Global profiling of protein lysine lactylation and potential target modified protein analysis in hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, often metastasizes to the lungs. The implications of lysine lactylation (Kla), a recently identified histone post-translational modification (PTM), in the pathology of HCC remain unclear. Here, we report the first proteomic survey of this specific modification in HCC (with no metastasis during 3 years of follow-up), normal liver tissues, and lung metastasis samples of HCC. Of the 2045 modification sites detected on 960 proteins, 1438 sites on 772 proteins contained quantitative information. Subsequently, we analyzed the differentially modified proteins among the different groups. Differentially lactylated proteins were found to be involved in several biological processes, including-but not limited to-amino acid metabolism, ribosomal protein synthesis, and fatty acid metabolism. In addition, we identified numerous highly valuable lactate-modified proteins from the literature. Among them, we verified the lactate modification levels of the following two tumor-related proteins and obtained similar results: USP14 and ABCF1. These two modified proteins will be further investigated in our future studies. This paper is the first report on the lactylome of HCC and it provides a reliable foundation for further research on Kla in HCC.     

In-depth discovery of protein lactylation in hepatocellular carcinoma

As a prevalent cancer type, hepatocellular carcinoma (HCC) accounts for a number of tumor-related deaths worldwide. Substantial efforts from various aspects, including RNA and proteins, have been devoted to understanding the mechanisms of HCC and proposing therapeutic schemes correspondingly. Within one of the important fields in cancer research – protein post-translational modifications (PTMs), recent discoveries revealed much broader landscapes of lysine lactylation (Kla) distributed in whole human proteome. Upon realizing the relation between Kla and cancers, Hong et al. (Proteomics 2023, 23, 2200432) comprehensively profiled lactylproteome in HCC tissues for the first time. All collected and processed samples were categorized into normal liver tissues, HCC without metastasis or HCC with lung metastasis tissues. As a result, 2045 Kla modification sites from 960 proteins were identified and 1438 sites from 772 proteins were quantifiably measured. Many differentially expressed Kla-proteins emerged and meant to contribute HCC formation and metastatsis. Among them, specific Kla sites from ubiquitin specific peptidase 14 (USP14) and ATP-binding cassette family 1 (ABCF1) were respectively verified as diagnostic indicators to characterize HCC and its metastasis. This work was of great significance, and made impacts in terms of further discovery of HCC rationale, as well as diagnosis of HCC status and targeted therapies.     

The Function and related Diseases of Protein Crotonylation

Crotonylation is a kind of newly discovered acylation modification. Thousands of crotonylation sites have been identified in histone and non-histone proteins over the past decade. As a modification closely related to acetylation, crotonylation was reported to share many universal enzymes with acetylation. Crotonylated proteins have important roles in the regulation of various biological processes, such as gene expression, process of spermatogenesis, cell cycle, and also in the pathogenesis of different diseases, which range from depression to cancer. In this review, we summarize the research processes of crotonylation and discuss the advances of regulation mechanism of both histone and non-histone proteins crotonylation in difference physiological processes. Also, we focus on the alteration of the crotonylation under certain pathological conditions and its role in the pathogenesis of each disease.     

Emerging roles of O-glycosylation in regulating protein aggregation,phase separation,and functions

Protein O-glycosylation is widely identified in various proteins involved in diverse biological processes. Recent studies have demonstrated that O-glycosylation plays crucial and multifaceted roles in modulating protein amyloid aggregation and liquid–liquid phase separation (LLPS) under physiological conditions. Dysregulation of these processes is closely associated with human diseases such as neurodegenerative diseases (NDs) and cancers. In this review, we first summarize the distinct roles of O-glycosylation in regulating pathological aggregation of different amyloid proteins related to NDs and elaborate the underlying mechanisms of how O-glycosylation modulates protein aggregation kinetics, induces new aggregated structures, and mediates the pathogenesis of amyloid aggregates under diseased conditions. Furthermore, we introduce recent discoveries on O-GlcNAc-mediated regulation of synaptic LLPS and phase separation potency of low-complexity domain-enriched proteins. Finally, we identify challenges in future research and highlight the potential for developing new therapeutic strategies of NDs by targeting protein O-glycosylation.     

