathology.

Heterogeneous splicing patterns resulting from KIF5A variants associated with amyotrophic lateral sclerosis.

Single nucleotide variants (SNVs) in the gene encoding Kinesin Family Member 5A (KIF5A), a neuronal motor protein involved in anterograde transport along microtubules, have been associated with amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive and fatal neurodegenerative disease that primarily affects motor neurons. Numerous ALS-associated KIF5A SNVs are clustered near splice site junctions of the penultimate exon 27 and are predicted to alter the carboxy-terminal (C-term) cargo-binding domain of KIF5A. Mis-splicing of exon 27, resulting in exon exclusion, is proposed to be the mechanism by which these SNVs cause ALS. Whether all SNVs proximal to exon 27 result in exon exclusion is unclear. To address this question, we designed an in vitro minigene splicing assay in HEK293 cells which revealed heterogeneous site-specific effects on splicing: only 5' splice site (5'ss) SNVs resulted in exon skipping. We also quantified splicing in select CRISPR-edited human stem cells differentiated to motor neurons and in neuronal tissues from a 5'ss SNV knock-in mouse, which showed the same result. Moreover, survival of representative 3' splice site, 5'ss, and truncated C-term variant KIF5A (v-KIF5A) motor neurons was severely reduced compared to wildtype motor neurons, and overt morphological changes were apparent. While total KIF5A mRNA levels were comparable across cell lines, total KIF5A protein levels were decreased for v-KIF5A lines, suggesting an impairment of protein synthesis or stability. Thus, despite the heterogeneous effect on RNA splicing, KIF5A SNVs similarly reduce the availability of the KIF5A protein, leading to axonal transport defects and motor neuron pathology.

The East Asian-specific LPL p.Ala288Thr (c.862G > A) missense variant exerts a mild effect on protein function

BackgroundLipoprotein lipase (LPL) is the key enzyme responsible for the hydrolysis of triglycerides. Loss-of-function variants in the LPL gene are associated with hypertriglyceridemia (HTG) and HTG-related diseases. Unlike nonsense, frameshift and canonical GT-AG splice site variants, a pathogenic role for clinically identified LPL missense variants should generally be confirmed by functional analysis. Herein, we describe the clinical and functional analysis of a rare LPL missense variant.MethodsChinese patients with HTG-associated acute pancreatitis (HTG-AP) were screened for rare nonsense, frameshift, missense or canonical GT-AG splice site variants in LPL and four other lipid metabolism-related genes (APOC2, APOA5, GPIHBP1 and LMF1) by Sanger sequencing. The functional consequences of the LPL missense variant of interest were characterized by in vitro expression in HEK-293T and COS-7 cells followed by Western blot and LPL activity assays.ResultsFive unrelated HTG-AP patients were found to be heterozygous for a rare East Asian-specific LPL missense variant, c.862G > A (p.Ala288Thr). All five patients were adult males, and all were overweight and had a long history of alcohol consumption. Transfection of LPL wild-type and c.862G > A expression vectors into two cell lines followed by Western blot analysis served to exclude the possibility that the p.Ala288Thr missense variant either impaired protein synthesis or increased protein degradation. Contrary to a previous functional study that claimed that p.Ala288Thr had a severe impact on LPL function (reportedly having 36% normal activity), our experiments consistently demonstrated that the variant had a comparatively mild effect on LPL functional activity, which was mediated through its impact upon LPL protein secretion (~ 20% reduced secretion compared to wild-type).ConclusionsIn this study, we identified the East Asian-specific LPL c.862G > A (p.Ala288Thr) missense variant in five unrelated HTG-AP patients. We demonstrated that this variant exerted only a relatively mild effect on LPL function in two cell lines. Heterozygosity for this LPL variant may have combined with alcohol consumption to trigger HTG-AP in these patients.

