Diverse Post-translational Modifications Research Articles

Discovery of Protein Modifications Using High Resolution Differential Mass Spectrometry Proteomics

Recent studies have revealed diverse amino acid, post-translational and non-canonical modifications of proteins in diverse organisms and tissues. However, their unbiased detection and analysis remain hindered by technical limitations. Here, we present a spectral alignment method for the identification of protein modifications using high-resolution mass spectrometry proteomics. Termed SAMPEI for Spectral Alignment-based Modified PEptide Identification, this open-source algorithm is designed for the discovery of functional protein and peptide signaling modifications, without prior knowledge of their identities. Using synthetic standards and controlled chemical labeling experiments, we demonstrate its high specificity and sensitivity for the discovery of sub-stoichiometric protein modifications in complex cellular extracts. SAMPEI mapping of mouse macrophage differentiation revealed diverse post-translational protein modifications, including distinct forms of cysteine itaconatylation. SAMPEI’s robust parameterization and versatility are expected to facilitate the discovery of biological modifications of diverse macromolecules. SAMPEI is available open-source from BioConda and GitHub.

Differential Impact of GABA A Receptors on Inhibitory Connectivity for Superficial Pyramidal Neuron Responsiveness In Vivo

A diverse set of GABAA receptors (GABAARs) enable synaptic plasticity adaptations at inhibitory postsynaptic sites in collaboration with the scaffolding protein gephyrin. Early studies helped to identify distinctions between GABAAR subsets allocated within specific functional circuits, but their contribution to the changing dynamics of a microcircuit remains unclear. Here, using the whisker-barrel system we assessed the contribution of specific GABAAR subtypes to sensory processing in vivo. We monitored spontaneous and evoked Ca2+ transients in layer 2/3 pyramidal cells with the genetically encoded Ca2+ sensor RCaMP1.07. Using Gabra1 or Gabra2 global and conditional knockout mice, we uncovered that α1- and α2-GABAARs determine the sparseness of L2/3 pyramidal neuron encoding. In a cell-type dependent manner, α1-GABAARs affected neuronal excitability while α2-GABAARs influenced the reliability of neuronal responses after whisker stimulation. We also discerned that gephyrin and its diverse post-translational modifications (PTMs) facilitate microcircuit homeostasis. Our results underscore the relevance of the diversity of GABAARs within a cortical microcircuit.

The Role of Post-Translational Modifications in the Phase Transitions of Intrinsically Disordered Proteins

Advances in genomics and proteomics have revealed eukaryotic proteomes to be highly abundant in intrinsically disordered proteins that are susceptible to diverse post-translational modifications. Intrinsically disordered regions are critical to the liquid–liquid phase separation that facilitates specialized cellular functions. Here, we discuss how post-translational modifications of intrinsically disordered protein segments can regulate the molecular condensation of macromolecules into functional phase-separated complexes.

High throughput discovery of functional protein modifications by Hotspot Thermal Profiling.

Mass spectrometry has revolutionized the ability to study posttranslationally modified proteoforms from biologic samples, yet we still lack methods to systematically predict, or even prioritize, which modification sites may perturb protein function. Here we describe a proteomic method to detect the effects of site-specific protein phosphorylation on the thermal stability of thousands of native proteins in live cells. This massively parallel biophysical assay unveiled shifts in overall protein stability in response to site-specific phosphorylation sites, as well as trends related to protein function and structure. This method can detect both intrinsic changes to protein structure as well as extrinsic changes to protein-protein, and protein-metabolite interactions resulting from the diminutive introduction of a phosphate onto large proteins. Finally, we show that functional “hotspot” protein modification sites can be discovered and prioritized for study in a high-throughput and unbiased fashion. This approach is applicable to diverse organisms, cell types and posttranslational modifications.

One-Step ARMS-PCR for the Detection of SNPs-Using the Example of the PADI4 Gene.