2-D protein maps of rat gastrocnemius and soleus muscles: a tool for muscle plasticity assessment

Functional characterization of muscle fibers relies on ATPase activity and on differential measurements of metabolic proteins, including mitochondrial and glycolytic enzymes, glucose, lactate and lactic acid transporters, calcium cycling proteins and components of the contractile machinery. The recent introduction of microarray technology has enabled detailed gene expression studies under different physiological and pathological conditions, thus generating novel hypotheses on muscle function. However, microarray approaches are limited by the incomplete genome coverage of currently available chips, and by poor correlation between mRNA concentration and protein expression level. We have used 2-DE and MS to build a reference map of proteins from rat mixed gastrocnemius and soleus muscle, and to assess qualitative and quantitative differences in protein distribution between these two functionally dissimilar muscles. More than 800 spots on each gel were detected by silver staining, of which 167 were excised, digested in-gel with trypsin and analyzed by ESI-MS/MS. One hundred and twenty eight distinct gene products were identified, including metabolic, transport and contractile proteins. Forty one spots displayed differences in relative expression level between mixed gastrocnemius and soleus samples. These data not only enable differentiation of functionally distinct slow-twitch and fast-twitch fiber types, but also provide tools for investigating muscle plasticity in response to physiological and environmental conditions such as aging or hypoxia.     

Functional consequences of actin nitration: in vitro and in disease states

To link the phenomena of inflammatory-induced increases in protein nitrotyrosine (NO₂Tyr) derivatives to protein dysfunction and consequent pathological conditions, the evaluation of discrete NO₂Tyr modifications on specific proteins must be undertaken. Mass spectrometric (MS) proteomics-based strategies allow for the identification of all individual proteins that are nitrated by separating tissue homogenates using 2D gel electrophoresis, detecting the nitrated proteins using an anti-NO₂Tyr antibody, and then identifying the peptides generated during an in-gel proteolytic digest using matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) MS. Actin, one of the most abundant proteins in eukaryotic cells, constitutes 5% or more of cell protein and serves with other cytoskeletal proteins as a critical target for nitration-induced functional impairment. Herein, examples of actin nitration detected under physiological conditions in various models of human disease or in clinically derived tissues are given and the impact that this post-translational protein modification can have on cell and organ function is discussed.     

The platelet microparticle proteome

Platelet-derived microparticles are the most abundant type of microparticle in human blood and contribute to many biologically significant processes. Here, we report the first proteomic analysis of microparticles generated from activated platelets. Using 1D SDS-PAGE and liquid chromatography coupled to a linear ion trap mass spectrometer, the identification of 578 proteins was accomplished using a minimum of 5 MS/MS detections of at least two different peptides for each protein. These microparticles displayed many proteins intrinsic to and well-characterized on platelets. For example, microparticles in these experiments were found to contain membrane surface proteins including GPIIIa, GPIIb, and P-selectin, as well other platelet proteins such as the chemokines CXCL4, CXCL7, and CCL5. In addition, approximately 380 of the proteins identified were not found in two previous studies of the platelet proteome. Since several of the proteins detected here have been previously implicated in microparticle formation and/or pathological function, it is hoped that this study will help fuel future work concerning the possible role of microparticles in various disease states.     

Crosstalk between glucose metabolism,lactate production and immune response modulation

Metabolites of glycolytic metabolism have been identified as signaling molecules and regulators of gene expression, in addition to their basic function as major energy and biosynthetic source. Immune cells reprogram metabolic pathways to cater to energy and biosynthesis demands upon activation. Most lymphocytes, including inflammatory M1 macrophages, mainly shift from oxidative phosphorylation to glycolysis, whereas regulatory T cells and M2 macrophages preferentially use the tricarboxylic acid (TCA) cycle and have reduced glycolysis. Recent studies have revealed the “non-metabolic” signaling functions of intermediates of the mitochondrial pathway and glycolysis. The roles of citrate, succinate and itaconate in immune response, including post-translational modifications of proteins and macrophages activation, have been highlighted. As an end product of glycolysis, lactate has received considerable interest from researchers. In this review, we specifically focused on studies exploring the integration of lactate into immune cell biology and associated pathologies. Lactate can act as a double-edged sword. On one hand, activated immune cells prefer to use lactate to support their function. On the other hand, accumulated lactate in the tissue microenvironment acts as a signaling molecule that restricts immune cell function. Recently, a novel epigenetic change mediated by histone lysine lactylation has been proposed. The burgeoning researches support the idea that histone lactylation participates in diverse cellular events. This review describes glycolytic metabolism, including the immunoregulation of metabolites of the TCA cycle and lactate. These latest findings strengthen our understanding on tumor and chronic inflammatory diseases and offer potential therapeutic options.     

