Proteomic Analysis of the Meniscus Cartilage in Osteoarthritis

The distribution of differential extracellular matrix (ECM) in the lateral and medial menisci can contribute to knee instability, and changes in the meniscus tissue can lead to joint disease. Thus, deep proteomic identification of the lateral and medial meniscus cartilage is expected to provide important information for treatment and diagnosis of various knee joint diseases. We investigated the proteomic profiles of 12 lateral/medial meniscus pairs obtained from excess tissue of osteoarthritis patients who underwent knee arthroscopy surgery using mass spectrometry-based techniques and measured 75 ECM protein levels in the lesions using a multiple reaction monitoring (MRM) assay we developed. A total of 906 meniscus proteins with a 1% false discovery rate (FDR) was identified through a tandem mass tag (TMT) analysis showing that the lateral and medial menisci had similar protein expression profiles. A total of 131 ECM-related proteins was included in meniscus tissues such as collagen, fibronectin, and laminin. Our data showed that 14 ECM protein levels were differentially expressed in lateral and medial lesions (p < 0.05). We present the proteomic characterization of meniscal tissue with mass spectrometry-based comparative proteomic analysis and developed an MRM-based assay of ECM proteins correlated with tissue regeneration. The mass spectrometry dataset has been deposited to the MassIVE repository with the dataset identifier MSV000087753.     

Indirect Immobilised Jagged-1 Enhances Matrisome Proteins Associated with Osteogenic Differentiation of Human Dental Pulp Stem Cells: A Proteomic Study

The indirect immobilisation of Jagged-1 (Jagged-1) promoted osteogenic differentiation of human dental pulp cells (hDPs). Furthermore, the analysis of the Reactome pathway of RNA sequencing data indicates the upregulated genes involved with the extracellular matrix (ECM). Hence, our objective was to investigate the effects of Jagged-1 on proteomic profiles of human dental pulp stem cells (hDPSC). hDPSCs were cultured on the surface coated with human IgG Fc fragment (hFc) and the surface coated with rhJagged1/Fc recombinant protein-coated surface. Cells were differentiated to the osteogenic lineage using an osteogenic differentiation medium (OM) for 14 days, and cells cultured in a growth medium were used as a control. The protein component of the cultured cells was extracted into the cytosol, membrane, nucleus, and cytoskeletal compartment. Subsequently, the proteomic analysis was performed using liquid chromatography–tandem mass spectrometry (LC-MS). Metascape gene list analysis reported that Jagged-1 stimulated the expression of the membrane trafficking protein (DOP1B), which can indirectly improve osteogenic differentiation. hDPSCs cultured on Jagged-1 surface under OM condition expressed COL27A1, MXRA5, COL7A1, and MMP16, which played an important role in osteogenic differentiation. Furthermore, common matrisome proteins of all cellular components were related to osteogenesis/osteogenic differentiation. Additionally, the gene ontology categorised by the biological process of cytosol, membrane, and cytoskeleton compartments was associated with the biomineralisation process. The gene ontology of different culture conditions in each cellular component showed several unique gene ontologies. Remarkably, the Jagged-1_OM culture condition showed the biological process related to odontogenesis in the membrane compartment. In conclusion, the Jagged-1 induces osteogenic differentiation could, mainly through the regulation of protein in the membrane compartment.     

Activity-Based Protein Profiling (ABPP) of Oxidoreductases

Over the last two decades, activity-based protein profiling (ABPP) has been established as a tremendously useful proteomic tool for measuring the activity of proteins in their cellular context, annotating the function of uncharacterized proteins, and investigating the target profile of small-molecule inhibitors. Unlike hydrolases and other enzyme classes, which exhibit a characteristic nucleophilic residue, oxidoreductases have received much less attention in ABPP. In this minireview, the state of the art of ABPP of oxidoreductases is described and the scope and limitations of the existing approaches are discussed. It is noted that several ABPP probes have been described for various oxidases, but none so far for a reductase, which gives rise to opportunities for future research.     

Characterization of New Proteomic Biomarker Candidates in Mucopolysaccharidosis Type IVA

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. Skeletal dysplasia and the related clinical features of MPS IVA are caused by disruption of the cartilage and its extracellular matrix, leading to a growth imbalance. Enzyme replacement therapy (ERT) with recombinant human GALNS has yielded positive results in activity of daily living and endurance tests. However, no data have demonstrated improvements in bone lesions and bone grow thin MPS IVA after ERT, and there is no correlation between therapeutic efficacy and urine levels of keratan sulfate, which accumulates in MPS IVA patients. Using qualitative and quantitative proteomics approaches, we analyzed leukocyte samples from healthy controls (n = 6) and from untreated (n = 5) and ERT-treated (n = 8, sampled before and after treatment) MPS IVA patients to identify potential biomarkers of disease. Out of 690 proteins identified in leukocytes, we selected a group of proteins that were dysregulated in MPS IVA patients with ERT. From these, we identified four potential protein biomarkers, all of which may influence bone and cartilage metabolism: lactotransferrin, coronin 1A, neutral alpha-glucosidase AB, and vitronectin. Further studies of cartilage and bone alterations in MPS IVA will be required to verify the validity of these proteins as potential biomarkers of MPS IVA.     

