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SLAS Discovery : Advancing Life... Jun 2024DNA-encoded small molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, it has been used to identify...
DNA-encoded small molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, it has been used to identify ligands against targets that are soluble or overexpressed on cell surfaces. Here, we report applying cell-based selection methods to profile surfaces of mouse C2C12 myoblasts and myotube cells in an unbiased, target agnostic manner. A panel of on-DNA compounds were identified and confirmed for cell binding selectivity. We optimized the cell selection protocol and employed a novel data analysis method to identify cell selective ligands against a panel of human B and T lymphocytes. We discuss the generality of using this workflow for DNA encoded small molecule library selection and data analysis against different cell types, and the feasibility of applying this method to profile cell surfaces for biomarker and target identification.
PubMed: 38917882
DOI: 10.1016/j.slasd.2024.100171 -
Microbiology Spectrum Jun 2024Protein acetylation and deacetylation are key epigenetic modifications that regulate the initiation and development of several diseases. In the context of infection with...
UNLABELLED
Protein acetylation and deacetylation are key epigenetic modifications that regulate the initiation and development of several diseases. In the context of infection with (), these processes are essential for host-pathogen interactions and immune responses. However, the specific effects of acetylation and deacetylation on cellular functions during infection are not fully understood. This study employed Tandem Mass Tag (TMT) labeling for quantitative proteomic profiling to examine the acetylproteome (acetylome) profiles of noninfected and -infected macrophages. We identified 715 acetylated peptides from 1,072 proteins and quantified 544 lysine acetylation sites (Kac) in 402 proteins in noninfected and -infected macrophages. Our research revealed a link between acetylation events and metabolic changes during infection. Notably, the deacetylation of heat shock protein 60 (HSP60), a key chaperone protein, was significantly associated with this process. Specifically, the deacetylation of HSP60 at K96 by sirtuin3 (SIRT3) enhances macrophage apoptosis, leading to the elimination of intracellular . These findings underscore the pivotal role of the SIRT3-HSP60 axis in the host immune response to . This study offers a new perspective on host protein acetylation and suggests that targeting host-directed therapies could be a promising approach for tuberculosis immunotherapy.
IMPORTANCE
Protein acetylation is crucial for the onset, development, and outcome of tuberculosis (TB). Our study comprehensively investigated the dynamics of lysine acetylation during infection, shedding light on the intricate host-pathogen interactions that underlie the pathogenesis of tuberculosis. Using an advanced quantitative lysine proteomics approach, different profiles of acetylation sites and proteins in macrophages infected with were identified. Functional enrichment and protein-protein network analyses revealed significant associations between acetylated proteins and key cellular pathways, highlighting their critical role in the host response to infection. Furthermore, the deacetylation of HSP60 and its influence on macrophage-mediated clearance of underscore the functional significance of acetylation in tuberculosis pathogenesis. In conclusion, this study provides valuable insights into the regulatory mechanisms governing host immune responses to infection and offers promising avenues for developing novel therapeutic interventions against TB.
PubMed: 38916288
DOI: 10.1128/spectrum.00749-24 -
BioRxiv : the Preprint Server For... Jun 2024Proteome changes associated with APOE4 variant carriage that are independent of Alzheimer's disease (AD) pathology and diagnosis are unknown. This study investigated...
INTRODUCTION
Proteome changes associated with APOE4 variant carriage that are independent of Alzheimer's disease (AD) pathology and diagnosis are unknown. This study investigated APOE4 proteome changes in people with AD, mild cognitive impairment, and no impairment.
METHODS
Clinical, APOE genotype, and cerebrospinal fluid (CSF) proteome and AD biomarker data was sourced from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database. Proteome profiling was done using supervised machine learning.
RESULTS
We found an APOE4-specific proteome signature that was independent of cognitive diagnosis and AD pathological biomarkers, and increased risk of progression to cognitive impairment. Proteins were enriched in brain regions including the caudate and cortex and cells including endothelial cells, oligodendrocytes, and astrocytes. Enriched peripheral immune cells included T cells, macrophages, and B cells.
DISCUSSION
APOE4 carriers have a unique CSF proteome signature associated with a strong brain and peripheral immune and inflammatory phenotype that likely underlies APOE4 carriers' vulnerability to cognitive decline and AD.
PubMed: 38915547
DOI: 10.1101/2024.04.18.590160 -
BioRxiv : the Preprint Server For... Jun 2024Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information...
Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information flow into and out of genetically modified organisms while (iii) allowing the biosynthesis of genetically encoded unnatural polymers . Achieving these three goals requires the reassignment of multiple of the 64 codons nature uses to encode proteins. However, synonymous codon replacement-recoding-is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using whole-genome synthesis, multiplexed directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple recoded genomes. Using this information, we assemble a synthetic genome in seven sections using only 57 codons to encode proteins. By discovering the rules responsible for the lethality of synonymous recoding and developing a data-driven multi-omics-based genome construction workflow that troubleshoots synthetic genomes, we overcome the lethal effects of 62,007 synonymous codon swaps and 11,108 additional genomic edits. We show that synonymous recoding induces transcriptional noise including new antisense RNAs, leading to drastic transcriptome and proteome perturbation. As the elimination of select codons from an organism's genetic code results in the widespread appearance of cryptic promoters, we show that synonymous codon choice may naturally evolve to minimize transcriptional noise. Our work provides the first genome-scale description of how synonymous codon changes influence organismal fitness and paves the way for the construction of functional genomes that provide genetic firewalls from natural ecosystems and safely produce biopolymers, drugs, and enzymes with an expanded chemistry.
PubMed: 38915524
DOI: 10.1101/2024.06.16.599206 -
BioRxiv : the Preprint Server For... Jun 2024Cellular functional pathways have evolved through selection based on fitness benefits conferred through protein intra- and inter-molecular interactions that comprise all...
Cellular functional pathways have evolved through selection based on fitness benefits conferred through protein intra- and inter-molecular interactions that comprise all protein conformational features and protein-protein interactions, collectively referred to as the interactome. While the interactome is regulated by proteome levels, it is also regulated independently by, post translational modification, co-factor, and ligand levels, as well as local protein environmental factors, such as osmolyte concentration, pH, ionic strength, temperature and others. In modern biomedical research, cultivatable cell lines have become an indispensable tool, with selection of optimal cell lines that exhibit specific functional profiles being critical for success in many cases. While it is clear that cell lines derived from different cell types have differential proteome levels, increased understanding of large-scale functional differences requires additional information beyond abundance level measurements, including how protein conformations and interactions are altered in certain cell types to shape functional landscapes. Here, we employed quantitative protein cross-linking coupled to mass spectrometry to probe large-scale protein conformational and interaction changes among three commonly employed human cell lines, HEK293, MCF-7, and HeLa cells. Isobaric quantitative Protein Interaction Reporter (iqPIR) technologies were used to obtain quantitative values of cross-linked peptides across three cell lines. These data illustrated highly reproducible (R values larger than 0.8 for all biological replicates) quantitative interactome levels across multiple biological replicates. We also measured protein abundance levels in these cells using data independent acquisition quantitative proteomics methods. Combining quantitative interactome and proteomics information allowed visualization of cell type- specific interactome changes mediated by proteome level adaptations as well as independently regulated interactome changes to gain deeper insight into possible drivers of these changes. Among the biggest detected alterations in protein interactions and conformations are changes in cytoskeletal proteins, RNA-binding proteins, chromatin remodeling complexes, mitochondrial proteins, and others. Overall, these data demonstrate the utility and reproducibility of quantitative cross-linking to study systems-level interactome variations. Moreover, these results illustrate how combined quantitative interactomics and proteomics can provide unique insight on cellular functional landscapes.
PubMed: 38915502
DOI: 10.1101/2024.06.12.598691 -
Scientific Reports Jun 2024Floating seedling cultivation technique is a novel seedling method in cotton and it provides an ideal model to study cotton growing under waterlogging stress....
Floating seedling cultivation technique is a novel seedling method in cotton and it provides an ideal model to study cotton growing under waterlogging stress. Morphological character and proteomic profile of the primary root from the seedling cultured by the new technology were evaluated in this study. Compared to seedlings cultured by the traditional method, the diameter of the taproot from floating technology is small at all five seedling stages from one-leaf stage to five-leaf stage. There are similar changes between the thickness of cortex and diameter of stele, which increased from the one- to the two-leaf stage but decreased from the two- to the five-leaf stage. At the one-leaf stage, the number and volume of mitochondria in the primary root-tip cells were less than those in the control. At the two-leaf stage, there was significantly less electron-dense material in the primary root-tip cells than those in the control group. From the one- to the two-leaf stage, the vacuole volume was significantly smaller than that in the control. Total 28 differentially expressed proteins were revealed from aquatic and control group roots of cotton seedlings at the three-leaf stage by two-dimensional electrophoresis, which included 24 up-regulated and four down-regulated proteins. The relative expression of the phosphoglycerate kinase (PGK) gene in aquatic roots increased from the one- to the four-leaf stage but declined rapidly from the four- to the five-leaf stage. The relative expression of the 14-3-3b gene tended to decrease from the one- to the five-leaf stage. The PGK and 14-3-3b genes were specifically expressed in the aquatic roots at the three-leaf stage. In brief, these changes induced waterlogging resistance in the aquatic roots of cotton seedlings in the floating nursery, thereby causing the roots to adapt to the aquatic environment, promoting the growth and development of cotton seedlings.
