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The synthetic steroid mifepristone blocks the growth of ovarian cancer cells, yet the mechanism driving such effect is not entirely understood. Unbiased genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to platinum led to the identification of two key genes upregulated by mifepristone and involved in the unfolded protein response (UPR): the master chaperone of the endoplasmic reticulum (ER), glucose regulated protein (GRP) of 78 kDa, and the CCAAT/enhancer binding protein homologous transcription factor (CHOP). GRP78 and CHOP were upregulated by mifepristone in ovarian cancer cells regardless of p53 status and platinum sensitivity. Further studies revealed that the three UPR-associated pathways, PERK, IRE1α, and ATF6, were activated by mifepristone. Also, the synthetic steroid acutely increased mRNA translation rate, which, if prevented, abrogated the splicing of XBP1 mRNA, a non-translatable readout of IRE1α activation. Moreover, mifepristone increased LC3-II levels due to increased autophagic flux. When the autophagic-lysosomal pathway was inhibited with chloroquine, mifepristone was lethal to the cells. Lastly, doses of proteasome inhibitors that are inadequate to block the activity of the proteasomes, caused cell death when combined with mifepristone; this phenotype was accompanied by accumulation of poly-ubiquitinated proteins denoting proteasome inhibition. The stimulation by mifepristone of ER stress and autophagic flux offers a therapeutic opportunity for utilizing this compound to sensitize ovarian cancer cells to proteasome or lysosome inhibitors.

Previous trials have shown the effects of fluvoxamine alone and inhaled budesonide alone for prevention of disease progression among outpatients with COVID-19. This placebo-controlled, randomized trial, conducted at 12 clinical sites in Brazil, sought to determine whether the combined effect of both drugs would increase treatment effects. Visual Abstract. Oral Fluvoxamine With Inhaled Budesonide for Treatment of Early-Onset COVID-19: Previous trials have shown the effects of fluvoxamine alone and inhaled budesonide alone for prevention of disease progression among outpatients with COVID-19. This placebo-controlled, randomized trial, conducted at 12 clinical sites in Brazil, sought to determine whether the combined effect of both drugs would increase treatment effects. Background: Previous trials have demonstrated the effects of fluvoxamine alone and inhaled budesonide alone for prevention of disease progression among outpatients with COVID-19. Objective: To determine whether the combination of fluvoxamine and inhaled budesonide would increase treatment effects in a highly vaccinated population. Design: Randomized, placebo-controlled, adaptive platform trial. (ClinicalTrials.gov: NCT04727424) Setting: 12 clinical sites in Brazil. Participants: Symptomatic adults with confirmed SARS-CoV-2 infection and a known risk factor for progression to severe disease. Intervention: Patients were randomly assigned to either fluvoxamine (100 mg twice daily for 10 days) plus inhaled budesonide (800 mcg twice daily for 10 days) or matching placebos. Measurements: The primary outcome was a composite of emergency setting retention for COVID-19 for more than 6 hours, hospitalization, and/or suspected complications due to clinical progression of COVID-19 within 28 days of randomization. Secondary outcomes included health care attendance (defined as hospitalization for any cause or emergency department visit lasting >6 hours), time to hospitalization, mortality, patient-reported outcomes, and adverse drug reactions. Results: Randomization occurred from 15 January to 6 July 2022. A total of 738 participants were allocated to oral fluvoxamine plus inhaled budesonide, and 738 received placebo. The proportion of patients observed in an emergency setting for COVID-19 for more than 6 hours or hospitalized due to COVID-19 was lower in the treatment group than the placebo group (1.8% [95% credible interval {CrI}, 1.1% to 3.0%] vs. 3.7% [95% CrI, 2.5% to 5.3%]; relative risk, 0.50 [95% CrI, 0.25 to 0.92]), with a probability of superiority of 98.7%. No relative effects were found between groups for any of the secondary outcomes. More adverse events occurred in the intervention group than the placebo group, but no important differences between the groups were detected. Limitation: Low event rate overall, consistent with contemporary trials in vaccinated populations. Conclusion: Treatment with oral fluvoxamine plus inhaled budesonide among high-risk outpatients with early COVID-19 reduced the incidence of severe disease requiring advanced care. Primary Funding Source: Latona Foundation, FastGrants, and Rainwater Charitable Foundation. [ABSTRACT FROM AUTHOR]

