Ribosomal frameshifting used in influenza A virus expression occurs within the sequence UCC_UUU_CGU and is in the +1 direction

Programmed ribosomal frameshifting is used in the expression of many virus genes and some cellular genes. In eukaryotic systems, the most well-characterized mechanism involves –1 tandem tRNA slippage on an X_XXY_YYZ motif. By contrast, the mechanisms involved in programmed +1 (or −2) slippage are more varied and often poorly characterized. Recently, a novel gene, PA-X, was discovered in influenza A virus and found to be expressed via a shift to the +1 reading frame. Here, we identify, by mass spectrometric analysis, both the site (UCC_UUU_CGU) and direction (+1) of the frameshifting that is involved in PA-X expression. Related sites are identified in other virus genes that have previously been proposed to be expressed via +1 frameshifting. As these viruses infect insects (chronic bee paralysis virus), plants (fijiviruses and amalgamaviruses) and vertebrates (influenza A virus), such motifs may form a new class of +1 frameshift-inducing sequences that are active in diverse eukaryotes

Demonstration of Protein-Based Human Identification Using the Hair Shaft Proteome

YesHuman identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects’ DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European–American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.The Technology Commercialization Innovation Program (Contracts #121668, #132043) of the Utah Governors Office of Commercial Development, the Scholarship Activitie

The 3T3-L1 adipocyte glycogen proteome

BACKGROUND: Glycogen is a branched polysaccharide of glucose residues, consisting of α-1-4 glycosidic linkages with α-1-6 branches that together form multi-layered particles ranging in size from 30 nm to 300 nm. Glycogen spatial conformation and intracellular organization are highly regulated processes. Glycogen particles interact with their metabolizing enzymes and are associated with a variety of proteins that intervene in its biology, controlling its structure, particle size and sub-cellular distribution. The function of glycogen in adipose tissue is not well understood but appears to have a pivotal role as a regulatory mechanism informing the cells on substrate availability for triacylglycerol synthesis. To provide new molecular insights into the role of adipocyte glycogen we analyzed the glycogen-associated proteome from differentiated 3T3-L1-adipocytes. RESULTS: Glycogen particles from 3T3-L1-adipocytes were purified using a series of centrifugation steps followed by specific elution of glycogen bound proteins using α-1,4 glucose oligosaccharides, or maltodextrins, and tandem mass spectrometry. We identified regulatory proteins, 14-3-3 proteins, RACK1 and protein phosphatase 1 glycogen targeting subunit 3D. Evidence was also obtained for a regulated subcellular distribution of the glycogen particle: metabolic and mitochondrial proteins were abundant. Unlike the recently analyzed hepatic glycogen proteome, no endoplasmic proteins were detected, along with the recently described starch-binding domain protein 1. Other regulatory proteins which have previously been described as glycogen-associated proteins were not detected, including laforin, the AMPK beta-subunit and protein targeting to glycogen (PTG). CONCLUSIONS: These data provide new molecular insights into the regulation of glycogen-bound proteins that are associated with the maintenance, organization and localization of the adipocyte glycogen particle

Tandem Mass Tagging Based Identification of Proteome Signatures for Reductive Stress Cardiomyopathy

Nuclear factor erythroid 2-related factor 2 (NRF2), a redox sensor, is vital for cellular redox homeostasis. We reported that transgenic mice expressing constitutively active Nrf2 (CaNrf2-TG) exhibit reductive stress (RS). In this study, we identified novel protein signature for RS-induced cardiomyopathy using Tandem Mass Tag (TMT) proteomic analysis in heart tissues of TG (CaNrf2-TG) mice at 6–7 months of age. A total of 1,105 proteins were extracted from 22,544 spectra. About 560 proteins were differentially expressed in TG vs. NTg hearts, indicating a global impact of RS on the myocardial proteome. Over 32 proteins were significantly altered in response to RS -20 were upregulated and 12 were downregulated in the hearts of TG vs. NTg mice, suggesting that these proteins could be putative signatures of RS. Scaffold analysis revealed a clear distinction between TG vs. NTg hearts. The majority of the differentially expressed proteins (DEPs) that were significantly altered in RS mice were found to be involved in stress related pathways such as antioxidants, NADPH, protein quality control, etc. Interestingly, proteins that were involved in mitochondrial respiration, lipophagy and cardiac rhythm were dramatically decreased in TG hearts. Of note, we identified the glutathione family of proteins as the significantly changed subset of the proteome in TG heart. Surprisingly, our comparative analysis of NGS based transcriptome and TMT-proteome indicated that ~50% of the altered proteins in TG myocardium was found to be negatively correlated with their transcript levels. In association with the altered proteome the TG mice displayed pathological cardiac remodeling. Copyright © 2022 Sunny, Jyothidasan, David, Parsawar, Veerappan, Jones, Pogwizd and Rajasekaran.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Evaluating changes in firefighter urinary metabolomes after structural fires: an untargeted, high resolution approach

