Protein Metabolic Pathways Research Articles

Review of Alzheimer's disease and treatments

Alzheimer's disease (AD) is a neurodegenerative disease that primarily affects older adults and is characterized by progressive memory loss and cognitive dysfunction. The disease is the most common type of dementia worldwide and imposes a significant burden on patients, families and society. More than 50 million people worldwide are affected by Alzheimer's disease, placing enormous financial strain on healthcare systems and families. The main molecular metabolic pathways of Alzheimer's disease include amyloid beta protein metabolic pathway and tau protein phosphorylation pathway. Abnormal accumulation of amyloid beta protein in the brain to form plaques, and excessive phosphorylation of tau protein to form neurofibrillary tangles, these pathological changes lead to nerve cell damage and death, resulting in the gradual loss of cognitive function. Current treatments can only slow the progression of the disease or alleviate symptoms and cannot completely cure AD. Finding effective treatment and intervention strategies can not only significantly improve the quality of life of Alzheimer's patients, but also reduce the burden of the disease on society and families. This article will review the background and epidemiology of Alzheimer's disease, discuss its main molecular metabolic pathways in detail, and review the current status of treatment, with a view to providing references for future research and treatment strategies.

Deciphering Rickettsia conorii metabolic pathways: a treasure map to therapeutic targets

Indian tick typhus is an infectious disease caused by intracellular gram-negative bacteria Rickettsia conorii (R. conorii). The bacterium is transmitted to humans through bite of infected ticks and sometimes by lice, fleas or mites. The disease is restricted to some areas with few cases but in last decade it is re-emerging with large number of cases from different areas of India. The insight in to genetic makeup of bacterial pathogens can be derived from their metabolic pathways. In the current study 18 metabolic pathways were found to be unique to the pathogen (R. conorii). A comprehensive analysis revealed 163 proteins implicated in 18 unique metabolic pathways of R. conorii. 140 proteins were reported to be essential for the bacterial survival, 46 were found virulent and 10 were found involved in resistance which can enhance the bacterial pathogenesis. The functional analysis of unique metabolic pathway proteins showed the abundance of plasmid conjugal transfer TrbL/VirB6, aliphatic acid kinase short chain, signal transduction response regulator receiver and components of type IV transporter system domains. The proteins were classified into six broad categories on the basis of predicted domains, i.e., metabolism, transport, gene expression and regulation, antimicrobial resistance, cell signalling and proteolysis. Further, in silico analysis showed that 88 proteins were suitable therapeutic targets which do not showed homology with host proteins. The 43 proteins showed hits with the DrugBank database showing their druggable nature and remaining 45 proteins were classified as novel drug targets that require further validation. The study will help to provide the better understanding of pathogens survival and embark on the development of successful therapies for the management of Indian tick typhus.

Expression of nuclear receptors and glucose metabolic pathway proteins in sebaceous carcinoma: Androgen receptor and monocarboxylate transporter 1 have a key role in disease progression.

Standard systemic treatments are not consistently effective for treating unresectable or advanced sebaceous carcinoma (SC). The present study investigated the pathogenic roles of nuclear receptors (NRs), glucose metabolic dysregulation and immune checkpoint proteins in SC as prognostic markers or therapeutic targets. Patients with pathologically confirmed SC between January 2002 and December 2019 at three university hospitals in South Korea were included in the present study. Immunohistochemistry was performed on paraffin-embedded tumor tissues for glucocorticoid receptors (GR), androgen receptors (AR), estrogen receptors (ER), progesterone receptors (PR), glucose transporter 1 (GLUT1), monocarboxylate transporters (MCT1 and MCT4), CD147, phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) and the immune checkpoint protein, programmed cell death-ligand 1 (PD-L1). The results were semi-quantitatively assessed and the associations of these proteins with various clinicopathological parameters were determined. A total of 39 cases of SC comprising 19 periocular and 20 extraocular tumors were enrolled. NRs were frequently detected in the tumor nuclei, with GR having the highest frequency (89.7%), followed by AR, ER (both 51.3%) and PR (41.0%). Regarding glucose metabolism, CD147, GLUT1 and MCT1 were highly expressed at 100, 89.7 and 87.2%, respectively, whereas MCT4 and pAMPK expression levels were relatively low at 38.5 and 35.9%, respectively. Membranous expression of PD-L1 was detected in five cases (12.8%), four of which were extraocular. In the multivariate analysis, advanced stage, low AR positivity and high MCT1 expression were independent poor prognostic factors for metastasis-free survival (all P<0.05). The present results suggested that hormonal and metabolic dysregulation may be associated with the pathogenesis of SC, and that AR and MCT1 in particular may serve as prognostic indicators and potential therapeutic targets. Additionally, ~10% of SC cases exhibited PD-L1 expression within the druggable range, and these patients are expected to benefit from treatment with immune checkpoint inhibitors.

