Data mining of protein families using common peptides

Predicting the function of a protein from its sequence is typically addressed using sequence-similarity. Here we propose a motif-based approach, using supervised motif extraction from protein sequences belonging to one functional family. The resulting deterministic motifs form Common Peptides (CPs) that characterize this family, allow for data mining of its proteins and facilitate further partition of the family into cluster

In vivo and in vitro characterization of Staphylococcus aureus and Bacillus subtilis polyglycerolphosphate lipoteichoic acid synthases

Staphylococcus aureus lipoteichoic acid (LTA) consists of a 1,3-linked polyglycerolphosphate chain retained in the bacterial membrane by a glycolipid anchor. The LTA backbone is produced by the lipoteichoic acid synthase LtaS, a membrane protein with five transmembrane helices and a large extracellular enzymatic domain (eLtaS). Proteomic studies revealed that LtaS is efficiently cleaved, and here it was demonstrated that the eLtaS domain is released into the culture supernatant as well as partially retained within the cell wall fraction. However, using an in vivo LtaS activity assay, it was shown that only the full-length LtaS enzyme is able to synthesize LTA. Neither expression of a secreted eLtaS variant, created by replacing the N-terminal membrane domain with a conventional signal sequence, nor expression of eLtaS fused to a single or multi-transmembrane domains of other staphylococcal proteins resulted in the production of LTA. These data indicate that the transmembrane domain of LtaS play an essential, yet unknown, role in LtaS enzyme function. In addition, the protease responsible for LtaS cleavage was identified. It was found that a S. aureus strain in which the gene encoding for the essential signal peptidase SpsB was cloned under inducible expression control showed an accumulation of the full-length LtaS enzyme in the absence of the inducer. These data suggest that SpsB is involved in LtaS cleavage. Four LtaS orthologues, YflE, YfnI, YqgS and YvgJ, are present in Bacillus subtilis. Using an in vitro enzyme assay and purified protein, it was determined that all four B. subtilis proteins are Mn2+-dependent metal enzymes that use the lipid phosphatidylglycerol as substrate. It was shown that YflE, YfnI and YqgS are bonafide LTA synthases capable of producing polyglycerolphosphate chains, while YvgJ appears to function as an LTA primase, as indicated by the accumulation of a glycolipid with the expected chromatographic mobility of GroP-Glc2-DAG. Taken together, experimental evidence for the enzyme function of all four B. subtilis LtaStype proteins is provided in this work and it was shown that all four enzymes are involved in the LTA synthesis process

Single Muscle Fiber Proteomics Reveals Fiber-Type-Specific Features of Human Muscle Aging

Skeletal muscle is a key tissue in human aging, which affects different muscle fiber types unequally. We developed a highly sensitive single muscle fiber proteomics workflow to study human aging and show that the senescence of slow and fast muscle fibers is characterized by diverging metabolic and protein quality control adaptations. Whereas mitochondrial content declines with aging in both fiber types, glycolysis and glycogen metabolism are upregulated in slow but downregulated in fast muscle fibers. Aging mitochondria decrease expression of the redox enzyme monoamine oxidase A. Slow fibers upregulate a subset of actin and myosin chaperones, whereas an opposite change happens in fast fibers. These changes in metabolism and sarcomere quality control may be related to the ability of slow, but not fast, muscle fibers to maintain their mass during aging. We conclude that single muscle fiber analysis by proteomics can elucidate pathophysiology in a sub-type-specific manner

Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics.