Topics: Gossypium; Proteomics; Plant Roots; Seedlings; Plant Proteins; Gene Expression Regulation, Plant; Stress, Physiological; Proteome
PubMed: 38914604
DOI: 10.1038/s41598-024-64322-y -
Scientific Reports Jun 2024In recent decades, the food system has been faced with the significant problem of increasing food waste. Therefore, the feed industry, supported by scientific research,...
In recent decades, the food system has been faced with the significant problem of increasing food waste. Therefore, the feed industry, supported by scientific research, is attempting to valorise the use of discarded biomass as co-products for the livestock sector, in line with EU objectives. In parallel, the search for functional products that can ensure animal health and performances is a common fundamental goal for both animal husbandry and feeding. In this context, camelina cake (CAMC), cardoon cake (CC) and cardoon meal (CM), due valuable nutritional profile, represent prospective alternatives. Therefore, the aim of this work was to investigate the antioxidant activity of CAMC, CC and CM following in vitro digestion using 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Total phenolic content (TPC) and angiotensin converting enzyme (ACE) inhibitory activity, actively involved in modulating antioxidant properties, were also studied. Further, a peptidomic analysis was adopted to substantiate the presence of bioactive peptides after in vitro digestion. The results obtained confirmed an interesting nutritional profile of CAMC, CC and CM and relevant antioxidant and ACE inhibitory activities. In particular, considering antioxidant profile, CM and CC revealed a significantly higher (10969.80 ± 18.93 mg TE/100 g and 10451.40 ± 149.17 mg TE/100 g, respectively; p < 0.05) ABTS value than CAMC (9511.18 ± 315.29 mg TE/100 g); a trend also confirmed with the FRAP assay (306.74 ± 5.68 mg FeSO/100 g; 272.84 ± 11.02 mg FeSO/100 g; 103.84 ± 3.27 mg FeSO/100 g, for CC, CM and CAMC, respectively). Similar results were obtained for TPC, demonstrating the involvement of phenols in modulating antioxidant activity. Finally, CAMC was found to have a higher ACE inhibitory activity (40.34 ± 10.11%) than the other matrices. Furthermore, potentially bioactive peptides associated with ACE inhibitory, anti-hypertensive, anti-cancer, antimicrobial, antiviral, antithrombotic, DPP-IV inhibitory and PEP-inhibitory activities were identified in CAMC. This profile was broader than that of CC and CM. The presence of such peptides corroborates the antioxidant and ACE profile of the sample. Although the data obtained report the important antioxidant profile of CAMC, CC, and CM and support their possible use, future investigations, particularly in vivo trials will be critical to evaluate and further investigate their effects on the health and performance of farm animals.
Topics: Antioxidants; Cynara; Brassicaceae; Angiotensin-Converting Enzyme Inhibitors; Phenols; Peptides; Animals; Plant Extracts; Animal Feed; Proteomics
PubMed: 38914602
DOI: 10.1038/s41598-024-64989-3 -
ELife Jun 2024Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Most cases of preterm birth occur spontaneously and result from preterm labor with...
BACKGROUND
Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Most cases of preterm birth occur spontaneously and result from preterm labor with intact (spontaneous preterm labor [sPTL]) or ruptured (preterm prelabor rupture of membranes [PPROM]) membranes. The prediction of spontaneous preterm birth (sPTB) remains underpowered due to its syndromic nature and the dearth of independent analyses of the vaginal host immune response. Thus, we conducted the largest longitudinal investigation targeting vaginal immune mediators, referred to herein as the immunoproteome, in a population at high risk for sPTB.
METHODS
Vaginal swabs were collected across gestation from pregnant women who ultimately underwent term birth, sPTL, or PPROM. Cytokines, chemokines, growth factors, and antimicrobial peptides in the samples were quantified via specific and sensitive immunoassays. Predictive models were constructed from immune mediator concentrations.
RESULTS
Throughout uncomplicated gestation, the vaginal immunoproteome harbors a cytokine network with a homeostatic profile. Yet, the vaginal immunoproteome is skewed toward a pro-inflammatory state in pregnant women who ultimately experience sPTL and PPROM. Such an inflammatory profile includes increased monocyte chemoattractants, cytokines indicative of macrophage and T-cell activation, and reduced antimicrobial proteins/peptides. The vaginal immunoproteome has improved predictive value over maternal characteristics alone for identifying women at risk for early (<34 weeks) sPTB.
CONCLUSIONS
The vaginal immunoproteome undergoes homeostatic changes throughout gestation and deviations from this shift are associated with sPTB. Furthermore, the vaginal immunoproteome can be leveraged as a potential biomarker for early sPTB, a subset of sPTB associated with extremely adverse neonatal outcomes.