In eukaryotes, cellular functions are tightly controlled by diverse post-translational modifications (PTMs) of proteins. One such PTM affecting many proteins is the deimination of arginine to citrulline. This process, called citrullination is catalyzed by a group of hydrolases called protein arginine deiminases (PADs), of which five isoforms have been identified. Hypercitrullination, as a result of increased PAD expression or activity, is associated with autoimmune diseases e.g., rheumatoid arthritis, lupus, Alzheimer’s disease, ulcerative colitis, multiple sclerosis, and certain cancers. Three common single nucleotide polymorphisms (SNPs) in the PADI4 gene have been described, namely rs874881, rs11203366, and rs11203367, which are thought to affect PAD4 expression and activity. We here compared the suitability of four methods for the screening of SNPs in the PADI4 gene: (i) SYBR-green based real-time polymerase chain reaction followed by high resolution melting curve analysis (HRM-PCR); (ii) PCR followed by detection of restriction fragment length polymorphisms (PCR-RFLP); (iii) conventional tetra-primer amplification refractory mutation system PCR (ARMS-PCR); and (iv) real-time PCR based on the one-step ARMS-PCR. Of these, ARMS-PCR proved to be the most suitable method regarding handling, duration, and cost of experiments. Using the method with SYBR-green based real-time PCR reagents further diminished handling steps and thus potential sources of error.

Function determinants of TET proteins: the arrangements of sequence motifs with specific codes.

The ten-eleven translocation (TET) proteins play a crucial role in promoting locus-specific reversal of DNA methylation, a type of chromatin modification. Considerable evidences have demonstrated that the sequence motifs with specific codes are important to determine the functions of domains and active sites. Here, we surveyed major studies and reviews regarding the multiple functions of the TET proteins and established the patterns of the motif arrangements that determine the functions of TET proteins. First, we summarized the functional sequence basis of TET proteins and identified the new functional motifs based on the phylogenetic relationship. Next, we described the sequence characteristics of the functional motifs in detail and provided an overview of the relationship between the sequence motifs and the functions of TET proteins, including known functions and potential functions. Finally, we highlighted that sequence motifs with diverse post-translational modifications perform unique functions, and different selection pressures lead to different arrangements of sequence motifs, resulting in different paralogs and isoforms.

Mitochondrial Function, Metabolic Regulation, and Human Disease Viewed through the Prism of Sirtuin 4 (SIRT4) Functions.

As cellular metabolic hubs, mitochondria are the main energy producers for the cell. These organelles host essential energy producing biochemical processes, including the TCA cycle, fatty acid oxidation, and oxidative phosphorylation. An accumulating body of literature has demonstrated that a majority of mitochondrial proteins are decorated with diverse posttranslational modifications (PTMs). Given the critical roles of these proteins in cellular metabolic pathways and response to environmental stress or pathogens, understanding the role of PTMs in regulating their functions has become an area of intense investigation. A major family of enzymes that regulate PTMs within the mitochondria are sirtuins (SIRTs). Albeit until recently the least understood sirtuin, SIRT4 has emerged as an enzyme capable of removing diverse PTMs from its substrates, thereby modulating their functions. SIRT4 was shown to have ADP-ribosyltransferase, deacetylase, lipoamidase, and deacylase enzymatic activities. As metabolic dysfunction is linked to human disease, SIRT4 levels and activities have been implicated in modulating susceptibility to hyperinsulinemia and diabetes, liver disease, cancer, neurodegeneration, heart disease, aging, and pathogenic infections. Therefore, SIRT4 has emerged as a possible candidate for targeted therapeutics. Here, we discuss the diverse enzymatic activities and substrates of SIRT4 and its roles in human health and disease.

Computational Approach for Structural Feature Determination of Grapevine NHX Antiporters.