Post-Translational Modifications in sperm Proteome: The Chemistry of Proteome diversifications in the Pathophysiology of male factor infertility

BackgroundThe spermatozoa undergo a series of changes in the epididymis to mature after their release from the testis and subsequently in the female reproductive tract after ejaculation to get capacitated and achieve fertilization potential. Despite having a silenced protein synthesis machinery, the dynamic change in protein profile of the spermatozoa is attributed either to acquisition of new proteins via vescicular transport or to several post-translational modifications (PTMs) occurring on the already expressed protein complement.Scope of reviewIn this review emphasis is given on the PTMs already reported on the human sperm proteins under normal and pathologic conditions with particular reference to sperm function such as motility and fertilization. An attempt has been made to summarize different protocols and methods used for analysis of PTMs on sperm proteins and the newer trends those were emerging.Major conclusionsDeciphering the differential occurrence of PTM on protein at ultrastructural level would give us a better insight of structure-function relationship of the particular protein. Protein with multiple PTMs could be used to generate the complex interaction network involved in a physiological function of a sperm. It can be speculated that crosstalk between different PTMs occurring either on same/ other proteins actually regulate the protein stability and activity both in physiological and pathological states.General significanceThe analytical prospective of various PTMs reported in human spermatozoa and their relevance to sperm function particularly in various pathophysiological states, would pave way for development of biomarkers for diagnosis, prognosis and therapeutic intervention of male infertility.     

Proteomic Profile of Human Schwann Cells

Schwann cells (SC) are essential for the growth, maintenance, and regeneration of peripheral nerves, but the proteome of normal human SC is poorly defined. Here, a proteomic analysis by LC–MS/MS is performed to define the protein expression profile of primary human SC. A total of 19 557 unique peptides corresponding to 1553 individual proteins are identified. Ingenuity Pathway Analysis (IPA), Gene Ontology (GO), and Database for Annotation, Visualization, and Integrated Discovery (DAVID) are used to assign protein localization and function, and to define enriched pathways. EIF2, mTOR, and integrin signaling are among the most enriched pathways and the most enriched biological function is cell–cell adhesion, which is in agreement with the supportive role of SC in peripheral nerves. In addition, several nociceptors and synaptic proteins are identified and may contribute to the recently discovered role of SC in pain sensation and cancer progression. This proteome analysis of normal human SC constitutes a reference for future molecular explorations of physiological and pathological processes where SC are involved.     

Osteogenic differentiation of adipose derived stem cells promoted by overexpression of osterix

Secreted proteins, which may be involved in the regulation of various biological processes, are the potential targets for diagnosis and treatment of diverse diseases. In this study, to identify the human hepatoma HepG2 cells-derived secreted proteins more extensively, we applied the protein sample preparations using the combinations of denaturation methods and molecular-mass cutoff via ultrafiltration to the two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D LC–MS/MS) analysis. We were able to identify a total of 86 proteins containing widely known secreted proteins of HepG2 such as alpha-fetoprotein, of which 73 proteins including 27 signal peptide-containing proteins have never been reported to be secreted from HepG2 cells in other proteomic studies. Among the identified signal peptide-containing proteins, ten proteins such as growth differentiation factor 15, osteopontin and stanniocalcin 2 were discovered as new secreted proteins of HepG2 cells. These observations suggest that the combinations of different sample preparation methods and 2D LC–MS/MS analysis are useful for identifying a wider range of low-abundance proteins and that the secreted proteins from HepG2 identified in this study may be useful as liver-specific biomarkers for diagnosis and treatment.     

Structural study of GCDFP-15/gp17 in disease versus physiological conditions using a proteomic approach