The transcriptional regulatory protein Rgg coordinates amino acid catabolism and virulence factor expression in Streptococcus pyogenes. We used a proteomic approach to compare cytoplasmic proteins isolated from S. pyogenes wild-type strain NZ131 (serotype M49) to proteins isolated from an rgg mutant strain during the exponential and stationary phases of growth. Proteins were separated by two-dimensional gel electrophoresis, and 125 protein spots of interest were identified by tandem mass spectrometry. Comparative analysis of proteins isolated from the isogenic strains revealed that growth phase-associated regulation of enzymes involved in the metabolism of arginine (ArcABC), histidine (HutI), and serine (SdhA) was abrogated in the rgg mutant strain, which synthesized the proteins in the exponential phase of growth. In contrast, the enzymes were detected only among wild-type proteins isolated from organisms in the stationary phase of growth. The differences in protein composition were correlated with previously described metabolic changes. In addition, proteins associated with thermal and oxidative stress responses, including ClpE and ClpL, were present in samples isolated from the rgg mutant strain but not in samples isolated from the wild-type strain. The rgg mutant strain was more tolerant to elevated temperature and puromycin than the wild-type strain; however, the mutant was less tolerant to paraquat. We concluded that Rgg is a global regulatory factor that contributes to growth phase-dependent synthesis of proteins associated with secondary metabolism and oxidative and thermal stress responses.

Streptococcus pyogenes (group A Streptococcus [GAS]) is a human pathogen that causes a wide range of diseases. For successful colonization within a variety of host niches, GAS must sense and respond to environmental changes. Intercellular communication mediated by peptides is one way GAS coordinates gene expression in response to diverse environmental stressors, which enhances bacterial survival and contributes to virulence. Using peptidomics, we identified SpoV (streptococcal peptide controlling virulence) in culture supernatant fluids. SpoV is a secreted peptide encoded near the gene (slo) encoding the extracellular cholesterol-dependent cytolysin streptolysin O (SLO). The addition of synthetic SpoV peptide derivatives, but not control peptides, increased slo transcript abundance in an M49 isolate but not in an M3 isolate. Deletion of spoV decreased slo transcript abundance, extracellular SLO protein levels, and SLO-specific hemolytic activity. Complementation of the spoV mutant increased slo transcript abundance. Lastly, a spoV mutant was deficient in the ability to survive in murine blood compared to the parental strain. Moreover, preincubation of the spoV mutant with synthetic SpoV peptide derivatives increased GAS survival. Our findings show that slo expression is regulated, in part, by the GAS-specific signaling peptide SpoV. IMPORTANCE GAS secretes signaling peptides that can alter gene expression and impact virulence. We used peptidomics to identify a signaling peptide designated SpoV. Further, we showed that SpoV altered the expression of the cholesterol-dependent cytolysin SLO. Peptide signaling plays an important regulatory role during disease progression among several bacterial pathogens, including GAS. The therapeutic potential of manipulating peptide-controlled regulatory networks makes it an attractive option for the development of novel therapeutic strategies that disrupt virulence gene expression. [ABSTRACT FROM AUTHOR]