A Proteomic Approach for Systematic Mapping of Substrates of Human Deubiquitinating Enzymes

The human genome contains nearly 100 deubiquitinating enzymes (DUBs) responsible for removing ubiquitin moieties from a large variety of substrates. Which DUBs are responsible for targeting which substrates remain mostly unknown. Here we implement the ^bioUb approach to identify DUB substrates in a systematic manner, combining gene silencing and proteomics analyses. Silencing of individual DUB enzymes is used to reduce their ubiquitin deconjugating activity, leading to an increase of the ubiquitination of their substrates, which can then be isolated and identified. We report here quantitative proteomic data of the putative substrates of 5 human DUBs. Furthermore, we have built a novel interactive database of DUB substrates to provide easy access to our data and collect DUB proteome data from other groups as a reference resource in the DUB substrates research field.     

Protein Lipidation Types: Current Strategies for Enrichment and Characterization

Post-translational modifications regulate diverse activities of a colossal number of proteins. For example, various types of lipids can be covalently linked to proteins enzymatically or non-enzymatically. Protein lipidation is perhaps not as extensively studied as protein phosphorylation, ubiquitination, or glycosylation although it is no less significant than these modifications. Evidence suggests that proteins can be attached by at least seven types of lipids, including fatty acids, lipoic acids, isoprenoids, sterols, phospholipids, glycosylphosphatidylinositol anchors, and lipid-derived electrophiles. In this review, we summarize types of protein lipidation and methods used for their detection, with an emphasis on the conjugation of proteins with polyunsaturated fatty acids (PUFAs). We discuss possible reasons for the scarcity of reports on PUFA-modified proteins, limitations in current methodology, and potential approaches in detecting PUFA modifications.     

Proteomic Study of Differential Protein Expression in Mouse Lung Tissues after Aerosolized Ricin Poisoning

Ricin is one of the most poisonous natural toxins from plants and is classified as a Class B biological threat pathogen by the Centers for Disease Control and Prevention (CDC) of U.S.A. Ricin exposure can occur through oral or aerosol routes. Ricin poisoning has a rapid onset and a short incubation period. There is no effective treatment for ricin poisoning. In this study, an aerosolized ricin-exposed mouse model was developed and the pathology was investigated. The protein expression profile in the ricin-poisoned mouse lung tissue was analyzed using proteomic techniques to determine the proteins that were closely related to the toxicity of ricin. 2D gel electrophoresis, mass spectrometry and subsequent biological functional analysis revealed that six proteins including Apoa1 apolipoprotein, Ywhaz 14-3-3 protein, Prdx6 Uncharacterized Protein, Selenium-binding protein 1, HMGB1, and DPYL-2, were highly related to ricin poisoning.     

A Proteomic Approach to Elucidate the Changes in Saliva and Serum Proteins of Pigs with Septic and Non-Septic Inflammation

Sepsis is a systemic inflammatory response triggered by an infectious agent and is recognized by the World Health Organization as a global concern, since it is one of the major causes of severe illness in humans and animals. The study of the changes that can occur in saliva and serum in sepsis can contribute to a better understanding of the pathophysiological mechanisms involved in the process and also to discover potential biomarkers that can help in its diagnosis and monitoring. The objective of this study was to characterize the changes that occur in the salivary and serum proteome of pigs with experimentally-induced sepsis. The study included five pigs with sepsis induced by LPS administration and five pigs with non-septic inflammation induced by turpentine for comparative purposes. In saliva, there were eighteen salivary proteins differentially expressed in the sepsis condition and nine in non-septic inflammation. Among these, significant increments in aldolase A and serpin B12 only occurred in the sepsis model. Changes in aldolase A were validated in a larger population of pigs with sepsis due to Streptococcus suis infection. In serum, there were 30 proteins differentially expressed in sepsis group and 26 proteins in the non-septic group, and most of the proteins that changed in both groups were related to non-specific inflammation. In the saliva of the septic animals there were some specific pathways activated, such as the organonitrogen compound metabolic process and lipid transport, whereas, in the serum, one of the main activated pathways was the regulation of protein secretion. Overall, saliva and serum showed different proteome variations in response to septic inflammation and could provide complementary information about the pathophysiological mechanisms occurring in this condition. Additionally, salivary aldolase A could be a potential biomarker of sepsis in pigs that should be confirmed in a larger population.     