Human hnRNPA1 reorganizes telomere-bound replication protein A.

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection.

Antifungal Drug Resistance in Candida albicans: Identifying Novel Targets for the Development of Effective Antifungal Agents.

Candidiasis, a fungal infection initiated primarily through Candida albicans, affects various portions of the human body and is particularly prevalent in immunocompromised individuals. The pathogenicity of C. albicans is facilitated by numerous virulence causes, including adhesins, morphogenesis, and phenotypic switching. The organism’s capability in the direction of switching between yeast and hyphal forms contributes to the severity of infections. The appearance of resistant strains has rendered current treatments less effective, necessitating the exploration of new drug targets and the progress of novel antifungal agents. Antifungal drug resistance is a multifaceted phenomenon involving genetic mutations, overexpression of efflux pumps, epigenetic changes, and biofilm formation, all regulated by complex genetic and transcriptional networks. These resistance mechanisms can cause treatment failures, highlighting the need for new antifungal agents and improved diagnostic tools. The identification of potential drug targets in C. albicans is critical due to increasing resistance to existing antifungal agents. Recent studies have identified promising targets, for example the riboflavin metabolic pathway and unique protein kinases involved in regulating virulence and pathogenicity. Developing new antifungals is difficult due to C. albicans’ eukaryotic nature and resistance. Ongoing research is essential to find novel targets and strategies, especially with the limited antifungal drug classes available.

Abstract C043: Hyperinsulinemia promotes pancreatic cancer progression by altering tumor metabolism

Abstract Metabolic diseases, such as type 2 diabetes (T2D), insulin resistance, and obesity, often accompany pancreatic ductal adenocarcinoma (PDAC), and they are associated with reduced survival. Hyperinsulinemia is a common hallmark symptom shared by those disorders and is independently associated with reduced survival of PDAC patients. While it has been established that endogenous hyperinsulinemia accelerates PDAC initiation by promoting the formation of KRAS-driven pre-cancerous lesions, its role in the progression of established tumors remains poorly understood. This represents a major knowledge gap in our understanding of whether insulin needs to be monitored and controlled during PDAC treatment, as it is not typically considered in current treatment paradigms. We hypothesized that hyperinsulinemia promotes the progression of PDAC tumors. Using patient-derived PDAC organoids (PDOs) and a mouse model of metabolic disorders, we found that insulin may promote PDOs’ growth by reprogramming tumor metabolism. A High-fat diet (HFD) treatment that induces hyperinsulinemia, but not hyperglycemia in mice accelerated the growth of PDO-derived orthotopic xenografts. Importantly, the fasting insulin level showed a significant positive correlation with the endpoint tumor volume, suggesting that endogenous insulin may positively contribute to PDOs’ growth in vivo. To assess the direct effect of insulin on the growth and molecular profile of PDOs, we treated an early passage PDO line with different concentrations of insulin and glucose at physiologically relevant levels. We observed a ∼1.2-fold increase in cell number after 7 days of culture in high insulin (10 nM) conditions with high (15 mM) or physiological (6 mM) levels of glucose, relative to the low insulin control. Phosphoproteomic analysis demonstrated significantly greater PI3K/AKT/mTOR, but not RAF/MEK/ERK, activity under high insulin with high or physiological levels of glucose compared to the control, directing the investigation toward the metabolic effects of insulin. Consistently, unbiased proteomics profiling revealed that metabolic proteins were significantly enriched in both high insulin conditions, and interestingly insulin enriched different sets of proteins in various metabolic pathways depending on glucose levels. Together, our preliminary results suggest that hyperinsulinemia may enhance the growth and progression of PDAC by altering tumor metabolism to support cell growth. Further experiments are needed to functionally link the insulin-mediated metabolic changes with increased PDO growth, as well as testing the direct effect of endogenous insulin on PDAC cells in vivo. Citation Format: Jeffrey Lin, James D Johnson, David Schaeffer, Vincent Richard, Christoph Borchers, Janel L Kopp. Hyperinsulinemia promotes pancreatic cancer progression by altering tumor metabolism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C043.