Firefighters have elevated rates of urinary tract cancers and other adverse health outcomes, which may be attributable to environmental occupational exposures. Untargeted metabolomics was applied to characterize this suite of environmental exposures and biological changes in response to occupational firefighting. 200 urine samples from 100 firefighters collected at baseline and two to four hours post-fire were analyzed using untargeted liquid-chromatography and high-resolution mass spectrometry. Changes in metabolite abundance after a fire were estimated with fixed effects linear regression, with false discovery rate (FDR) adjustment. Partial least squares discriminant analysis (PLS-DA) was also used, and variable important projection (VIP) scores were extracted. Systemic changes were evaluated using pathway enrichment for highly discriminating metabolites. Metabolome-wide-association-study (MWAS) identified 268 metabolites associated with firefighting activity at FDR q < 0.05. Of these, 20 were annotated with high confidence, including the amino acids taurine, proline, and betaine; the indoles kynurenic acid and indole-3-acetic acid; the known uremic toxins trimethylamine n-oxide and hippuric acid; and the hormone 7a-hydroxytestosterone. Partial least squares discriminant analysis (PLS-DA) additionally implicated choline, cortisol, and other hormones. Significant pathways included metabolism of urea cycle/amino group, alanine and aspartate, aspartate and asparagine, vitamin b3 (nicotinate and nicotinamide), and arginine and proline. Firefighters show a broad metabolic response to fires, including altered excretion of indole compounds and uremic toxins. Implicated pathways and features, particularly uremic toxins, may be important regulators of firefighter’s increased risk for urinary tract cancers. © 2023, The Author(s).Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Neutrophil cathepsin G proteolysis of protease-activated receptor 4 generates a novel, functional tethered ligand

Platelet-neutrophil interactions regulate ischemic vascular injury. Platelets are activated by serine proteases that cleave protease-activated receptor (PAR) amino termini, resulting in an activating tethered ligand. Neutrophils release cathepsin G (CatG) at sites of injury and inflammation, which activates PAR4 but not PAR1, although the molecular mechanism of CatG-induced PAR4 activation is unknown. We show that blockade of the canonical PAR4 thrombin cleavage site did not alter CatG-induced platelet aggregation, suggesting CatG cleaves a different site than thrombin. Mass spectrometry analysis using PAR4 N-terminus peptides revealed CatG cleavage at Ser67-Arg68. A synthetic peptide, RALLLGWVPTR, representing the tethered ligand resulting from CatG proteolyzed PAR4, induced PAR4-dependent calcium flux and greater platelet aggregation than the thrombin-generated GYPGQV peptide. Mutating PAR4 Ser67or Arg68 reduced CatG-induced calcium flux without affecting thrombin-induced calcium flux. Dog platelets, which contain a conserved CatG PAR4 Ser-Arg cleavage site, aggregated in response to human CatG and RALLLGWVPTR, while mouse (Ser-Gln) and rat (Ser-Glu) platelets were unresponsive. Thus, CatG amputates the PAR4 thrombin cleavage site by cleavage at Ser67-Arg68 and activates PAR4 by generating a new functional tethered ligand. These findings support PAR4 as an important CatG signaling receptor and suggest a novel therapeutic approach for blocking platelet-neutrophil-mediated pathophysiologies. © 2022 by The American Society of Hematology.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Imputed nsSNP profile probabilities in European and African populations.

<p><b>A</b>) The probability of an overall individual nsSNP profile in the population (Pr(profile|population)) was estimated by determining the probability of detected nsSNP alleles, or allele combination, in each gene (Pr(nsSNP gene profile|population)), and then using the product rule to multiply these probabilities together (Pr(overall profile|population)). <b>B</b>) The probability of overall imputed nsSNP profiles occurring in the European population (Pr(profile|EUR population)) was calculated using imputed nsSNP alleles from individuals in the two European-American cohorts (EA1 and EA2) and the product rule. Values are presented as a logarithm (log₁₀(Pr(profile|EUR population))). Confidence intervals (90% CI) are estimated using parametric bootstrapping. <b>C</b>) The overall imputed nsSNP profile probability in the African population was also calculated (Pr(profile|AFR population)) and plotted versus the probability of the profile occurring in the European population (Pr(profile|EUR population)). Confidence intervals (90% CI) were estimated using parametric bootstrapping. In addition to European–American subjects (red), imputed nsSNP profile probabilities were also estimated from proteomic datasets derived from an African-American (green) and Kenyan (blue) cohort. The line of equal profile probability in the European and African population is indicated (dotted line). <b>D</b>) The likelihood of hair samples coming from a European relative to African genetic background was calculated as the ratio of overall imputed nsSNP profile probabilities in the European and African populations (EUR/AFR = Pr(profile|EUR population)/Pr(profile|AFR population)); European-American subjects (red), African-American subjects (green), and Kenyan subjects (blue) are indicated.</p