Abstract Background The production of Streptococcus pyogenes exoproteins, many of which contribute to virulence, is regulated in response to nutrient availability. CodY is a transcriptional regulator that controls gene expression in response to amino acid availability. The purpose of this study was to identify differences in the expression of streptococcal exoproteins associated with deletion of the codY gene. Results We compared the secreted proteins produced by wild-type S. pyogenes to a codY mutant in the post-exponential phase of growth. We used both one and two-dimensional gel electrophoresis to separate exoproteins. Proteins that were significantly different in abundance upon repeated analysis were identified with tandem mass spectrometry. The production of the secreted cysteine protease SpeB, a secreted chromosomally encoded nuclease (SdaB), and a putative adhesion factor (Spy49_0549) were more abundant in supernatant fluids obtained from the codY mutant. In addition, hyaluronidase (HylA), CAMP factor (Cfa), a prophage encoded nuclease (Spd-3), and an uncharacterized extracellular protein (Spy49_0015) were less abundant in supernatant fluids obtained from the codY mutant strain. Enzymatic assays showed greater DNase activity in culture supernatants isolated in the post-exponential phase of growth from the codY mutant strain compared to the wild-type strain. Because extracellular nucleases and proteases can influence biofilm formation, we also measured the ability of the strains to form biofilms during growth with both rich medium (Todd Hewitt yeast extract; THY) and chemically defined media (CDM). No difference was observed with rich media but with CDM the biofilms formed by the codY mutant strain had less biomass compared to the wild-type strain. Conclusions Overall, the results indicate that CodY alters the abundance of a select group of S. pyogenes exoproteins, including DNases, a protease, and hylauronidase, which together may alleviate starvation by promoting dissemination of the pathogen to nutrient rich environments and by hydrolysis of host macromolecules.

Abstract In the rat, the decidual tissue is an important component for maternal recognition of pregnancy. Decidualization can be induced by either the implantation of the blastocyst or by artificial stimuli. The process of decidua formation or decidualization, is characterized by growth and differentiation of endometrial stromal cells. Prostaglandin F2alpha (PGF2α) has been shown to be involved in inhibition of implantation, alteration of embryo development, induction of luteal regression, and the mediation of pregnancy loss induced by microorganism infections. In order to establish a direct role for PGF2α in decidual function, we have evaluated its effects on the expression of an extensive array of genes using primary decidual cell culture. Upon treatment with PGF2α sixty genes were significantly down-regulated whereas only six genes were up-regulated (from a total of 1176 genes studied). Interestingly, the majority of the genes inhibited by PGF2α are either directly or indirectly involved in the turnover of the extracellular matrix (ECM). Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (tPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase, all of which were significantly up-regulated by PGF2α. The results strongly suggest that the abortificient role of elevated levels of PGF2α after implantation is due, in large part, to inhibition of genes involved in the normal turnover of the extracellular matrix necessary for decidual formation.

The biochemical properties as well as the nucleotide and amino acid sequences of arabinofuranosidase 3 (ABF3) from the soft rot fungus Penicillium purpurogenum are described. ABF3 is the first modular family 43 enzyme from a fungal source, which has both [alpha]-L-arabinofuranosidase and xylobiohydrolase functionalities.

Penicillium purpurogenum is a filamentous fungus, which grows on a variety of natural carbon sources and secretes a large number of enzymes involved in cellulose, hemicelluloses and pectin biodegradation. The purpose of this work has been to identify potential lignocellulolytic enzymes and to compare the secreted enzymes produced when the fungus is grown on sugar beet pulp (rich in cellulose and pectin) and corn cob (rich in cellulose and xylan). Culture supernatants were subjected to two-dimensional nano-liquid chromatography/tandem mass spectrometry. Using MASCOT and a genome-derived protein database, the proteins present in the supernatant were identified. The putative function in the degradation of the polysaccharides was determined using dbCAN software. The results show that there is a good correlation between the polysaccharide composition of the carbon sources and the function of the secreted enzymes: both cultures are rich in cellulases, while sugar beet pulp induces pectinases and corncob, xylanases. The eventual biochemical characterisation of these enzymes will be of value for a better understanding of the biodegradation process performed by the fungus and increase the availability of enzymes for biotechnological methods associated with this process. [ABSTRACT FROM AUTHOR]