Activated AMP-protein kinase (pAMPK) is overexpressed in human somatotroph pituitary adenomas

IntroductionAMPK (AMP-activated protein kinase) is an enzyme that acts as a metabolic sensor and regulates multiple pathways via phosphorylating proteins in metabolic and proliferative pathways. The aim of this work was to study the activated cellular AMPK (phosphorylated-AMPK at Thr172, pAMPK) levels in pituitary tumor samples from patients with sporadic and familial acromegaly, as well as in samples from normal human pituitary gland. MethodsWe studied pituitary adenoma tissue from patients with sporadic somatotroph adenomas, familial acromegaly with heterozygote germline variants in the aryl hydrocarbon receptor interacting protein (AIP) gene (p.Q164*, p.R304* and p.F269_H275dup) and autopsy from normal pituitary glands without structural alterations. ResultsCellular levels of pAMPK were significantly higher in patients with sporadic acromegaly compared to normal pituitary glands (p < 0.0001). Tissues samples from patients with germline AIP mutations also showed higher cellular levels of pAMPK compared to normal pituitary glands. We did not observe a significant difference in cellular levels of pAMPK according to the cytokeratin (CAM5.2) pattern (sparsely or densely granulated) for tumor samples of sporadic acromegaly. ConclusionOur data show, for the first time in human cells, an increase of cellular levels of pAMPK in sporadic somatotropinomas, regardless of cytokeratin pattern, as well as in GH-secreting adenomas from patients with germline AIP mutations.

Nucleotide metabolism-related host proteins RNA polymerase II subunit and uridine phosphorylase 1 interacting with porcine epidemic diarrhea virus N proteins affect viral replication.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that targets pig intestines to cause disease. It is globally widespread and causes huge economic losses to the pig industry. PEDV N protein is the protein that constitutes the core of PEDV virus particles, and most of it is expressed in the cytoplasm, and a small part can also be expressed in the nucleus. However, the role of related proteins in host nucleotide metabolic pathways in regulating PEDV replication have not been fully elucidated. In this study, PEDV-N-labeled antibodies were co-immunoprecipitated and combined with LC-MS to screen for host proteins that interact with N proteins. Bioinformatics analyses showed that the selected host proteins were mainly enriched in metabolic pathways. Moreover, co-immunoprecipitation and confocal microscopy confirmed that the second-largest subunit of RNA polymerase II (RPB2) and uridine phosphorylase 1 (UPP1) interacted with the N protein. RPB2 is the main subunit of RNA polymerase II and plays an important role in eukaryotic transcription. UPP1 is an enzyme that catalyzes reversible phosphorylation of uridine to uracil and ribo-1-phosphate to promote catabolism and bio anabolism. RPB2 overexpression significantly promoted viral replication, whereas UPP1 overexpression significantly inhibited viral replication. Studies on interactions between the PEDV N and host proteins are helpful in elucidating the pathogenesis and immune escape mechanism of PEDV.