Gross cystic disease fluid protein (GCDFP-15), also known as prolactin-inducible protein (PIP), is a specific breast tumor marker. GCDFP-15/PIP is also identified as gp17 and/or seminal actin-binding protein (SABP) from seminal vesicles and as extraparotid glycoprotein (EP-GP) from salivary glands. It is an aspartyl proteinase able to specifically cleave fibronectin (FN), suggesting a possible involvement in mammary tumor progression and fertilization. Other functions were attributed to this protein(s) on the basis of its ability to interact with an array of molecules such as CD4, actin, and fibrinogen. We investigated the structure of the protein(s) under disease versus physiological conditions by RP-HPLC chromatography, ProteinChip technology, and QStar MS/MS mass spectrometry. The proteins behaved differently when examined by RP-HPLC chromatography and surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) mass spectrometry, suggesting different conformations and/or tissue-specific posttranslational modifications of the proteins, although their primary structure was identical by MS/MS analysis. Both showed a single N-glycosylation site. A different N-linked glycosylation pattern was observed in pathological GCDFP-15/PIP as compared with physiological gp17/SABP protein by coupling enzymatic digestion and ProteinChip technology. Furthermore, taking advantage of ProteinChip technology, we analyzed the interaction of both proteins with CD4 and FN. We observed that the physiological form was mainly involved in the binding to CD4. Moreover, we defined the specific FN binding-domain of this protein. These data suggested that, depending on its conformational state, the protein could differently bind to its various binding molecules and change its function(s) in the microenviroments where it is expressed.     

Targeting of Bacillus anthracis interaction factors for human macrophages using two-dimensional gel electrophoresis

Bacillus anthracis, a gram-positive, endospore-forming, aerobic rod-shaped bacterium, interacts with macrophages at various stages of the disease. Spore germination and the outgrowth of vegetative bacilli are crucial steps enabling the bacteria to proliferate actively and to synthesize the virulence factors leading to a massive septicemia. In this study, we performed a proteomic analysis and MALDI-TOF/MS were carried out to identify proteins using human macrophages infected with the spores of B. anthracis live-Sterne or inactivated-Sterne. We identified 21 proteins which are related to the infection of B. anthracis spores on human macrophages at the early stage events. These proteins function in processes such as cytoskeleton regulation, apoptosis, cell division, and protein degradation. Proteins such as PAK 2 revealed a relationship to apoptosis in human macrophages. These proteins play an important role in the macrophage survival and death on human macrophages with infected B. anthracis spores.     

Proteomic analysis of the tetraspanin web using LC-ESI-MS/MS and MALDI-FTICR-MS

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. Tetraspanins play a role as organizers of a molecular network of interactions, the "tetraspanin web". Here, we have performed a proteomic characterization of the tetraspanin web using a model of human colon cancer consisting of two cell lines derived from primary tumor and metastasis from the same patient. The tetraspanin complexes were isolated after immunoaffinity purification and the proteins were identified by MS using LC-ESI-MS/MS and MALDI-FTICR. The high resolution and mass accuracy of FTICR MS allowed reliable identification using mass finger printing with only two peptides. Thus, it could be used to resolve the composition of complex peptide mixtures from membrane proteins. Different types of membrane proteins were identified, including adhesion molecules (integrins, Lu/B-CAM, GA733 proteins), receptors and signaling molecules (BAI2, PKC, G proteins), proteases (ADAM10, TADG15), and membrane fusion proteins (syntaxins) as well as poorly characterized proteins (CDCP1, HEM-1, CTL1, and CTL2). Some components were differentially detected in the tetraspanin web of the two cell lines. These differences may be relevant for tumor progression and metastasis.     

Protein-protein interactions: principles, techniques, and their potential role in new drug development

A vast network of genes is inter-linked through protein-protein interactions and is critical component of almost every biological process under physiological conditions. Any disruption of the biologically essential network leads to pathological conditions resulting into related diseases. Therefore, proper understanding of biological functions warrants a comprehensive knowledge of protein-protein interactions and the molecular mechanisms that govern such processes. The importance of protein-protein interaction process is highlighted by the fact that a number of powerful techniques/methods have been developed to understand how such interactions take place under various physiological and pathological conditions. Many of the key protein-protein interactions are known to participate in disease-associated signaling pathways, and represent novel targets for therapeutic intervention. Thus, controlling protein-protein interactions offers a rich dividend for the discovery of new drug targets. Availability of various tools to study and the knowledge of human genome have put us in a unique position to understand highly complex biological network, and the mechanisms involved therein. In this review article, we have summarized protein-protein interaction networks, techniques/methods of their binding/kinetic parameters, and the role of these interactions in the development of potential tools for drug designing.     