The purpose of this study was to investigate the influence of increasing sulfate concentrations on chromium removal, to evaluate the effect of the presence of Cr(VI) on sulfate removal by Streptomyces sp. MC1 and to analyze the differential protein expression profile in the presence of this metal for the identification of proteins repressed or overexpressed. In the presence of Cr(VI) but in the absence of sulfate ions, bacterial growth was negligible, showing the Cr(VI) toxicity for this bacterium. However, the sulfate presence stimulated bacterium growth and Cr(VI) removal, regardless of its concentrations. Streptomyces sp. MC1 showed ability to remove chromium and sulfate simultaneously. Also, the sulfate presence favored the decrease of total chromium concentration from supernatants reaching a decrease of 50% at 48 h. In presence of chromium, seven proteins were down-expressed and showed homology to proteins involved in protein biosynthesis, energy production and free radicals detoxification while two proteins involved in oxidation-reduction processes identified as dihydrolipoamide dehydrogenase and S-adenosyl- l-methionine synthase were overexpressed. [ABSTRACT FROM AUTHOR]

Arabinan is a component of pectin, which is one of the polysaccharides present in lignocelluose. The enzymes degrading the main chain of arabinan are the endo- (EC 3.2.1.99) and exo-arabinanases (3.2.1.-). Only three exo-arabinanases have been biochemically characterized; they belong to glycosyl hydrolase family 93. In this work, the cDNA of an exo-arabinanase (Arap2) from Penicillium purpurogenum has been heterologously expressed in Pichia pastoris. The gene is 1310 bp long, has three introns and codes for a protein of 380 amino acid residues; the mature protein has a calculated molecular mass of 39 823 Da. The heterologously expressed Arap2 has a molecular mass in the range of 60–80 kDa due to heterogeneous glycosylation. The enzyme is active on debranched arabinan with optimum pH of 4–5.5 and optimal temperature of 40 °C, and has an exo-type action mode, releasing arabinobiose from its substrates. The expression profile of arap2 in corncob and sugar beet pulp follows a different pattern and is not related to the presence of arabinan. This is the first exo-arabinanase studied from P. purpurogenum and the first expressed in yeast. The availability of heterologous Arap2 may be useful for biotechnological applications requiring acidic conditions. [ABSTRACT FROM AUTHOR]

Context: Glutaredoxins (GRX) are involved in the regulation of thiol redox state. GRX-1 is a cytosolic enzyme responsible for the catalysis of deglutathionylation of proteins. To date, very few inhibitors of GRX-1 have been reported. Objective: The objective of this paper is to report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethyl-sulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as an inhibitor of human GRX-1. Materials and methods: The mechanism of inhibition of GRX-1 was investigated using dialysis, substrate protection, and mass spectrometry. Results: 2-AAPA inhibits GRX-1 in a time and concentration dependent manner. The activity did not return following dialysis indicating that inhibition is irreversible. Results of substrate protection and mass spectrometry indicate that the inhibition is occurring at the active site. The compound also produced GRX inhibition in human ovarian cancer cells. Discussion: 2-AAPA is an irreversible GRX-1 inhibitor with similar or greater potency compared to previously reported inhibitors. Conclusion: The inhibition of GRX-1 by 2-AAPA could be used as a tool to study thiol redox state. [ABSTRACT FROM AUTHOR]

Huntington disease (HD), a fatal neurodegenerative disorder, is caused by a lengthening of the polyglutamine tract in the huntingtin (Htt) protein. Despite considerable effort, thus far there is no cure or treatment available for the disorder. Using the approach of tandem affinity purification we recently discovered that prothymosin-α (ProTα), a small highly acidic protein, interacts with mutant Htt (mHtt). This was confirmed by coimmunoprecipitation and a glutathione S-transferase (GST) pull-down assay. Overexpression of ProTα remarkably reduced mHtt-induced cytotoxicity in both non-neuronal and neuronal cell models expressing N-terminal mHtt fragments, whereas knockdown of ProTα expression in the cells enhanced mHttcaused cell death. Deletion of the central acidic domain of ProTα abolished not only its interaction with mHtt but also its protective effect on mHtt-caused cytotoxicity. Additionally, overexpression of ProTα inhibited caspase-3 activation but enhanced aggregation of mHtt. Furthermore, when added to cultured cells expressing mHtt, the purified recombinant ProTα protein not only entered the cells but it also significantly suppressed the mHtt-caused cytotoxicity. Taken together, these data suggest that ProTα might be a novel therapeutic target for treating HD and other polyglutamine expansion disorders. [ABSTRACT FROM AUTHOR]