Organic matter degradation in the deep, sulfidic waters of the Black Sea: insights into the ecophysiology of novel anaerobic bacteria

BackgroundRecent studies have reported the identity and functions of key anaerobes involved in the degradation of organic matter (OM) in deep (> 1000 m) sulfidic marine habitats. However, due to the lack of available isolates, detailed investigation of their physiology has been precluded. In this study, we cultivated and characterized the ecophysiology of a wide range of novel anaerobes potentially involved in OM degradation in deep (2000 m depth) sulfidic waters of the Black Sea.ResultsWe have successfully cultivated a diverse group of novel anaerobes belonging to various phyla, including Fusobacteriota (strain S5), Bacillota (strains A1T and A2), Spirochaetota (strains M1T, M2, and S2), Bacteroidota (strains B1T, B2, S6, L6, SYP, and M2P), Cloacimonadota (Cloa-SY6), Planctomycetota (Plnct-SY6), Mycoplasmatota (Izemo-BS), Chloroflexota (Chflx-SY6), and Desulfobacterota (strains S3T and S3-i). These microorganisms were able to grow at an elevated hydrostatic pressure of up to 50 MPa.Moreover, this study revealed that different anaerobes were specialized in degrading specific types of OM. Strains affiliated with the phyla Fusobacteriota, Bacillota, Planctomycetota, and Mycoplasmatota were found to be specialized in the degradation of cellulose, cellobiose, chitin, and DNA, respectively, while strains affiliated with Spirochaetota, Bacteroidota, Cloacimonadota, and Chloroflexota preferred to ferment less complex forms of OM. We also identified members of the phylum Desulfobacterota as terminal oxidizers, potentially involved in the consumption of hydrogen produced during fermentation. These results were supported by the identification of genes in the (meta)genomes of the cultivated microbial taxa which encode proteins of specific metabolic pathways. Additionally, we analyzed the composition of membrane lipids of selected taxa, which could be critical for their survival in the harsh environment of the deep sulfidic waters and could potentially be used as biosignatures for these strains in the sulfidic waters of the Black Sea.ConclusionsThis is the first report that demonstrates the cultivation and ecophysiology of such a diverse group of microorganisms from any sulfidic marine habitat. Collectively, this study provides a step forward in our understanding of the microbes thriving in the extreme conditions of the deep sulfidic waters of the Black Sea.Exyq52VguUrmavAnxe2qURVideo

Estrogen Regulation of PAD2 and Citrullination in the Aging Female Heart

Advanced age is the primary risk factor for heart failure, an enormous public health burden. With aging, men tend to develop systolic dysfunction whereas women are predisposed to develop diastolic dysfunction. Despite these known phenotype differences, underlying molecular mechanisms of sex differences in cardiac aging are unknown. Citrullination, a post-translational modification catalyzed by the peptidylarginine deiminases (PAD) enzyme family, is positively regulated by the sex hormone estrogen (E2) suggesting a potential novel mechanism of age-related cardiac functional changes in women. We hypothesized that PAD expression and citrullination are sexually dimorphic with aging such that as E2 levels decline, so too does citrullination, thus contributing to diastolic dysfunction. We quantified expression of PAD2, the primary cardiac isoform, in female young reproductively competent adult (3 months) and aged (21 months) C57Bl6 mice. Consistent with clinical reports, in our model of aging, female mice developed diastolic dysfunction, with a prolonged linear phase relaxation (54.0 ± 1.7 msec, adult and 66.0 ± 3.3 msec aged, p&lt;0.05). PAD2 expression decreased with age in the female heart, concomitant with decreases in myocardial protein citrullination. To identify potential mechanisms of differential citrullination by age, we performed cit-mass spectrometry. Adult female mice had higher overall citrullination patterns than aged females with enrichment of citrullinated proteins in metabolic, mitochondrial, and sarcomeric pathways. In reproductively competent adult females, cardiac PAD2 expression was higher during estrus compared to diestrus. To confirm direct regulation of PAD2 by E2, a cohort of adult mice underwent ovariectomy (OVX) with or without E2 replacement, provided orally in the water (0.5μM E2) for three weeks. Exogenous E2 supplementation post-OVX increased uterine weight and PAD2 expression, further confirming positive regulation by E2. Together, we suggest that hormonally regulated changes in PAD2 and citrullination with age contribute to sex-specific cardiac aging. Ongoing experiments will directly test the role of PAD2 in the heart by generation of a novel myocyte specific PAD2 deleted mouse across the female lifespan. This abstract was made possible by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under 2P20GM103432. This work was supported by the National Institutes of Health [K01 AG058810 DRB, [K01 AG066845 KCW]. This is the full abstract presented at the American Physiology Summit 2024 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.