FUNDING
This research was conducted by the Perinatology Research Branch, Division of Obstetrics and Maternal-Fetal Medicine, Division of Intramural Research, National Institute of Child Health and Human Development, National Institutes of Health, U.S. Department of Health and Human Services (NICHD/NIH/DHHS) under contract HHSN275201300006C. ALT, KRT, and NGL were supported by the Wayne State University Perinatal Initiative in Maternal, Perinatal and Child Health.
Topics: Humans; Female; Longitudinal Studies; Pregnancy; Vagina; Premature Birth; Adult; Retrospective Studies; Proteome; Cytokines; Fetal Membranes, Premature Rupture; Young Adult; Immunoproteins
PubMed: 38913421
DOI: 10.7554/eLife.90943 -
ACS Omega Jun 2024The study aimed to elucidate the significance of CLEC4G, CAMK2β, SLC22A1, CBFA2T3, and STAB2 in the prognosis of hepatocellular carcinoma (HCC) patients and their...
The study aimed to elucidate the significance of CLEC4G, CAMK2β, SLC22A1, CBFA2T3, and STAB2 in the prognosis of hepatocellular carcinoma (HCC) patients and their associated molecular biological characteristics. Additionally, the research sought to identify new potential biomarkers with therapeutic and diagnostic relevance for clinical applications. We utilized a publicly available high throughput phosphoproteomics and proteomics data set of HCC to focus on the analysis of 12 downregulated phosphoproteins in HCC. Our approach integrates bioinformatic analysis with pathway analysis, encompassing gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and the construction of a protein-protein interaction (PPI) network. In total, we quantified 11547 phosphorylation sites associated with 4043 phosphoproteins from a cohort of 159 HCC patients. Within this extensive data set, our specific focus was on 19 phosphorylation sites displaying significant downregulation (log FC ≤ -2 with -values < 0.0001). Remarkably, our investigation revealed distinct pathways exhibiting differential regulation across multiple dimensions, including the genomic, transcriptomic, proteomic, and phosphoproteomic levels. These pathways encompass a wide range of critical cellular processes, including cellular component organization, cell cycle control, signaling pathways, transcriptional and translational control, and metabolism. Furthermore, our bioinformatics analysis unveiled noteworthy insights into the subcellular localizations, biological processes, and molecular functions associated with these proteins and phosphoproteins. Within the context of the PPI network, we identified 12 key genes CLEC4G, STAB2, ADH1A, ADH1B, CAMK2B, ADH4, CHGB, PYGL, ADH1C, AKAP12, CBFA2T3, and SLC22A1 as the top highly interconnected hub genes. The findings related to CLEC4G, ADH1B, SLC22A1, CAMK2β, CBFA2T3, and STAB2 indicate their reduced expression in HCC, which is associated with an unfavorable prognosis. Furthermore, the results of KEGG and GO pathway analyses suggest that these genes may impact liver cancer by engaging various targets and pathways, ultimately promoting the progression of hepatocellular carcinoma. These results underscore the significant potential of CLEC4G, ADH1B, SLC22A1, CAMK2β, CBFA2T3, and STAB2 as key contributors to HCC development and advancement. This insight holds promise for identifying therapeutic targets and charting research avenues to enhance our understanding of the intricate molecular mechanisms underlying hepatocellular carcinoma.
PubMed: 38911766
DOI: 10.1021/acsomega.4c01496 -
Frontiers in Neuroscience 2024Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare incurable neurodegenerative disease caused by mutations in the gene, which codes for...
INTRODUCTION
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare incurable neurodegenerative disease caused by mutations in the gene, which codes for sacsin, a large protein involved in protein homeostasis, mitochondrial function, cytoskeletal dynamics, autophagy, cell adhesion and vesicle trafficking. However, the pathogenic mechanisms underlying sacsin dysfunction are still largely uncharacterized, and so attempts to develop therapies are still in the early stages.
METHODS
To achieve further understanding of how processes are altered by loss of sacsin, we used untargeted proteomics to compare protein profiles in ARSACS fibroblasts versus controls.
RESULTS
Our analyses confirmed the involvement of known biological pathways and also implicated calcium and lipid homeostasis in ARSACS skin fibroblasts, a finding further verified in SH-SY5Y cells. Validation through mass spectrometry-based analysis and comparative quantification of lipids by LC-MS in fibroblasts revealed increased levels of ceramides coupled with a reduction of diacylglycerols.
DISCUSSION
In addition to confirming aberrant Ca homeostasis in ARSACS, this study described abnormal lipid levels associated with loss of sacsin.
PubMed: 38911600
DOI: 10.3389/fnins.2024.1375299