Comparison of Aflatoxin Yield from Peanut Isolate of Aspergillus flavus with Other Isolates

Background: Mouldy growth in food commodities by aflatoxin producing fungi is a serious health concern to animals as well as human beings due to the harmful effects of aflatoxins like hepatotoxicity, carcinogenicity, impairment of protein synthesis, formation of DNA adducts, etc. even in minute concentrations. A tropical climate with moderate temperature, high humidity and heavy rainfall provides optimal conditions for the growth of aflatoxin producing fungi like Aspergillus flavus in different food items. The current study was to examine if the substrate(s) in a food item has any role to play in aflatoxin production by A. flavus. Methods: The fungus was isolated from ten different edible materials and identified using standard procedure. The isolates were cultured in potato dextrose agar slants keeping the culture conditions identical in triplicates. The toxin extracted using chloroform as solvent and downstream processing to get crude toxin was quantified on the basis of dry weight. Thin layer chromatography was done and the fluorescence of the toxins from different isolates was visualised under high wavelength of ultra-violet light with standard Aflatoxin B1 chromatographed vis-a-vis to confirm presence of the toxin in all the extracted toxins. Result: Results of the extracted toxin dry weight comparison between different isolates as well as the fluorescence intensity in the chromatograms revealed that contaminated peanut isolate of A. flavus yielded the highest amount of crude toxin. Thus, the results indicate that there are some factor(s) in peanut that promote(s) aflatoxin production by A. flavus. This information was further used for formulating potato dextrose agar media for isolation of aflatoxin producing fungi by incorporating peanut extract in the media and comparing the amount of crude toxin yield.

Nucleolar HEAT Repeat Containing 1 Up-regulated by the Mechanistic Target of Rapamycin Complex 1 Signaling Promotes Hepatocellular Carcinoma Growth by Dominating Ribosome Biogenesis and Proteome Homeostasis

Hyperactivation of ribosome biogenesis leads to hepatocyte transformation and plays pivotal roles in hepatocellular carcinoma (HCC) development. We aimed to identify critical ribosome biogenesis proteins that are overexpressed and crucial in HCC progression. HEAT repeat containing 1 (HEATR1) expression and clinical correlations were analyzed using The Cancer Genome Atlas and Gene Expression Omnibus databases and further evaluated by immunohistochemical analysis of an HCC tissue microarray. Gene expression was knocked down by small interfering RNA. HEATR1-knockdown cells were subjected to viability, cell cycle, and apoptosis assays and used to establish subcutaneous and orthotopic tumor models. Chromatin immunoprecipitation and quantitative polymerase chain reaction were performed to detect the association of candidate proteins with specific DNA sequences. Endogenous coimmunoprecipitation combined with mass spectrometry was used to identify protein interactions. We performed immunoblot and immunofluorescence assays to detect and localize proteins in cells. The nucleolus ultrastructure was detected by transmission electron microscopy. Click-iT (Thermo Fisher Scientific) RNA imaging and puromycin incorporation assays were used to measure nascent ribosomal RNA and protein synthesis, respectively. Proteasome activity, 20S proteasome foci formation, and protein stability were evaluated in HEATR1-knockdown HCC cells. HEATR1 was the most up-regulated gene in a set of ribosome biogenesis mediators in HCC samples. High expression of HEATR1 was associated with poor survival and malignant clinicopathologic features in patients with HCC and contributed to HCC growth invitro and invivo. HEATR1 expression was regulated by the transcription factor specificity protein 1, which can be activated by insulin-like growth factor 1-mammalian target of rapamycin complex 1 signaling in HCC cells. HEATR1 localized predominantly in the nucleolus, bound to ribosomal DNA, and was associated with RNA polymerase I transcription/processing factors. Knockdown of HEATR1 disrupted ribosomal RNA biogenesis and impaired nascent protein synthesis, leading to reduced cytoplasmic proteasome activity and inhibitory-κB/nuclear factor-κB signaling. Moreover, HEATR1 knockdown induced nucleolar stress with increased nuclear proteasome activity and inactivation of the nucleophosmin 1-MYC axis. Our study revealed that HEATR1 is up-regulated by insulin-like growth factor 1-mammalian target of rapamycin complex 1-specificity protein 1 signaling in HCC and functions as a crucial regulator of ribosome biogenesis and proteome homeostasis to promote HCC development.

Run, Ribosome, Run: From Compromised Translation to Human Health.