The soft rot fungus Penicillium purpurogenum grows on a variety of natural substrates and secretes various isoforms of xylanolytic enzymes, including three arabinofuranosidases. This work describes the biochemical properties as well as the nucleotide and amino acid sequences of arabinofuranosidase 3 (ABF3). This enzyme has been purified to homogeneity. It is a glycosylated monomer with a molecular weight of 50,700 and can bind cellulose. The enzyme is active with p-nitrophenyl β-D-L-arabinofuranoside and p-nitrophenyl β-D-xylopyranoside with κm of 0.65 mM and 12 mM, respectively. The enzyme is active on xylooligosaccharides, yielding products of shorter length, including xylose. However, it does not hydrolyze arabinooligosaccharides. When assayed with polymeric substrates, little arabinose is liberated from arabinan and debranched arabinan; however, it hydrolyzes arabinose and releases xylooligosaccharides from arabinoxylan. Sequencing both ABF3 cDNA and genomic DNA reveals that this gene does not contain introns and that the open reading frame is 1,380 nucleotides in length. The deduced mature protein is composed of 433 amino acids residues and has a calculated molecular weight of 47,305. The deduced amino acid sequence has been validated by mass spectrometry analysis of peptides from purified ABF3. A total of 482 bp of the promoter were sequenced; putative binding sites for transcription factors such as CreA (four), XInR (one), and AreA (three) and two CCAAT boxes were found. The enzyme has two domains, one similar to proteins of glycosyl hydrolase family 43 at the amino-terminal end and a family 6 carbohydrate binding module at the carboxyl end. ABF3 is the first described modular family 43 enzyme from a fungal source, having both a-i-arabinofuranosidase and xylobiohydrolase functionalities. [ABSTRACT FROM AUTHOR]

Enteric bacterial pathogens cause food borne disease, which constitutes an enormous economic and health burden. Enterohemorrhagic Escherichia coli (EHEC) causes a severe bloody diarrhea following transmission to humans through various means, including contaminated beef and vegetable products, water, or through contact with animals. EHEC also causes a potentially fatal kidney disease (hemolytic uremic syndrome) for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g., enteropathogenic E. coli (EPEC), Salmonella, Shigella, Yersinia) utilize a type III secretion system (T3SS) to inject virulence proteins (effectors) into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, in many cases, the mechanisms by which these effectors bind to host proteins and disrupt the normal function of intestinal epithelial cells have not been completely characterized. In this study, we present evidence that the E. coli O157:H7 nleH1 and nleH2 genes encode T3SS effectors that bind to the human ribosomal protein S3 (RPS3), a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) transcriptional complexes. NleH1 and NleH2 co-localized with RPS3 in the cytoplasm, but not in cell nuclei. The Nterminal region of both NleH1 and NleH2 was required for binding to the N-terminus of RPS3. NleH1 and NleH2 are autophosphorylated Ser/Thr protein kinases, but their binding to RPS3 is independent of kinase activity. NleH1, but not NleH2, reduced the nuclear abundance of RPS3 without altering the p50 or p65 NF-kB subunits or affecting the phosphorylation state or abundance of the inhibitory NF-κB chaperone lκBα NleH1 repressed the transcription of a RPS3/ NF-κB-dependent reporter plasmid, but did not inhibit the transcription of RPS3-independent reporters. In contrast, NleH2 stimulated RPS3-dependent transcription, as well as an AP-1-dependent reporter. We identified a region of NleH1 (N40-K45) that is at least partially responsible for the inhibitory activity of NleH1 toward RPS3. Deleting nleH1 from E. coli O157:H7 produced a hypervirulent phenotype in a gnotobiotic piglet model of Shiga toxin-producing E. coli infection. We suggest that NleH may disrupt host innate immune responses by binding to a cofactor of host transcriptional complexes. [ABSTRACT FROM AUTHOR]