A Mitochondrial Localized Chaperone Regulator OsBAG6 Functions in Saline-Alkaline Stress Tolerance in Rice

B-cell lymphoma 2 (Bcl-2)-associated athanogene (BAG) family genes play prominent roles in regulating plant growth, development, and stress response. Although the molecular mechanism underlying BAG’s response to abiotic stress has been studied in Arabidopsis, the function of OsBAG underlying saline-alkaline stress tolerance in rice remains unclear. In this study, OsBAG6, a chaperone regulator localized to mitochondria, was identified as a novel negative regulator of saline-alkaline stress tolerance in rice. The expression level of OsBAG6 was induced by high concentration of salt, high pH, heat and abscisic acid treatments. Overexpression of OsBAG6 in rice resulted in significantly reduced plant heights, grain size, grain weight, as well as higher sensitivity to saline-alkaline stress. By contrast, the osbag6 loss-of-function mutants exhibited decreased sensitivity to saline-alkaline stress. The transcriptomic analysis uncovered differentially expressed genes related to the function of “response to oxidative stress”, “defense response”, and “secondary metabolite biosynthetic process” in the shoots and roots of OsBAG6-overexpressing transgenic lines. Furthermore, cytoplasmic levels of Ca2+ increase rapidly in plants exposed to saline-alkaline stress. OsBAG6 bound to calcium sensor OsCaM1-1 under normal conditions, which was identified by comparative interactomics, but not in the presence of elevated Ca2+. Released OsCaM1-1 saturated with Ca2+ is then able to regulate downstream stress-responsive genes as part of the response to saline-alkaline stress. OsBAG6 also interacted with energy biosynthesis and metabolic pathway proteins that are involved in plant growth and saline-alkaline stress response mechanisms. This study reveals a novel function for mitochondrial localized OsBAG6 proteins in the saline-alkaline stress response alongside OsCaM1-1.

Unbalanced redox status network as an early pathological even in congenital cataracts

Aims/Purpose: The lens proteome undergoes dramatic composition changes during development and maturation. Defective developmental processes lead to congenital cataracts that account for about 30% of cases of childhood blindness. Gene mutations are associated with approximately 50% of early‐onset forms of lens opacity, with the remainder being of unknown aetiology. To gain a better understanding of congenital cataractogenesis, we utilized a transgenic mouse model expressing a mutant ubiquitin protein in the lens (K6W‐Ub) that recapitulates many of the early pathological changes seen in human congenital cataracts.Methods: We performed MS‐based tandem‐mass‐tag quantitative proteomics (TMT), computational molecular phenotyping (CMP), high‐performance liquid chromatography (HPLC), western blotting and immunohistochemical analysis in E15, P1 and P30 control or K6W‐Ub lenses.Results: TMT identified proteins that were altered during normal lens differentiation, some of which were also altered in cataractous lenses. These included proteins in glutathione and amino acid metabolic pathways. CMP revealed that glutathione and taurine were reduced spatially altered in the K6W‐Ub cataractous lens. HPLC revealed that both the ratio GSH/GSSG and taurine, two indicators of redox status, were differentially compromised in K6W‐Ub cataractous lenses.Conclusions: Our findings shed light on the molecular mechanisms associated with congenital cataracts and point out that unbalanced redox status due to reduced levels of taurine and glutathione, metabolites already linked to age‐related cataract, could be a major underlying mechanism behind lens opacities that appear early in life.Funding: Funding was provided by grants RYC 2018‐024434‐I, MINECO PID 2020‐119466RB‐I00, FUSP‐PPC‐19‐B53C4C64, NIH RO1EY026979 (to AT), USDA 8050‐51000‐089‐01S (to AT).

The Molecular Frequency, Conservation and Role of Reactive Cysteines in Plant Lipid Metabolism.

