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1. Identification of histone post-translational modifications in three tissues of Mozambique tilapia (Oreochromis mossambicus)

   Mojica, EA* ; Fu, Y ; Kültz, D ( January 2021 , SOCIETY FOR INTEGRATIVE AND COMPARATIVE BIOLOGY 2021 VIRTUAL ANNUAL MEETING)

   Histone post-translational modifications (PTMs) are epigenetic marks that modify the state of chromatin and lead to alterations in gene expression. Advances in mass spectrometry have enabled the high-throughput analysis of histone PTMs without the need for prior knowledge of individual PTMs of interest. In this study, the global histone PTM landscape was analyzed in the gills, kidney, and testes of Mozambique tilapia (Oreochromis mossambicus) through tandem mass spectrometry using data dependent acquisition (DDA-LCMS2) and PTM mapping approaches. PTM assignment to a specific amino acid was validated using A-score and localization probability scores that are based on the detection of diagnostic MSMS ions. These values signify the robustness of PTM assignment to a specific residue within the protein sequence. For PTMs that were represented by both modified and unmodified versions of the corresponding peptide, the stoichiometry was calculated and compared between tissues. We have identified multiple types of histone PTMs and assigned them to specific residues in each tissue. These PTMs include acetylation, methylation, demethylation, trimethylation, phosphorylation/ dehydration, and ubiquitination. Our results indicate that the gills, kidney, and testes each display a unique profile of histone PTMs. These data provide a strong basis for the generation of spectral libraries that enablemore »high-throughput quantitative analyses of histone PTM stoichiometry on a global scale in tilapia exposed to diverse environmental and developmental contexts.« less
2. Developmentally regulated mitochondrial biogenesis and cell death competence in maize pollen

   https://doi.org/10.1186/s12870-022-03897-y  

   Chamusco, Karen C. ; Milazzo, May N. ; Bhan, Kanchan S. ; Kamps, Terry L. ; Smith, Prestina ; Durojaiye, Modupeoluwa ; Moreira, Cristina D. ; Gallo, Maria ; Chase, Christine D. ( November 2022 , BMC Plant Biology)

   Cytoplasmic male sterility (CMS) is a maternally inherited failure to produce functional pollen that most commonly results from expression of novel, chimeric mitochondrial genes. InZea mays, cytoplasmic male sterility type S (CMS-S) is characterized by the collapse of immature, bi-cellular pollen. Molecular and cellular features of developing CMS-S and normal (N) cytoplasm pollen were compared to determine the role of mitochondria in these differing developmental fates.

   Terminal deoxynucleotidyl transferase dUTP nick end labeling revealed both chromatin and nuclear fragmentation in the collapsed CMS-S pollen, demonstrating a programmed cell death (PCD) event sharing morphological features with mitochondria-signaled apoptosis in animals. Maize plants expressing mitochondria-targeted green fluorescent protein (GFP) demonstrated dynamic changes in mitochondrial morphology and association with actin filaments through the course of N-cytoplasm pollen development, whereas mitochondrial targeting of GFP was lost and actin filaments were disorganized in developing CMS-S pollen. Immunoblotting revealed significant developmental regulation of mitochondrial biogenesis in both CMS-S and N mito-types. Nuclear and mitochondrial genome encoded components of the cytochrome respiratory pathway and ATP synthase were of low abundance at the microspore stage, but microspores accumulated abundant nuclear-encoded alternative oxidase (AOX). Cytochrome pathway and ATP synthase components accumulated whereas AOX levels declined during the maturationmore »of N bi-cellular pollen. Increased abundance of cytochrome pathway components and declining AOX also characterized collapsed CMS-S pollen. The accumulation and robust RNA editing of mitochondrial transcripts implicated translational or post-translational control for the developmentally regulated accumulation of mitochondria-encoded proteins in both mito-types.

   CMS-S pollen collapse is a PCD event coincident with developmentally programmed mitochondrial events including the accumulation of mitochondrial respiratory proteins and declining protection against mitochondrial generation of reactive oxygen species.

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3. Engineering heterologous enzyme secretion in Yarrowia lipolytica

   https://doi.org/10.1186/s12934-022-01863-9  

   Wang, Weigao ; Blenner, Mark A. ( July 2022 , Microbial Cell Factories)

   Eukaryotic cells are often preferred for the production of complex enzymes and biopharmaceuticals due to their ability to form post-translational modifications and inherent quality control system within the endoplasmic reticulum (ER). A non-conventional yeast species,Yarrowia lipolytica, has attracted attention due to its high protein secretion capacity and advanced secretory pathway. Common means of improving protein secretion inY. lipolyticainclude codon optimization, increased gene copy number, inducible expression, and secretory tag engineering. In this study, we develop effective strategies to enhance protein secretion using the model heterologous enzyme T4 lysozyme.