Yersinia enterocolitica (Ye) inserts outer proteins (Yops) into cytoplasm to infect host cells. However, in spite of considerable progress, the mechanisms implicated in this process, including the association of Yops with host proteins, remain unclear. Here, we evaluated the functional role of Galectin-1 (Gal1), an endogenous β-galactoside-binding protein, in modulating Yop interactions with host cells. Our results showed that Gal1 binds to Yops in a carbohydrate-dependent manner. Interestingly, Gal1 binding to Yops protects these virulence factors from trypsin digestion. Given that early control of Ye infection involves activation of macrophages, we evaluated the role of Gal1 and YopP in the modulation of macrophage function. Although Gal1 and YopP did not influence production of superoxide anion and/or TNF by Ye-infected macrophages, they coordinately inhibited nitric oxide (NO) production. Notably, recombinant Gal1 (rGal1) did not rescue NO increase observed in Lgals1−/− macrophages infected with the YopP mutant Ye ∆yopP. Whereas NO induced apoptosis in macrophages, no significant differences in cell death were detected between Gal1-deficient macrophages infected with Ye ∆yopP, and WT macrophages infected with Ye wt. Strikingly, increased NO production was found in WT macrophages treated with MAPK inhibitors and infected with Ye wt. Finally, rGal1 administration did not reverse the protective effect in Peyer Patches (PPs) of Lgals1−/− mice infected with Ye ∆yopP. Our study reveals a cooperative role of YopP and endogenous Gal1 during Ye infection. [ABSTRACT FROM AUTHOR]

Background: Diabetes-related foot complications have been identified as the most common isolated cause of morbidity among patients with diabetes and the leading cause of amputation. Therefore, new strategies to stimulate skin regeneration may provide a novel therapeutic approach to reduce non-healing ulcer disease. Recently, we demonstrated in proof-of-concept in humans that administration of allogeneic bone marrow mesenchymal stromal cellss derivatives (allo-hBM-MSCDs) is effective in a similar way to the use of allogeneic bone marrow mesenchymal stromal cellss (allo-hBM-MSCs) in grade 2 diabetic foot ulcers (DFUs). Aim: To assess the safety and efficacy profile of the allo-hBM-MSCDs relative to the conventional approach (PolyMen® dressing) in 1/2 clinical trial phases in patients with grade 1 and 2 DFUs. Methods: In the present study, we used 2 doses of allo-hBM-MSCDs (1 mL) or 1 dose of allo-hBM-MSCs (1 × 106 cells) intradermally injected around wounds and assessed their safety and effectiveness, relative to the conventional approach (PolyMem dressing). Allo-hBM-MSCDs and allo-hBM-MSCs were produced in a certified Good Manufacturing Practice-type Laboratory. Patients with grade 1 and 2 DFUs were randomized to receive allo-hBM-MSCDs (n=12), allo-hBM-MSCs (n=6) or conventional treatment (PolyMem dressing) (n=10). The wound-healing process was macroscopically evaluated until the complete closure of the ulcers. Results: No adverse events were reported. Patients with grade 1 and 2 DFUs treated with either allo-hBM-MSCDs or allo-hBM-MSCs, achieved greater percentages of wound closure, enhanced skin regeneration in shorter times and a greater ulcer-free survival relative to the patients who received conventional treatment. Finally, through proteomic analysis, we elucidated the proteins and growth factors that are secreted by allo-hBM-MSCs and relevant to the wound-healing process. In addition, by combining proteomics with Gene Ontology analysis, we comprehensively classified secreted proteins on both biological process and molecular function. Conclusions: In this phase 1/2 trial, our cumulative results suggest that 2 doses of allo-hBM-MSCDs combined with a wound dressing are a safe and effective treatment for grade 1 and 2 DFUs. [ABSTRACT FROM AUTHOR]