Plant NHX antiporters are responsible for monovalent cation/H+ exchange across cellular membranes and play therefore a critical role for cellular pH regulation, Na+ and K+ homeostasis, and salt tolerance. Six members of grapevine NHX family (VvNHX1-6) have been structurally characterized. Phylogenetic analysis revealed their organization in two groups: VvNHX1-5 belonging to group I (vacuolar) and VvNHX6 belonging to group II (endosomal). Conserved domain analysis of these VvNHXs indicates the presence of different kinds of domains. Out of these, two domains function as monovalent cation-proton antiporters and one as the aspartate-alanine exchange; the remaining are not yet with defined function. Overall, VvNHXs proteins are typically made of 11-13 putative transmembrane regions at their N-terminus which contain the consensus amiloride-binding domain in the 3rd TM domain and a cation-binding site in between the 5th and 6th TM domain, followed by a hydrophilic C-terminus that is the target of several and diverse regulatory posttranslational modifications. Using a combination of primary structure analysis, secondary structure alignments, and the tertiary structural models, the VvNHXs revealed mainly 18 α helices although without β sheets. Homology modeling of the 3D structure showed that VvNHX antiporters are similar to the bacterial sodium proton antiporters MjNhaP1 (Methanocaldococcus jannaschii) and PaNhaP (Pyrococcus abyssi).

Tat basic domain: A "Swiss army knife" of HIV-1 Tat?

Tat (transactivator of transcription) regulates transcription from the HIV provirus. It plays a crucial role in disease progression, supporting efficient replication of the viral genome. Tat also modulates many functions in the host genome via its interaction with chromatin and proteins. Many of the functions of Tat are associated with its basic domain rich in arginine and lysine residues. It is still unknown why the basic domain exhibits so many diverse functions. However, the highly charged basic domain, coupled with the overall structural flexibility of Tat protein itself, makes the basic domain a key player in binding to or associating with cellular and viral components. In addition, the basic domain undergoes diverse posttranslational modifications, which further expand and modulate its functions. Here, we review the current knowledge of Tat basic domain and its versatile role in the interaction between the virus and the host cell.

In Silico and In Vitro Considerations of Keratinocyte Nuclear Receptor Protein Structural Order for Improving Experimental Analysis.

Nuclear receptors (NR) regulate gene expression critical in keratinocyte replication and differentiation. In addition to a ligand-binding domain, NR like other transcription factor families have a DNA-binding domain that must attain a particular conformation for effective interaction with the three-dimensional structure in promoters of target genes for control of their expression. Such protein-DNA assemblies extend the classic "lock and key" idea typified by protein-protein interactions. However, it is becoming increasingly clear that multi-subdomain transcription factors like NR frequently range along the length of the protein from structured, ordered regions expected for interaction with a preset partner to more flexible, intrinsically disordered regions which are more available for diverse posttranslational modifications and/or interaction with differing partners. The extended amino terminus of NR (the A/B subdomain) is one such intrinsically disordered region. Here we provide a primer on in silico-based recognition of amino acid composition and order associated with such conformational flexibility along with adaptations of readily accessible laboratory techniques (e.g., considerations for recombinant expression, sensitivity to protease and proteasome digestion) to facilitate initial prediction and testing for intrinsic disorder in various proteins of interest to keratinocyte biologists, like NR and other transcription factors.

HDAC3 and HDAC8 are required for cilia assembly and elongation.

ABSTRACTCilia are extended from mother centrioles in quiescent G0/G1 cells and retracted in dividing cells. Diverse post-translational modifications play roles in the assembly and disassembly of the cilium. Here, we examined class I histone deacetylases (HDACs) as positive regulators of cilia assembly in serum-deprived RPE1 and HK2 cells. We observed that the number of cells with cilia was significantly reduced in HDAC3- and HDAC8-depleted cells. The ciliary length also decreased in HDAC3- and HDAC8-depleted cells compared to that in control cells. A knockdown-rescue experiment showed that wild-type HDAC3 and HDAC8 rescued the cilia assembly and ciliary length in HDAC3- and HDAC8-depleted cells, respectively; however, deacetylase-dead HDAC3 and HDAC8 mutants did not. This suggests that deacetylase activity is critical for both HDAC3 and HDAC8 function in cilia assembly and ciliary length control. This is the first study to report that HDACs are required for the assembly and elongation of the primary cilia.

Differential Ion Mobility Separations/Mass Spectrometry with High Resolution in Both Dimensions.