Significance: Translation is an essential cellular process, and diverse signaling pathways have evolved to deal with problems arising during translation. Erroneous stalls and unresolved ribosome collisions are implicated in many pathologies, including neurodegeneration and metabolic dysregulation. Recent Advances: Many proteins involved in detection and clearance of stalled and collided ribosomes have been identified and studied in detail. Ribosome profiling techniques have revealed extensive and nonprogrammed ribosome stalling and leaky translation into the 3' untranslated regions of mRNAs. Impairment of protein synthesis has been linked to aging in yeast and mice. Critical Issues: Ribosomes act as sensors of cellular states, but the molecular mechanisms, as well as physiological relevance, remain understudied. Most of our current knowledge stems from work in yeast and simple multicellular organisms such as Caenorhabditis elegans, while we are only beginning to comprehend the role of ribosome surveillance in higher organisms. As an example, the ribotoxic stress response, a pathway responding to global translational stress, has been studied mostly in response to small translation inhibitors and ribotoxins, and has only recently been explored in physiological settings. This review focuses on ribosome-surveillance pathways and their importance for cell and tissue homeostasis upon naturally occurring insults such as oxidative stress, nutrient deprivation, and viral infections. Future Directions: A better insight into the physiological roles of ribosome-surveillance pathways and their crosstalk could lead to an improved understanding of human pathologies and aging. Antioxid. Redox Signal. 39, 336-350.

Effects of intermittent fasting on brain tissue in male Fisher-344 rats

Introduction: Intermittent fasting (IF), time-restricted food intake without any caloric restrictions, has been shown to improve cognition in animal models. Several pathways have been identified in IF-induced neuroprotection; here, we observed the effect of IF on neuroprotection targeting molecular markers of protein synthesis (mTOR), autophagy (ATG5/ LC3A), and antioxidant expression (SOD2). We hypothesized that IF would improve autophagy and antioxidant expression and reduce protein synthesis in male fisher-344 rats. Methods: Twenty-four, 10-week-old male Fisher-344 rats were randomly assigned into two groups (ad-libitum, or IF) and housed 4 per cage, on a 12:12 light: dark cycle. Ad-libitum group had 24/7 access to food and water while the IF group had 24/7 access to water and full access to food on an every-other-day basis (i.e. alternate day fasting) for ten weeks. At 20 weeks of age, animals were euthanized and brain tissues (hippocampus and cortex) were sectioned and snap-frozen in liquid nitrogen. Following homogenization, western blots were completed via Jess Protein Simple automated western blotting (Bio-Techne, Minneapolis, MN). Specific proteins of interested included, LC3 (Novas, NB100-2331), ATG5 (Novas, NB110-538185S), mTOR (Santa Cruz, sc-517464) and SOD2 (Abcam, ab68155). Compass software generated area under the curve values were used to determine protein expression. Group differences were analyzed with a two-tailed T-test per Prism Graph Pad software. Results: In cortex tissue, a significantly lower mTOR expression was observed in the IF group compared to the AL group (p<.001), however, mTOR expression was not significantly different in the hippocampus. Antioxidant enzyme, SOD2, was significantly higher in IF cortex tissues (p<.05) compared to AL controls. In contrast, SOD2 was significantly lower in IF hippocampal tissues (p<.01) compared to controls. No significant effect was documented in the LC3 and ATG5 autophagy markers. Conclusion: In the present study, IF significantly impaired protein synthesis (via mTOR expression) in the cortex only. Previous work has also concluded the downregulation of mTOR with both calorie restriction and fasting. However, the fact that no decline was present in the hippocampus is intriguing and will be the focus of follow-up investigations. As expected, IF increased antioxidant enzyme (SOD2) expression in the cortex and interestedly, IF decreased antioxidant expression in the hippocampus. While the hippocampus is noted for its high antioxidant content, this decreased expression induced with IF warrants more research. P20GM113109 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Small molecule bio-signature in childhood intra-thoracic tuberculosis identified by metabolomics.