Summary: Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize. The recombinant precursor of this protein, pre-EF-Tu, has been found to exhibit chaperone activity and protect heat-labile proteins, such as citrate synthase and malate dehydrogenase, from thermal aggregation. Chloroplast EF-Tu is highly conserved and it is possible that the chaperone activity of this protein is not species-specific. In this study, we investigated the effect of native wheat pre-EF-Tu on thermal aggregation of rubisco activase. Additionally, we investigated the effect of native and recombinant maize pre-EF-Tu on activase aggregation. Activase was chosen because it displays an exceptional sensitivity to thermal aggregation and constrains photosynthesis at high temperature. The native precursors of both wheat and maize EF-Tu displayed chaperone activity, as shown by the capacity of both proteins to reduce thermal aggregation of rubisco activase in vitro. Similarly, the recombinant maize pre-EF-Tu protected activase from thermal aggregation. This is the first report on chaperone activity of native pre-EF-Tu and the first evidence for thermal protection of a photosynthetic enzyme by this putative chaperone. The results are consistent with the hypothesis that chloroplast EF-Tu plays a functional role in heat tolerance by acting as a molecular chaperone. [Copyright &y& Elsevier]

GABA B receptors (GABA B Rs) are heterodimeric seven-transmembrane receptors that interact with a range of proteins and form large protein complexes on cholesterol-rich membrane microdomains. As the brain ages, membrane cholesterol levels exhibit alterations, although it remains unclear how these changes impact protein-protein interactions and downstream signaling. Herein, we studied the structural bases for the interaction between GABA B R and the KCC2 transporter, including their protein expression and distribution, and we compared data between young and aged rat cerebella. Also, we analyzed lipid profiles for both groups, and we used molecular dynamics simulations on three plasma membrane systems with different cholesterol concentrations, to further explore the GABA B R-transporter interaction. Based on our results, we report that a significant decrease in GABA B2 subunit expression occurs in the aged rat cerebella. After performing a comparative co-immunoprecipitation analysis, we confirm that GABA B R and KCC2 form a protein complex in adult and aged rat cerebella, although their interaction levels are reduced substantially as the cerebellum ages. On the other hand, our lipid analyses reveal a significant increase in cholesterol and sphingomyelin levels of the aged cerebella. Finally, we used the Martini coarse-grained model to conduct molecular dynamics simulations, from which we observed that membrane cholesterol concentrations can dictate whether the GABA B R tail domains physically establish G protein-independent contacts with a transporter, and the timing when those associations eventually occur. Taken together, our findings illustrate how age-related alterations in membrane cholesterol levels affect protein-protein interactions, and how they could play a crucial role in regulating GABA B R's interactome-mediated signaling., Significance Statement: This study elucidates age-related changes in cerebellar GABA B receptors (GABA B Rs), KCC2, and plasma membrane lipids, shedding light on mechanisms underlying neurological decline. Molecular dynamics simulations reveal how membrane lipids influence protein-protein interactions, offering insights into age-related neurodegeneration. The findings underscore the broader impact of cerebellar aging on motor functions, cognition, and emotional processing in the elderly. By elucidating plasma membrane regulation and GABAergic dynamics, this research lays the groundwork for understanding aging-related neurological disorders and inspires further investigation into therapeutic interventions., Competing Interests: Competing Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Proteomics-based bottoms-up, at a big scale applied to the protein identification and relative quantification present in complex mixtures (cell lysates, tissues, biological fluids, secretome, etc.) is a useful strategy to identify proteins and analyze their changes. Samples processed through a gel-free approach provide a simple method for protein separation and profile comparison of different conditions, such as using fewer steps in the protocol, reducing excessive sample handling, and covering an extended range of molecular weights and isoelectric points. However, it presents a great limitation related to the management of large dynamic ranges of proteins. There are numerous protocols that allow handling the problem or limitations generated by a high dynamic range of the proteins present in the sample. The Gel-LC technique is a complementary alternative of the gel-free approach available to solve the issue of protein samples with a high dynamic range. The different steps of the protocol involve sample processing through Gel-LC (1D-SDS-PAGE) prior to digestion, 1D-nanoUHPLC coupled to high-resolution/mass accuracy tandem mass spectrometry analysis (1D-nanoUHPLC-HR/MA-MS /MS analysis) and afterward, the protein identification and relative quantification analysis using bioinformatics tools for the data conversion, organization, and interpretation., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)