Cysteines (Cys) are chemically reactive amino acids containing sulfur that play diverse roles in plant biology. Recent proteomics investigations in Arabidopsis thaliana have revealed the presence of thiol post-translational modifications (PTMs) in several Cys residues. These PTMs are presumed to impact protein structure and function, yet mechanistic data regarding the specific Cys susceptible to modification and their biochemical relevance remain limited. To help address these limitations, we have conducted a wide-ranging analysis by integrating published datasets encompassing PTM proteomics (comparing S-sulfenylation, persulfidation, S-nitrosylation and S-acylation), genomics and protein structures, with a specific focus on proteins involved in plant lipid metabolism. The prevalence and distribution of modified Cys residues across all analyzed proteins is diverse and multifaceted. Nevertheless, by combining an evaluation of sequence conservation across 100+ plant genomes with AlphaFold-generated protein structures and physicochemical predictions, we have unveiled structural propensities associated with Cys modifications. Furthermore, we have identified discernible patterns in lipid biochemical pathways enriched with Cys PTMs, notably involving beta-oxidation, jasmonic acid biosynthesis, fatty acid biosynthesis and wax biosynthesis. These collective findings provide valuable insights for future investigations targeting the mechanistic foundations of Cys modifications and the regulation of modified proteins in lipid metabolism and other metabolic pathways.

FDX1 Is Required for the Biogenesis of Mitochondrial Cytochrome c Oxidase in Mammalian Cells

Ferredoxins (FDXs) are evolutionarily conserved iron-sulfur (Fe-S) proteins that function as electron transfer proteins in diverse metabolic pathways. Mammalian mitochondria contain two ferredoxins, FDX1 and FDX2, which share a high degree of structural similarity but exhibit different functionalities. Previous studies have established the unique role of FDX2 in the biogenesis of Fe-S clusters; however, FDX1 seems to have multiple targets in vivo, some of which are only recently emerging. Using CRISPR-Cas9-based loss-of-function studies in rat cardiomyocyte cell line, we demonstrate an essential requirement of FDX1 in mitochondrial respiration and energy production. We attribute reduced mitochondrial respiration to a specific decrease in the abundance and assembly of cytochrome c oxidase (CcO), a mitochondrial heme-copper oxidase and the terminal enzyme of the mitochondrial respiratory chain. FDX1 knockout cells have reduced levels of copper and heme a/a3, factors that are essential for the maturation of the CcO enzyme complex. Copper supplementation failed to rescue CcO biogenesis, but overexpression of heme a synthase, COX15, partially rescued COX1 abundance in FDX1 knockout cells. This finding links FDX1 function to heme a biosynthesis, and places it upstream of COX15 in CcO biogenesis like its ancestral yeast homolog. Taken together, our work has identified FDX1 as a critical CcO biogenesis factor in mammalian cells.

The regulatory effect of Angelicae Sinensis Radix on neuroendocrine-immune network and sphingolipid metabolism in CUMS-induced model of depression

Ethnopharmacological relevanceConventional antidepressants therapy remains unsatisfactory due to the disadvantages of delayed clinical onset of action and side effects. Traditional Chinese Medicine (TCM) with good efficacy and higher safety have received much attention. Angelicae Sinensis Radix (AS), a well-known TCM, has been proved to exhibit the efficacy of antidepression recently. Aim of the studyThe purpose of this study was to investigate the potential anti-depressant mechanisms of AS based on chronic unpredictable mild stress (CUMS) rat model. Materials and methodsIn this study, behavioral experiments, molecular biology techniques, and ultra performance liquid chromatography-triple-time of flight mass spectrometer (UPLC-Triple-TOF/MS) were combined to explore the potential antidepressant mechanisms of AS based on CUMS rat model. ResultsThe results demonstrated that AS could reduce the contents of serum hypothalamic-pituitary-adrenal (HPA) axis hormones in CUMS rats, including corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol (CORT). In addition, AS regulated the percentage of CD4+ T lymphocytes, the ratio of CD4+/CD8+, and the levels of serum cytokines such as IL-1β, IL-4, IL-6, and TNF-α in CUMS rats. Lipidomics showed that 31 lipids were related to depression and AS could regulate the lipid metabolism alteration induced by CUMS, particularly sphingolipid metabolism. Finally, the key proteins in sphingolipid metabolic pathways in hippocampus of CUMS rats could be back-regulated by AS, including serine palmitoyl transferase (SPTLC2), ceramide synthase (CerS2), sphingomyelinase (SPHK1), and neutral sphingomyelinase (nSMase). ConclusionAS could alleviate NEI network disorder and restore the levels of sphingolipid metabolites and key proteins in CUMS rats. The underlying mechanism by which AS relieved depression-like behavior in CUMS rats may be through modulation of NEI and disturbances in sphingolipid metabolism.