Strong orthogonality to mass spectrometry makes differential ion mobility spectrometry (FAIMS) a powerful tool for isomer separations. However, high FAIMS resolution has been achieved overall only with buffers rich in He or H2. That obstructed coupling to Fourier transform mass spectrometers operating under ultrahigh vacuum, but exceptional m/ z resolution and accuracy of FTMS are indispensable for frontline biological and environmental applications. By raising the waveform amplitude to 6 kV, we enabled high FAIMS resolution using solely N2 and thus straightforward integration with any MS platform: here Orbitrap XL with the electron transfer dissociation (ETD) option. The initial evaluation for complete histone tails (50 residues) with diverse post-translational modifications on alternative sites demonstrates a broad capability to separate and confidently identify the PTM localization variants in the middle-down range.

Redox-Responsive Protein Design: Design of a Small Protein Motif Dependent on Glutathionylation.

Cysteine S-glutathionylation is a protein post-translational modification that promotes cellular responses to changes in oxidative conditions. The design of protein motifs that directly depend on defined changes to protein side chains provides new methods for probing diverse protein post-translational modifications. A canonical, 12-residue EF-hand motif was redesigned to be responsive to cysteine glutathionylation. The key design principle was the replacement of the metal-binding Glu12 carboxylate of an EF-hand with a motif capable of metal binding via a free carboxylate in the glutathione-conjugated peptide. In the optimized peptide (DKDADGWCG), metal binding and terbium luminescence were dependent on glutathionylation, with weaker metal binding in the presence of reduced cysteine but increased metal affinity and a 3.5-fold increase in terbium luminescence at 544 nm when cysteine was glutathionylated. Nuclear magnetic resonance spectroscopy indicated that the structure at all residues of the glutathionylated peptide changed in the presence of metal, with chemical shift changes consistent with the adoption of an EF-hand-like structure in the metal-bound glutathionylated peptide. This small protein motif consists of canonical amino acids and is thus genetically encodable, for its potential use as a localized tag to probe protein glutathionylation.

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue.

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array of diverse posttranslational modifications (PTMs) that mostly occur in the amino termini of histone proteins (i.e., histone tail) and regulate the accessibility and functional state of the underlying DNA. Histone tails extend from the core of the nucleosome and are subject to the addition of acetyl groups by histone acetyltransferases (HATs) and the removal of acetyl groups by histone deacetylases (HDACs) during cellular growth and differentiation. Specific acetylation patterns on lysine (K) residues on histone tails determine a dynamic homeostasis between transcriptionally active or transcriptionally repressed chromatin by (1) influencing the core histone assembly and (2) recruiting synergistic or antagonistic chromatin-associated proteins to the transcription site. The fundamental regulatory mechanism of the complex nature of histone tail PTMs influences the majority of chromatin-templated processes and results in changes in cell maturation and differentiation in both normal and pathological development. The goal of the current report is to provide novices with an efficient method to purify core histone proteins from cells and brain tissue and to reliably quantify acetylation marks on histones H3 and H4.

Regulated Disorder: Posttranslational Modifications Control the RIN4 Plant Immune Signaling Hub

RIN4 is an intensively studied immune regulator in Arabidopsis and is involved in perception of microbial features outside and bacterial effectors inside plant cells. Furthermore, RIN4 is conserved in land plants and is targeted for posttranslational modifications by several virulence proteins from the bacterial pathogen Pseudomonas syringae. Despite the important roles of RIN4 in plant immune responses, its molecular function is not known. RIN4 is an intrinsically disordered protein (IDP), except at regions where pathogen-induced posttranslational modifications take place. IDP act as hubs for protein complex formation due to their ability to bind to multiple client proteins and, thus, are important players in signal transduction pathways. RIN4 is known to associate with multiple proteins involved in immunity, likely acting as an immune-signaling hub for the formation of distinct protein complexes. Genetically, RIN4 is a negative regulator of immunity, but diverse posttranslational modifications can either enhance its negative regulatory function or, on the contrary, render it a potent immune activator. In this review, we describe the structural domains of RIN4 proteins, their intrinsically disordered regions, posttranslational modifications, and highlight the implications that these features have on RIN4 function. In addition, we will discuss the potential role of plasma membrane subdomains in mediating RIN4 protein complex formations.