The diagnosis of pediatric tuberculosis (TB) remains a major challenge, hence the evaluation of new tools for improved diagnostics is urgently required. We investigated the serum metabolic profile of children with culture-confirmed intra-thoracic TB (ITTB) (n = 23) and compared it with those of non-TB controls (NTCs) (n = 13) using proton NMR spectroscopy-based targeted and untargeted metabolomics approaches. In targeted metabolic profiling, five metabolites (histidine, glycerophosphocholine, creatine/phosphocreatine, acetate, and choline) differentiated TB children from NTCs. Additionally, seven discriminatory metabolites (N-α-acetyl-lysine, polyunsaturated fatty acids, phenylalanine, lysine, lipids, glutamate + glutamine, and dimethylglycine) were identified in untargeted metabolic profiling. The pathway analysis revealed alterations in six metabolic pathways. The altered metabolites were associated with impaired protein synthesis, hindered anti-inflammatory and cytoprotective mechanisms, abnormalities in energy generation processes and membrane metabolism, and deregulated fatty acid and lipid metabolisms in children with ITTB. The diagnostic significance of the classification models obtained from significantly distinguishing metabolites showed sensitivity, specificity, and area under the curve of 78.2%, 84.6%, and 0.86, respectively, in the targeted profiling and 92.3%, 100%, and 0.99, respectively, in the untargeted profiling. Our findings highlight detectable metabolic changes in childhood ITTB; however, further validation is warranted in a large cohort of the pediatric population.

Investigating Eukaryotic Elongation Factor 2 Kinase/Eukaryotic Translation Elongation Factor 2 Pathway Regulation and Its Role in Protein Synthesis Impairment during Disuse-Induced Skeletal Muscle Atrophy

The principal mechanism underlying the reduced rate of protein synthesis in atrophied skeletal muscle is largely unknown. eEF2k impairs the ability of eEF2 to bind to the ribosome via T56 phosphorylation. Perturbations in the eEF2k/eEF2 pathway during various stages of disuse muscle atrophy have been investigated utilizing a rat hindlimb suspension (HS) model. Two distinct components of eEF2k/eEF2 pathway misregulation were demonstrated, observing a significant (P<0.01) increase in eEF2k mRNA expression as early as 1-day HS and in eEF2k protein level after 3-day HS. We set out to determine whether eEF2k activation is a Ca2+-dependent process with involvement of Cav1.1. The ratio of T56-phosphorylated:total eEF2 was robustly elevated after 3-day HS, which was completely reversed by BAPTA-AM and decreased by 1.7-fold (P<0.05) by nifedipine. Transfection of C2C12 with pCMV-eEF2k and administration with small molecules were used to modulate eEF2k and eEF2 activity. Importantly, pharmacological enhancement of eEF2 phosphorylation induced P-p70S6k (T389) upregulation and restoration of global protein synthesis in the HS rats. Taken together, the eEF2k/eEF2 pathway is upregulated during disuse muscle atrophy involving calcium-dependent activation of eEF2k partly via Cav1.1. The study provides evidence, in vitro and in vivo, of the eEF2k/eEF2 pathway impact on p70S6k activity as well as protein expression of key atrophy biomarkers, MAFbx/atrogin-1 and MuRF-1.

Tandem mass tag-based proteomics technology provides insights into multi-targeted mechanism of peptide MOp2 from Moringa oleifera seeds against Staphylococcus aureus

Our previous study proved that the peptide MOp2 from Moringa oleifera seeds exhibited membrane-damaging effects on Staphylococcus aureus. In this study, TMT-based proteomics technology was mainly used to investigate the antimicrobial mechanism of MOp2 against S. aureus. A total of 541 differentially expressed proteins (DEPs) were identified in S. aureus treated with MOp2, among which 256 and 285 DEPs were upregulated and downregulated, respectively. They were mainly involved in inositol phosphate metabolism, S. aureus infection, citrate cycle (TCA cycle), and phosphotransferase system (PTS) and acted as ABC transporters and ribosomes. Moreover, the decreasing AKP activity indicated that MOp2 affected cell wall biosynthesis, and excessive ROS accumulation caused apoptosis, resulting in DNA fragmentation, nuclear morphological changes, and phosphatidylserine externalization. Additionally, molecular docking showed that MOp2 could interact with eight DEPs, including ProC, QoxB, SOD2, DnaK, GroEL, RplY, AcpS, and FabG, indicating that MOp2 might act on these molecular targets, leading to increased cell permeability, oxidative damage, impaired protein synthesis, cell wall synthesis obstruction, and energy metabolism disorder. Overall, these findings provided new insights into the multi-targeted mechanism of MOp2 against S. aureus, and could provide a theoretical basis for its application as a novel food preservative and antibiotic substitute.