Sexual Dimorphism of the Mouse Plasma Metabolome Is Associated with Phenotypes of 30 Gene Knockout Lines.

Although metabolic alterations are observed in many monogenic and complex genetic disorders, the impact of most mammalian genes on cellular metabolism remains unknown. Understanding the effect of mouse gene dysfunction on metabolism can inform the functions of their human orthologues. We investigated the effect of loss-of-function mutations in 30 unique gene knockout (KO) lines on plasma metabolites, including genes coding for structural proteins (11 of 30), metabolic pathway enzymes (12 of 30) and protein kinases (7 of 30). Steroids, bile acids, oxylipins, primary metabolites, biogenic amines and complex lipids were analyzed with dedicated mass spectrometry platforms, yielding 827 identified metabolites in male and female KO mice and wildtype (WT) controls. Twenty-two percent of 23,698 KO versus WT comparison tests showed significant genotype effects on plasma metabolites. Fifty-six percent of identified metabolites were significantly different between the sexes in WT mice. Many of these metabolites were also found to have sexually dimorphic changes in KO lines. We used plasma metabolites to complement phenotype information exemplified for Dhfr, Idh1, Mfap4, Nek2, Npc2, Phyh and Sra1. The association of plasma metabolites with IMPC phenotypes showed dramatic sexual dimorphism in wildtype mice. We demonstrate how to link metabolomics to genotypes and (disease) phenotypes. Sex must be considered as critical factor in the biological interpretation of gene functions.

In vivo Polycystin-1 interactome using a novel Pkd1 knock-in mouse model.

PKD1 is the most commonly mutated gene causing autosomal dominant polycystic kidney disease (ADPKD). It encodes Polycystin-1 (PC1), a putative membrane protein that undergoes a set of incompletely characterized post-transcriptional cleavage steps and has been reported to localize in multiple subcellular locations, including the primary cilium and mitochondria. However, direct visualization of PC1 and detailed characterization of its binding partners remain challenging. We now report a new mouse model with HA epitopes and eGFP knocked-in frame into the endogenous mouse Pkd1 gene by CRISPR/Cas9. Using this model, we sought to visualize endogenous PC1-eGFP and performed affinity-purification mass spectrometry (AP-MS) and network analyses. We show that the modified Pkd1 allele is fully functional but the eGFP-tagged protein cannot be detected without signal amplification by secondary antibodies. Using nanobody-coupled beads and large quantities of tissue, AP-MS identified an in vivo PC1 interactome, which is enriched for mitochondrial proteins and components of metabolic pathways. These studies suggest this mouse model and interactome data will be useful to understand PC1 function, but that new methods and brighter tags will be required to track endogenous PC1.

Proteomics-based analysis of the stress response of Bacillus cereus spores under ultrasound and electrolyzed water treatment

Ultrasound is a green nonthermal technology with promising applications in microbial inactivation. Electrolyzed water has been investigated and found to have a synergistic inactivation effect of ultrasound on spores. This study used a data-independent-acquisition method to analyze the stress response of Bacillus cereus spores following ultrasound combined with electrolyzed water treatment. We identified 197 differentially expressed proteins under ultrasound combined with an electrolyzed water treatment for which the ratio in the metabolic pathway was the highest. Spores downregulated key proteins in energy metabolic and transportation pathways, in particular in phosphotransferase systems and ATP synthase under ultrasound, electrolyzed water, and combined stress. The results of this study revealed that the key proteins in intracellular metabolism decreased after ultrasound treatment, and the expression of small acid-soluble spore protein and cell wall biosynthesis protein increased. Meanwhile, DNA integration, recombination, and inversion protein and small acid-soluble spore protein were upregulated after electrolyzed water treatment. In general, the spores exhibited stress resistance under external stress. The inactivation of spores by further stress was reduced, which we called “cross-protection.”