Morphological and quantitative analysis on α-tubulin modifications in glioblastoma cells

Glioblastoma is one of the primary tumors of the brain with high invasiveness and lethality. In the study of its pathophysiology in recent years much attention has been paid to the microtubular network, but exclusively to β-III tubulin, whose presence in glioblastoma cells is associated with the degree of malignancy and diffusion. As is well known, the microtubular network performs its many functions thanks to various post-translational modifications, most of which affect the α-tubulin subunit. These modifications, able to coexist in the same microtubule, bind certain driving and cargo proteins, deeply influencing cellular functions. Since there are no data in the literature about the diverse post-translational modifications of tubulin in glioblastoma cells, this work aims to fill this gap. In the present work, through immunofluorescence, morphological analysis and Western blot, we studied the pattern of tyrosinated, detyrosinated, delta 2 (Δ2), acetylated and polyglutamylated tubulin. We detected good immunopositivity in fluorescence for almost all the modifications examined. Only Δ2 showed a very low signal. Western blot displayed that the most abundant tubulin modifications in glioblastoma cells were tyrosinated, acetylated and polyglutamylated. Morphological evaluation revealed that these modifications were more present along the cytoplasmic extensions, less evident around the cell body and always strongly evident in the mitotic spindles of the dividing cells. For the first time, the most abundant post-translational modifications and their cellular compartmentalization in glioblastoma cells have been highlighted, suggesting a novel approach in the study of their microtubular network and in the search of new experimental therapeutic strategies.

Obesity-mediated regulation of cardiac protein acetylation: parallel analysis of total and acetylated proteins via TMT-tagged mass spectrometry.

Lysine residues undergo diverse and reversible post-translational modifications (PTMs). Lysine acetylation has traditionally been studied in the epigenetic regulation of nucleosomal histones that provides an important mechanism for regulating gene expression. Histone acetylation plays a key role in cardiac remodeling and function. However, recent studies have shown that thousands of proteins can be acetylated at multiple acetylation sites, suggesting the acetylome rivals the kinome as a PTM. Based on this, we examined the impact of obesity on protein lysine acetylation in the left ventricle (LV) of male c57BL/6J mice. We reported that obesity significantly increased heart enlargement and fibrosis. Moreover, immunoblot analysis demonstrated that lysine acetylation was markedly altered with obesity and that this phenomenon was cardiac tissue specific. Mass spectral analysis identified 2515 proteins, of which 65 were significantly impacted by obesity. Ingenuity Pathway Analysis® (IPA) further demonstrated that these proteins were involved in metabolic dysfunction and cardiac remodeling. In addition to total protein, 189 proteins were acetylated, 14 of which were significantly impacted by obesity. IPA identified the Cardiovascular Disease Pathway as significantly regulated by obesity. This network included aconitate hydratase 2 (ACO2), and dihydrolipoyl dehydrogenase (DLD), in which acetylation was significantly increased by obesity. These proteins are known to regulate cardiac function yet, the impact for ACO2 and DLD acetylation remains unclear. Combined, these findings suggest a critical role for cardiac acetylation in obesity-mediated remodeling; this has the potential to elucidate novel targets that regulate cardiac pathology.

Food Bioactive HDAC Inhibitors in the Epigenetic Regulation of Heart Failure.

Approximately 5.7 million U.S. adults have been diagnosed with heart failure (HF). More concerning is that one in nine U.S. deaths included HF as a contributing cause. Current HF drugs (e.g., β-blockers, ACEi) target intracellular signaling cascades downstream of cell surface receptors to prevent cardiac pump dysfunction. However, these drugs fail to target other redundant intracellular signaling pathways and, therefore, limit drug efficacy. As such, it has been postulated that compounds designed to target shared downstream mediators of these signaling pathways would be more efficacious for the treatment of HF. Histone deacetylation has been linked as a key pathogenetic element for the development of HF. Lysine residues undergo diverse and reversible post-translational modifications that include acetylation and have historically been studied as epigenetic modifiers of histone tails within chromatin that provide an important mechanism for regulating gene expression. Of recent, bioactive compounds within our diet have been linked to the regulation of gene expression, in part, through regulation of the epi-genome. It has been reported that food bioactives regulate histone acetylation via direct regulation of writer (histone acetyl transferases, HATs) and eraser (histone deacetylases, HDACs) proteins. Therefore, bioactive food compounds offer unique therapeutic strategies as epigenetic modifiers of heart failure. This review will highlight food bio-actives as modifiers of histone deacetylase activity in the heart.