BackgroundAnimal venoms attract enormous interest given their potential for pharmacological discovery and understanding the evolution of natural chemistries. Next-generation transcriptomics and proteomics provide unparalleled, but underexploited, capabilities for venom characterization. We combined multi-tissue RNA-Seq with mass spectrometry and bioinformatic analyses to determine venom gland specific transcripts and venom proteins from the Western black widow spider (Latrodectus hesperus) and investigated their evolution.ResultsWe estimated expression of 97,217 L. hesperus transcripts in venom glands relative to silk and cephalothorax tissues. We identified 695 venom gland specific transcripts (VSTs), many of which BLAST and GO term analyses indicate may function as toxins or their delivery agents. ~38% of VSTs had BLAST hits, including latrotoxins, inhibitor cystine knot toxins, CRISPs, hyaluronidases, chitinase, and proteases, and 59% of VSTs had predicted protein domains. Latrotoxins are venom toxins that cause massive neurotransmitter release from vertebrate or invertebrate neurons. We discovered ≥ 20 divergent latrotoxin paralogs expressed in L. hesperus venom glands, significantly increasing this biomedically important family. Mass spectrometry of L. hesperus venom identified 49 proteins from VSTs, 24 of which BLAST to toxins. Phylogenetic analyses showed venom gland specific gene family expansions and shifts in tissue expression.ConclusionsQuantitative expression analyses comparing multiple tissues are necessary to identify venom gland specific transcripts. We present a black widow venom specific exome that uncovers a trove of diverse toxins and associated proteins, suggesting a dynamic evolutionary history. This justifies a reevaluation of the functional activities of black widow venom in light of its emerging complexity

Substrate specificity and structural investigation into PepO and PepW : two peptidases from Lactobacillus rhamnosus : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand

The proteolytic systems of lactic acid bacteria have important roles in the maturation and flavour development of cheese. Lactic acid bacteria pepetidases contribute to the taste of cheese through the production of low-molecular weight peptides and free amino acids. Although some lactic acid bacteria peptidases have been structurally and enzymatically characterised for their substrate specificity, there are some that are yet to be completely biochemically characterised. The aim of the present study was to investigate the substrate specificity and three-dimensional structure of two peptidases that could potentially be used as a tool to modify and control cheese bitterness and possibly other flavour attributes from Lactobacillus rhamnosus, PepO and PepW. The pepW gene was successfully cloned from L. rhamnosus into an E. coli expression system. Recombinant PepW was purified to homogeneity and was shown to exist as a hexamer of 50 kDa subunits. Recombinant PepO was expressed from a previously established L. lactis expression system and purified to homogeneity. PepO was shown to exist as a 70 kDa monomer, and function as a metallopeptidase. Pepo and PepW were shown to selectively hydrolyse chymosin-derived bovine β- and κ-casein peptides, and casein peptides extracted from Cheddar cheese. One conclusive PepO cleavage site that had not been previously characterised was identified. This was the β-casein peptide bond between Leu₆-Asn₇. Several possible PepO and PepW cleavage sites in αs₁-, β- and κ- casein were identified, suggesting that PepO has a broad endopeptidase activity, whilst PepW has a specific exopeptidase activity. Pepo and PepW crystals were successfully grown for structure determination by x-ray crystallography. Native data sets were collected for both PepO and PepW, and derivative data were collected for PepO. Structure determination was attempted using Multiple Isomorphous Replacement and Molecular Replacement techniques. Results from the substrate specificity and structural investigation of the L. rhamnosus peptidases, PepO and PepW, are presented in this thesis

Structure of the γ-D-glutamyl-L-diamino acid endopeptidase YkfC from Bacillus cereus in complex with L-Ala-γ-D-Glu: insights into substrate recognition by NlpC/P60 cysteine peptidases.

Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific γ-D-glutamyl-L-diamino acid endopeptidases. The crystal structure of a YkfC ortholog from Bacillus cereus (BcYkfC) at 1.8 Å resolution revealed that it contains two N-terminal bacterial SH3 (SH3b) domains in addition to the C-terminal catalytic NlpC/P60 domain that is ubiquitous in the very large family of cell-wall-related cysteine peptidases. A bound reaction product (L-Ala-γ-D-Glu) enabled the identification of conserved sequence and structural signatures for recognition of L-Ala and γ-D-Glu and, therefore, provides a clear framework for understanding the substrate specificity observed in dipeptidyl-peptidase VI, YkfC and other NlpC/P60 domains in general. The first SH3b domain plays an important role in defining substrate specificity by contributing to the formation of the active site, such that only murein peptides with a free N-terminal alanine are allowed. A conserved tyrosine in the SH3b domain of the YkfC subfamily is correlated with the presence of a conserved acidic residue in the NlpC/P60 domain and both residues interact with the free amine group of the alanine. This structural feature allows the definition of a subfamily of NlpC/P60 enzymes with the same N-terminal substrate requirements, including a previously characterized cyanobacterial L-alanine-γ-D-glutamate endopeptidase that contains the two key components (an NlpC/P60 domain attached to an SH3b domain) for assembly of a YkfC-like active site