Insights concerning advancing the agroecological sustainability of salinity tolerance through proteomics profiling of hexaploid wheat (Triticum aestivum L.)

Wheat plays a significant role in the provision of food and nutrition. However, rapid soil salinization poses a severe threat to its production worldwide. Salt stress stunts wheat growth and quality, resulting in low grain yields. The adaptation of wheat to salinity involves complex physio-biochemical and molecular mechanisms. This study aimed to identify the genetic approaches concerning salt stress-responsive proteins and protein pathways in wheat roots under controlled (0 mM) and NaCl stress (250 mM) solution using label-free proteomic quantification analysis. We found a significant accumulation of Na+ in the leaf and root compared with the controlled condition. Besides, we identified 2436 proteins enhanced under salt stress, with 198 differentially abundant proteins (DAPs), including 170 up-regulated and 28 down-regulated proteins. Many of these proteins were involved in salinity tolerance, including heat shock proteins, glutathione S-transferase, dehydrin, peroxidase, potassium channel beta subunit-type H+-ATPase, superoxide dismutase (Cu-Zn), 14-3-3 protein, peroxidase, malate dehydrogenase, and heat shock proteins. The abundance of a V-type H+ ATPase and 14-3-3 protein was also enhanced, facilitating Na+ compartmentalization in the vacuole through the salt overly sensitive (SOS) pathway. Additionally, many antioxidant enzymes, including peroxidase, glutathione-S-transferase, and thioredoxins, were up-regulated, playing a vital role in detoxifying reactive oxygen species (ROS) in salinity-stressed wheat roots. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses showed that salinity stress enhances the abundance of proteins in several metabolic pathways, such as the citrate cycle, ribosome, oxidative phosphorylation, glycolysis, carbon metabolism, and cytoplasm. We anticipate that the identified proteins under salinity conditions will provide us with a deeper understanding of their application in agriculture biotechnology.

The involvement of NLRP3 inflammasome in CUMS-induced AD-like pathological changes and related cognitive decline in mice

BackgroundNumerous studies have found that inhibiting the expression of NLRP3 inflammasome can significantly improve depressive-like behaviors in mice, but the research on its effect on cognitive decline in depression and its mechanism is still lacking. This study aimed to elucidate the role of NLRP3 inflammasome in cognitive decline in depression and explore the common neuro-immunological mechanisms of depression and Alzheimer’s disease (AD).MethodsMale C57BL/6 mice were subjected to chronic unpredictable mild stress (CUMS) for 5 weeks, treatment group was administered with the NLRP3 inhibitor MCC950 (10 mg/kg, i.p.), fluoxetine served as positive control. Then, the mice were assessed for cognitive behaviors and depression-like behaviors, and changes of microglia and neurons in hippocampus and levels of Aβ metabolic pathway and tau protein were measured. To explore the mechanism of NLRP3 activation on neurons, we performed in vitro studies using BV2 microglia and mouse primary neurons. Furthermore, we focused on the role of NLRP3 inflammasome in the function of neurons and the expression of AD pathological indicators.ResultsCUMS induced depressive-like behaviors and cognitive decline in mice, which could be reversed by inhibiting NLRP3 inflammasome. MCC950, a specific NLRP3 inhibitor, alleviated CUMS-induced neuron injury and AD-like pathological changes, including the abnormal expression of Aβ metabolic pathway and the hyper-phosphorylation of tau protein. LPS (1 μg/mL) + ATP (1 mM) treatment activated the expression of NLRP3 inflammasome and IL-1β in vitro. In vitro experiment also proved that inhibiting the expression of NLRP3 inflammasome in microglia can restore the Aβ metabolic pathway to normal, decrease neuronal tau protein phosphorylation and protect neurons.ConclusionsInhibition of NLRP3 inflammasome effectively alleviated CUMS-induced depressive-like behaviors and cognitive decline in mice, and inhibited the activation of AD physiological indicators.