Comparative Proteome Profile of Human Placenta from Normal and Preeclamptic Pregnancies

To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, we used LC-MS/MS to construct a large-scale comparative proteome profile of human placentas from normal and preeclamptic pregnancies. A total of 2636 proteins were detected in human placentas, and 171 different proteins were definitively identified between control and preeclamptic placentas. Further bioinformatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes which occur during pathological changes of preeclamptic placenta. 6 proteins were randomly selected to verify their expression patterns with Western blotting. Of which, 3 proteins’ cellular localizations were validated with immunohistochemistry. Elucidation of how protein-expression changes coordinate the pathological development would provide researchers with a better understanding of the critical biological processes of preeclampsia and potential targets for therapeutic agents to regulate placenta function, and eventually benefit the treatment of preeclampsia.     

Proteomic analysis of membrane proteins from Streptococcus pneumoniae with multiple separation methods plus high accuracy mass spectrometry

Abstract Streptococcus pneumoniae is a Gram-positive human pathogen that causes a variety of serious mucosal and invasive diseases in human. Bacterial membrane proteins play crucial roles in host-pathogen interactions and bacterial pathogenesis, and thus are potential drug targets or vaccine candidates. In this study, membranes from Streptococcus pneumoniae D39 were enriched by mechanical grinding and ultracentrifugation, and then the membrane proteins were extracted with trifluroethanol and chloroform. Around 60% of the extracted proteins were identified to be membrane proteins with 2-DE coupled with MALDI-MS/MS and 2D-LC-ESI-MS/MS. These identified membrane proteins can be functionally categorized into various groups involved in nutriment transport, signal transduction, protein folding or secretion, oxidation, carbohydrate metabolism, and other physiological processes. A protein interaction network was constructed for understanding the regulation relationship of the membrane proteins. This study represents the first global characterization of membrane proteome from Gram-positive streptococcus species of bacteria, providing valuable clues for further investigation aiming at identifying drug/vaccine targets for the bacterial infection.     

Systemic analysis of tyrosine phosphorylated proteins in angiopoietin-1 induced signaling pathway of endothelial cells

Angiogenesis is an essential process in physiological and pathological processes and is well-regulated to maintain the cellular homeostasis by balancing the endothelial cells in proliferation and apoptosis. Angiopoietin-1 (Ang1) regulates angiogenesis as a ligand of Tie 2 receptor tyrosine kinase. However, the regulation pathways are not well-understood. To date, only a few of the signaling molecules involved in the Tie 2 receptor tyrosine kinase-mediated angiogenesis have been identified. In this study, we systematically identified tyrosine-phosphorylated proteins in Ang1-induced signaling cascade in human umbilical vein endothelial cells (HUVECs), employing proteomic analyses combining two-dimensional gel electrophoresis, Western analysis using phosphotyrosine antibody and mass spectrometry (MALDI-TOF MS and nanoLC-ESI-q-TOF tandem MS). We report here the identification, semiquantitative analysis, and kinetic changes of tyrosine-phosphorylated proteins in response to Ang1 in HUVECs and identified 66 proteins among 69 protein spots showing significant changes. Of these, p54nrb was validated as a molecule involved in cell migration. These results suggest that Ang1 induces stabilization of neo-vessel network by regulating the phosphorylations of metabolic and structural proteins.     

Elastases and elastokines: elastin degradation and its significance in health and disease

Abstract

Elastin is an important protein of the extracellular matrix of higher vertebrates, which confers elasticity and resilience to various tissues and organs including lungs, skin, large blood vessels and ligaments. Owing to its unique structure, extensive cross-linking and durability, it does not undergo significant turnover in healthy tissues and has a half-life of more than 70?years. Elastin is not only a structural protein, influencing the architecture and biomechanical properties of the extracellular matrix, but also plays a vital role in various physiological processes. Bioactive elastin peptides termed elastokines – in particular those of the GXXPG motif – occur as a result of proteolytic degradation of elastin and its non-cross-linked precursor tropoelastin and display several biological activities. For instance, they promote angiogenesis or stimulate cell adhesion, chemotaxis, proliferation, protease activation and apoptosis. Elastin-degrading enzymes such as matrix metalloproteinases, serine proteases and cysteine proteases slowly damage elastin over the lifetime of an organism. The destruction of elastin and the biological processes triggered by elastokines favor the development and progression of various pathological conditions including emphysema, chronic obstructive pulmonary disease, atherosclerosis, metabolic syndrome and cancer. This review gives an overview on types of human elastases and their action on human elastin, including the formation, structure and biological activities of elastokines and their role in common biological processes and severe pathological conditions.