Multi-omic Analyses of Extensively Decayed Pinus contorta Reveal Expression of a Diverse Array of Lignocellulose-Degrading Enzymes.

Fungi play a key role cycling nutrients in forest ecosystems, but the mechanisms remain uncertain. To clarify the enzymatic processes involved in wood decomposition, the metatranscriptomics and metaproteomics of extensively decayed lodgepole pine were examined by RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. Following de novo metatranscriptome assembly, 52,011 contigs were searched for functional domains and homology to database entries. Contigs similar to basidiomycete transcripts dominated, and many of these were most closely related to ligninolytic white rot fungi or cellulolytic brown rot fungi. A diverse array of carbohydrate-active enzymes (CAZymes) representing a total of 132 families or subfamilies were identified. Among these were 672 glycoside hydrolases, including highly expressed cellulases or hemicellulases. The CAZymes also included 162 predicted redox enzymes classified within auxiliary activity (AA) families. Eighteen of these were manganese peroxidases, which are key components of ligninolytic white rot fungi. The expression of other redox enzymes supported the working of hydroquinone reduction cycles capable of generating reactive hydroxyl radicals. These have been implicated as diffusible oxidants responsible for cellulose depolymerization by brown rot fungi. Thus, enzyme diversity and the coexistence of brown and white rot fungi suggest complex interactions of fungal species and degradative strategies during the decay of lodgepole pine.IMPORTANCE The deconstruction of recalcitrant woody substrates is a central component of carbon cycling and forest health. Laboratory investigations have contributed substantially toward understanding the mechanisms employed by model wood decay fungi, but few studies have examined the physiological processes in natural environments. Herein, we identify the functional genes present in field samples of extensively decayed lodgepole pine (Pinus contorta), a major species distributed throughout the North American Rocky Mountains. The classified transcripts and proteins revealed a diverse array of oxidative and hydrolytic enzymes involved in the degradation of lignocellulose. The evidence also strongly supports simultaneous attack by fungal species employing different enzymatic strategies

Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms

Fluorescence Correlation Spectroscopy Reveals Efficient Cytosolic Delivery of Protein Cargo by Cell-Permeant Miniature Proteins.

New methods for delivering proteins into the cytosol of mammalian cells are being reported at a rapid pace. Differentiating between these methods in a quantitative manner is difficult, however, as most assays for evaluating cytosolic protein delivery are qualitative and indirect and thus often misleading. Here we make use of fluorescence correlation spectroscopy (FCS) to determine with precision and accuracy the relative efficiencies with which seven different previously reported "cell-penetrating peptides" (CPPs) transport a model protein cargo-the self-labeling enzyme SNAP-tag-beyond endosomal membranes and into the cytosol. Using FCS, we discovered that the miniature protein ZF5.3 is an exceptional vehicle for delivering SNAP-tag to the cytosol. When delivered by ZF5.3, SNAP-tag can achieve a cytosolic concentration as high as 250 nM, generally at least 2-fold and as much as 6-fold higher than any other CPP evaluated. Additionally, we show that ZF5.3 can be fused to a second enzyme cargo-the engineered peroxidase APEX2-and reliably delivers the active enzyme to the cell interior. As FCS allows one to realistically assess the relative merits of protein transduction domains, we anticipate that it will greatly accelerate the identification, evaluation, and optimization of strategies to deliver large, intact proteins to intracellular locales