   By engineering the commonly used native lip2prepro secretion signal, we have successfully improved secreted T4 lysozyme titer by 17-fold. Similar improvements were measured for other heterologous proteins, including hrGFP and$$\alpha$$$\alpha$-amylase. In addition to secretion tag engineering, we engineered the secretory pathway by expanding the ER and co-expressing heterologous enzymes in the secretion tag processing pathway, resulting in combined 50-fold improvement in T4 lysozyme secretion.

   Overall, our combined strategies not only proved effective in improving the protein production inYarrowia lipolytica, but also hint the possible existence of a different mechanism of secretion regulation in ER and Golgi body in this non-conventional yeast.
4. The Arabidopsis PeptideAtlas: Harnessing worldwide proteomics data to create a comprehensive community proteomics resource

   https://doi.org/10.1093/plcell/koab211  

   van Wijk, Klaas J ; Leppert, Tami ; Sun, Qi ; Boguraev, Sascha S ; Sun, Zhi ; Mendoza, Luis ; Deutsch, Eric W ( August 2021 , The Plant Cell)

   Abstract We developed a resource, the Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/), to solve central questions about the Arabidopsis thaliana proteome, such as the significance of protein splice forms and post-translational modifications (PTMs), or simply to obtain reliable information about specific proteins. PeptideAtlas is based on published mass spectrometry (MS) data collected through ProteomeXchange and reanalyzed through a uniform processing and metadata annotation pipeline. All matched MS-derived peptide data are linked to spectral, technical, and biological metadata. Nearly 40 million out of ∼143 million MS/MS (tandem MS) spectra were matched to the reference genome Araport11, identifying ∼0.5 million unique peptides and 17,858 uniquely identified proteins (only isoform per gene) at the highest confidence level (false discovery rate 0.0004; 2 non-nested peptides ≥9 amino acid each), assigned canonical proteins, and 3,543 lower-confidence proteins. Physicochemical protein properties were evaluated for targeted identification of unobserved proteins. Additional proteins and isoforms currently not in Araport11 were identified that were generated from pseudogenes, alternative start, stops, and/or splice variants, and small Open Reading Frames; these features should be considered when updating the Arabidopsis genome. Phosphorylation can be inspected through a sophisticated PTM viewer. PeptideAtlas is integrated with community resources including TAIR, tracks in JBrowse, PPDB, and UniProtKB. Subsequentmore »PeptideAtlas builds will incorporate millions more MS/MS data.« less
5. An in silico proteomics screen to predict and prioritize protein–protein interactions dependent on post-translationally modified motifs

   https://doi.org/10.1093/bioinformatics/bty434  

   Schmoker, Anna M. ; Driscoll, Heather E. ; Geiger, Stefanie R. ; Vincent, James J. ; Ebert, Alicia M. ; Ballif, Bryan A. ; Wren, ed., Jonathan ( June 2018 , Bioinformatics)

   The development of proteomic methods for the characterization of domain/motif interactions has greatly expanded our understanding of signal transduction. However, proteomics-based binding screens have limitations including that the queried tissue or cell type may not harbor all potential interacting partners or post-translational modifications (PTMs) required for the interaction. Therefore, we sought a generalizable, complementary in silico approach to identify potentially novel motif and PTM-dependent binding partners of high priority.

   We used as an initial example the interaction between the Src homology 2 (SH2) domains of the adaptor proteins CT10 regulator of kinase (CRK) and CRK-like (CRKL) and phosphorylated-YXXP motifs. Employing well-curated, publicly-available resources, we scored and prioritized potential CRK/CRKL–SH2 interactors possessing signature characteristics of known interacting partners. Our approach gave high priority scores to 102 of the >9000 YXXP motif-containing proteins. Within this 102 were 21 of the 25 curated CRK/CRKL–SH2-binding partners showing a more than 80-fold enrichment. Several predicted interactors were validated biochemically. To demonstrate generalized applicability, we used our workflow to predict protein–protein interactions dependent upon motif-specific arginine methylation. Our data demonstrate the applicability of our approach to, conceivably, any modular binding domain that recognizes a specific post-translationally modified motif.

   Supplementary data are available at Bioinformaticsmore »online.

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