In-Depth Proteomic Analysis of De Novo Proteome in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common dementia syndrome in the elderly characterized by synaptic failure and unique brain pathology. De novo protein synthesis is required for the maintenance of memory and synaptic plasticity. Mounting evidence links impaired neuronal protein synthesis capacity and overall protein synthesis deficits to AD pathogenesis. Meanwhile, identities of AD-associated dysregulation of "newly synthesized proteome" remain elusive. To investigate de novo proteome alterations in the hippocampus of aged Tg19959 AD model mice. In this study, we combined the bioorthogonal noncanonical amino acid tagging (BONCAT) method with the unbiased large-scale proteomic analysis in acute living brain slices (we name it "BONSPEC") to investigate de novo proteome alterations in the hippocampus of Tg19959 AD model mice. We further applied multiple bioinformatics methods to analyze in-depth the proteomics data. In total, 1,742 proteins were detected across the 10 samples with the BONSPEC method. After exclusion of those only detected in less than half of the samples in both groups, 1,362 proteins were kept for further analysis. 37 proteins were differentially expressed (based on statistical analysis) between the WT and Tg19959 groups. Among them, 19 proteins were significantly decreased while 18 proteins were significantly increased in the hippocampi of Tg19959 mice compared to WT mice. The results suggest that proteins involved in synaptic function were enriched in de novo proteome of AD mice. Our study could provide insights into the future investigation into the molecular signaling mechanisms underlying AD and related dementias (ADRDs).

Stimulation of soluble guanylate cyclase by vericiguat reduces skeletal muscle atrophy of mice following chemotherapy.

Background: The chemotherapeutic doxorubicin (DOX) promotes severe skeletal muscle atrophy, which induces skeletal muscle weakness and fatigue. Soluble guanylate cyclase (sGC) contributes to a variety of pathophysiological processes, but whether it is involved in DOX-induced skeletal muscle atrophy is unclear. The present study aimed to stimulate sGC by vericiguat, a new oral sGC stimulator, to test its role in this process. Methods: Mice were randomly divided into four groups: control group, vericiguat group, DOX group, and DOX + vericiguat group. Exercise capacity was evaluated before the mice were sacrificed. Skeletal muscle atrophy was assessed by histopathological and molecular biological methods. Protein synthesis and degradation were monitored in mice and C2C12 cells. Results: In this study, a significant decrease in exercise capacity and cross-sectional area (CSA) of skeletal muscle fibers was found in mice following DOX treatment. Furthermore, DOX decreased sGC activity in mice and C2C12 cells, and a positive correlation was found between sGC activity and CSA of skeletal muscle fibers in skeletal muscle. DOX treatment also impaired protein synthesis, shown by puromycin detection, and activated ubiquitin-proteasome pathway. Following sGC stimulation, the CSA of muscle fibers was elevated, and exercise capacity was enhanced. Stimulation of sGC also increased protein synthesis and decreased ubiquitin-proteasome pathway. In terms of the underlying mechanisms, AKT/mTOR and FoxO1 pathways were impaired following DOX treatment, and stimulation of sGC restored the blunted pathways. Conclusion: These results unravel sGC stimulation can improve skeletal muscle atrophy and increase the exercise capacity of mice in response to DOX treatment by enhancing protein synthesis and inhibiting protein degradation. Stimulation of sGC may be a potential treatment of DOX-induced skeletal muscle dysfunction.