Comparison of probability estimates based on imputed nsSNPs and mitochondrial DNA haplotype.

<p>The mitochondrial DNA haplotype and subgroup from one of the European-American cohorts (EA2, n = 15) were classified, compared to a database of subjects from an American sample population (Utah, n = 9,372), and the logarithm of haplotype probability was calculated (log₁₀(Pr(mtDNA haplotype|Utah population)), black bars). Genetically variant peptides containing single amino acid polymorphisms were identified in the hair shaft proteomic datasets of the same subjects, an overall profile of imputed nsSNP loci determined, and logarithm of the probability of each profile occurring in the European population was calculated as described in the Materials and Methods section (log₁₀(Pr(imputed nsSNP profile|EUR population)), red bars). Confidence intervals (90% CI) were estimated using parametric bootstrapping. Each measure is represented using the same axis (log₁₀(Pr(profile|population))).</p

Direct validation of imputed non-synonymous SNP alleles.

<p><b>A</b>) Genetically variant peptides (GVPs) that contained single amino-acid polymorphisms (SAPs) were identified in both European-American cohorts (EA1 and EA2) and collated for each subject. Imputed nsSNP alleles (Gene Name = GN, SNP accession number = rs#, allele nucleotide = nuc) were directly compared to the genotype resulting from direct Sanger sequencing (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160653#pone.0160653.s011" target="_blank">S1 Methods</a>). Correctly imputed nsSNP alleles (TP, true positives) are indicated by a blue square. Imputed alleles that were incorrectly predicted (FP, false positive) are indicated by red squares. Alleles that were identified using Sanger sequencing, but did not contain a resulting GVP in the matching proteomic dataset (FN, false negative) are indicated by light green squares. Alleles absent in both subjects DNA and in resulting proteomic datasets (TN, true negatives) are indicated by white squares[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160653#pone.0160653.ref049" target="_blank">49</a>]. Failed Sanger sequencing determination of nsSNP allelic status is indicated by grey. <b>B</b>) The effectiveness of each SAP-containing peptide to impute nsSNP alleles was also quantified. The sensitivity of each genetically variant peptide, measured as the proportion of nsSNP-alleles that are correctly detected and imputed (TP/(TP+FN)), was calculated as a percentage (log₁₀(%). The positive predictive value (PPV) of genetically variant peptide-based SNP imputations was calculated as the percentage of correct validated SNP imputations of all imputations (TP/(TP + FP); log₁₀(%))[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160653#pone.0160653.ref049" target="_blank">49</a>]. <b>C</b>)</p

Hair shaft proteomic profile in modern and archaeological samples.

<p><b>A</b>) Absolute protein abundance from all datasets corresponding to a cohort of European-American subjects (EA2, subjects 1 to 19) and archaeological subjects (S1 to S6) was measured (<a href="http://www.thegpm.org" target="_blank">www.thegpm.org</a>) and collated. Proteins that appeared in proteomic datasets of 15% or more of the subjects (n = 401) were aligned as a paralogous neighbor-joining tree in order to cluster detected proteins with higher levels of homology (<a href="http://www.uniprot.org" target="_blank">www.uniprot.org</a>.). The neighbor-joining tree based on protein paralogy is aligned on the vertical and subjects on the horizontal. Protein abundance is indicated by conditional formatting (maximum value = yellow, minimal value = black). <b>B</b>) The function of individual proteins was obtained (<a href="http://www.uniprot.org" target="_blank">www.uniprot.org</a>) and collated for both modern (EA2, 1 to 19) and archaeological (S1 to S6) hair shaft samples (categories = structural, metabolism, protein and RNA regulation, membrane proteins, and miscellaneous). The relative abundance of the different protein classes is indicated by area. The size of each circle is proportional to the relative abundance of total detected peptides in each sample class.</p