In silico insights on diverse interacting partners and phosphorylation sites of respiratory burst oxidase homolog (Rbohs) gene families from Arabidopsis and rice

BackgroundNADPH oxidase (Nox) is a critical enzyme involved in the generation of apoplastic superoxide (O2−), a type of reactive oxygen species (ROS) and hence regulate a wide range of biological functions in many organisms. Plant Noxes are the homologs of the catalytic subunit from mammalian NADPH oxidases and are known as respiratory burst oxidase homologs (Rbohs). Previous studies have highlighted their versatile roles in tackling different kind of stresses and in plant growth and development. In the current study, potential interacting partners and phosphorylation sites were predicted for Rboh proteins from two model species (10 Rbohs from Arabidopsis thaliana and 9 from Oryza sativa japonica). The present work is the first step towards in silico prediction of interacting partners and phosphorylation sites for Rboh proteins from two plant species.ResultsIn this work, an extensive range of potential partners (unique and common), leading to diverse functions were revealed from interaction networks and gene ontology classifications, where majority of AtRbohs and OsRbohs play role in stress-related activities, followed by cellular development. Further, 68 and 38 potential phosphorylation sites were identified in AtRbohs and OsRbohs, respectively. Their distribution, location and kinase specificities were also predicted and correlated with experimental data as well as verified with the other EF-hand containing proteins within both genomes.ConclusionsAnalysis of regulatory mechanisms including interaction with diverse partners and post-translational modifications like phosphorylation have provided insights regarding functional multiplicity of Rbohs. The bioinformatics-based workflow in the current study can be used to get insights for interacting partners and phosphorylation sites from Rbohs of other plant species.

PARP1 interacts with HMGB1 and promotes its nuclear export in pathological myocardial hypertrophy.

High-mobility group box 1 (HMGB1) exhibits various functions according to its subcellular location, which is finely conditioned by diverse post-translational modifications, such as acetylation. The nuclear HMGB1 may prevent from cardiac hypertrophy, whereas its exogenous protein is proven to induce hypertrophic response. This present study sought to investigate the regulatory relationships between poly(ADP-ribose) polymerase 1 (PARP1) and HMGB1 in the process of pathological myocardial hypertrophy. Primary-cultured neonatal rat cardiomyocytes (NRCMs) were respectively incubated with three cardiac hypertrophic stimulants, including angiotensin II (Ang II), phenylephrine (PE), and isoproterenol (ISO), and cell surface area and the mRNA expression of hypertrophic biomarkers were measured. the catalytic activity of PARP1 was remarkably enhanced, meanwhile HMGB1 excluded from the nucleus. PARP1 overexpression by infecting with adenovirus PARP1 (Ad-PARP1) promoted the nuclear export of HMGB1, facilitated its secretion outside the cell, aggravated cardiomyocyte hypertrophy, which could be alleviated by HMGB1 overexpression. PE treatment led to the similar results, while that effect was widely depressed by PARP1 silencing or its specific inhibitor AG14361. Moreover, SD rats were intraperitoneally injected with 3-aminobenzamide (3AB, 20 mg/kg every day, a well-established PARP1 inhibitor) 7 days after abdominal aortic constriction (AAC) surgery for 6 weeks, echocardiography and morphometry of the hearts were measured. Pre-treatment of 3AB relieved AAC-caused the translocation of nuclear HMGB1 protein, cardiac hypertrophy, and heart dysfunction. Our research offers a novel evidence that PARP1 combines with HMGB1 and accelerates its translocation from nucleus to cytoplasm, and the course finally causes cardiac hypertrophy.