Comorbid pathology in children with type II–III spinal muscular atrophy on the background of acquired deformities of the skeleton bones

Spinal muscular atrophy (SMA) is a severe genetic disease associated with impaired SMN protein synthesis and degeneration of alpha motor neurons in the spinal cord. Developing neurogenic kyphoscoliosis and deformity of the chest against the background of symmetrical muscular hypotension sharply limit the activity of patients, worsening the concomitant diseases.Purpose. The study aims at determining the comorbid background of children with type II–III spinal muscular atrophy who underwent inpatient treatment for acquired skeletal bone deformities.Material and methods. A retrospective analysis of the data was carried out for the period from 2017 to 2021 based on the medical records of 31 children. The study group included 10 girls and 21 boys; 16 children were with type II and 15 — with type III spinal muscular atrophy. The following were assessed: comorbidity, neurological status, hemodynamic parameters, echocardiography, spirometry, laboratory research data.Results. In our study, comorbidity was associated with nutritional status (19% of patients overweight, 29% underweight), mental retardation (3%), gastroesophageal reflux disease (19%), diseases of the ENT organs (16%), eyes (19%), heart and lungs (93%). For health reasons, 61% of children required the use of non-invasive ventilation, and 71% of insufflator-aspirators. Limited motor abilities were registered based on the HFMSE and GMFCS scales, dysphagia based on the EDACS scale. A biochemical blood test revealed a low level of creatinine.Conclusion. Patients with spinal muscular atrophy require multidisciplinary care in diagnosis, treatment and rehabilitation. The use of objective rating scales, instrumental and laboratory methods of examination allow for a comprehensive analysis of the potential of children with spinal muscular atrophy, to select effective, family-oriented treatment regimens. Serum creatinine as a biomarker for the severity of muscle denervation makes it possible to monitor the progression of spinal muscular atrophy and predict response to treatment.

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer’s disease mouse model: a role for ER-mitochondria interaction

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer’s disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

Weight gain during the menopause transition: Evidence for a mechanism dependent on protein leverage.

Weight gain during the menopause transition: Evidence for a mechanism dependent on protein leverage.

Transcriptomic and proteomic analysis of Staphylococcus aureus response to cuminaldehyde stress

The previous study indicated that cuminaldehyde (CUM) could be used as an antibacterial agent in sauced beef to reduce the propagation of Staphylococcus aureus (S. aureus). This research took sauced beef treated with 0.4 μL/mL CUM as the research object. Transcriptomic and proteomic methods were used to comprehensively analyze the changes in genes and proteins of S. aureus under CUM stress. A total of 258 differentially expressed genes (DEGs, 178 up-regulated and 80 down-regulated) and 384 differentially expressed proteins (DEPs, 61 up-regulated and 323 down-regulated) were found. It was observed that CUM destroyed the cell wall and cell membrane by inhibiting the synthesis of peptidoglycan and fatty acid. Low energy consumption strategies were formed by reducing glycolysis and ribosome de novo synthesis. The levels of genes and proteins associated with the glycine, serine, threonine, methionine, cysteine, and branched-chain amino acids were dramatically changed, which impaired protein synthesis and reduced bacterial viability. In addition, the up-regulated DEGs and DEFs involved in DNA replication, recombination and single-stranded DNA-binding contributed to DNA repair. Moreover, ATP-binding cassettes (ABC) transporters were also perturbed, such as the uptake of betaine and iron were inhibited. Thus, this study revealed the response mechanism of S. aureus under the stress of CUM, and provided a theoretical basis for the application of CUM in meat products.

Proteostasis Deregulation in Neurodegeneration and Its Link with Stress Granules: Focus on the Scaffold and Ribosomal Protein RACK1.

The role of protein misfolding, deposition, and clearance has been the dominant topic in the last decades of investigation in the field of neurodegeneration. The impairment of protein synthesis, along with RNA metabolism and RNA granules, however, are significantly emerging as novel potential targets for the comprehension of the molecular events leading to neuronal deficits. Indeed, defects in ribosome activity, ribosome stalling, and PQC—all ribosome-related processes required for proteostasis regulation—can contribute to triggering stress conditions and promoting the formation of stress granules (SGs) that could evolve in the formation of pathological granules, usually occurring during neurodegenerating effects. In this review, the interplay between proteostasis, mRNA metabolism, and SGs has been explored in a neurodegenerative context with a focus on Alzheimer’s disease (AD), although some defects in these same mechanisms can also be found in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are discussed here. Finally, we highlight the role of the receptor for activated C kinase 1 (RACK1) in these pathologies and note that, besides its well characterized function as a scaffold protein, it has an important role in translation and can associate to stress granules (SGs) determining cell fate in response to diverse stress stimuli.