Revealing the Changes in Saliva and Serum Proteins of Pigs with Meningitis Caused by Streptococcus Suis: A Proteomic Approach

Meningitis due to Streptococcus suis causes high mortality and morbidity on pig farms and has increasing zoonotic potential worldwide. Saliva proteome analysis would potentially be useful in elucidating pathophysiological changes and mining for new biomarkers to diagnose and monitor S. suis infection. The objective of this study was to investigate the changes in the salivary and serum proteome profile of piglets with meningitis. The LC-MS/MS TMT proteomic approach was used to analyze saliva and serum samples from 20 male piglets: 10 with meningitis and 10 healthy. In saliva, 11 proteins had higher and 10 had lower relative abundance in piglets with meningitis. The proteins with the highest relative abundance were metavinculin (VCL) and desmocollin-2 (DSC2). Adenosine deaminase (ADA) was selected for validation using a spectrophotometric assay and demonstrated excellent performance in the differentiation between healthy and pigs with meningitis due to S. suis. In serum, the most protruding changes occurred for one SERPIN and haptoglobin (HP). In saliva and serum, the highest number of proteins with altered abundance were linked, via the enrichment analysis, with platelet and neutrophil pathways. Overall, meningitis caused by S. suis resulted in specific proteome changes in saliva and serum, reflecting different pathophysiological mechanisms, and marking new potential biomarkers for this infection.     

Proteomic Tools for the Quantitative Analysis of Artificial Peptide Libraries: Detection and Characterization of Target-Amplified PD-1 Inhibitors

We report a quantitative proteomics data analysis pipeline, which coupled to protein-directed dynamic combinatorial chemistry (DDC) experiments, enables the rapid discovery and direct characterization of protein-protein interaction (PPI) modulators. A low-affinity PD-1 binder was incubated with a library of >100 D-peptides under thiol-exchange favoring conditions, in the presence of the target protein PD-1, and we determined the S-linked dimeric species that resulted, amplified in the protein samples versus the controls. We chemically synthesized the target dimer candidates and validated them by thermophoresis binding and protein-protein interaction assays. The results provide a proof-of-concept for using this strategy in the high-throughput search of improved drug-like peptide binders that block therapeutically relevant protein-protein interactions.     

Proteomic Landscape of Adeno-Associated Virus (AAV)-Producing HEK293 Cells

Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing.     

SARS-CoV-2 Membrane Protein: From Genomic Data to Structural New Insights

Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) is composed of four structural proteins and several accessory non-structural proteins. SARS-CoV-2’s most abundant structural protein, Membrane (M) protein, has a pivotal role both during viral infection cycle and host interferon antagonism. This is a highly conserved viral protein, thus an interesting and suitable target for drug discovery. In this paper, we explain the structural nature of M protein homodimer. To do so, we developed and applied a detailed and robust in silico workflow to predict M protein dimeric structure, membrane orientation, and interface characterization. Single Nucleotide Polymorphisms (SNPs) in M protein were retrieved from over 1.2 M SARS-CoV-2 genomes and proteins from the Global Initiative on Sharing All Influenza Data (GISAID) database, 91 of which were located at the predicted dimer interface. Among those, we identified SNPs in Variants of Concern (VOC) and Variants of Interest (VOI). Binding free energy differences were evaluated for dimer interfacial SNPs to infer mutant protein stabilities. A few high-prevalent mutated residues were found to be especially relevant in VOC and VOI. This realization may be a game-changer to structure-driven formulation of new therapeutics for SARS-CoV-2.     

Integrated Proteomic and Metabolomic Profiling of Phytophthora cinnamomi Attack on Sweet Chestnut (Castanea sativa) Reveals Distinct Molecular Reprogramming Proximal to the Infection Site and Away from It

Phytophthora cinnamomi is one of the most invasive tree pathogens that devastates wild and cultivated forests. Due to its wide host range, knowledge of the infection process at the molecular level is lacking for most of its tree hosts. To expand the repertoire of studied Phytophthora–woody plant interactions and identify molecular mechanisms that can facilitate discovery of novel ways to control its spread and damaging effects, we focused on the interaction between P. cinnamomi and sweet chestnut (Castanea sativa), an economically important tree for the wood processing industry. By using a combination of proteomics, metabolomics, and targeted hormonal analysis, we mapped the effects of P. cinnamomi attack on stem tissues immediately bordering the infection site and away from it. P. cinnamomi led to a massive reprogramming of the chestnut proteome and accumulation of the stress-related hormones salicylic acid (SA) and jasmonic acid (JA), indicating that stem inoculation can be used as an easily accessible model system to identify novel molecular players in P. cinnamomi pathogenicity.     

Proteomic Approaches to Uncover Salt Stress Response Mechanisms in Crops

Salt stress is an unfavorable outcome of global climate change, adversely affecting crop growth and yield. It is the second-biggest abiotic factor damaging the morphological, physio-biochemical, and molecular processes during seed germination and plant development. Salt responses include modulation of hormonal biosynthesis, ionic homeostasis, the antioxidant defense system, and osmoprotectants to mitigate salt stress. Plants trigger salt-responsive genes, proteins, and metabolites to cope with the damaging effects of a high salt concentration. Enhancing salt tolerance among crop plants is direly needed for sustainable global agriculture. Novel protein markers, which are used for crop improvement against salt stress, are identified using proteomic techniques. As compared to single-technique approaches, the integration of genomic tools and exogenously applied chemicals offers great potential in addressing salt-stress-induced challenges. The interplay of salt-responsive proteins and genes is the missing key of salt tolerance. The development of salt-tolerant crop varieties can be achieved by integrated approaches encompassing proteomics, metabolomics, genomics, and genome-editing tools. In this review, the current information about the morphological, physiological, and molecular mechanisms of salt response/tolerance in crops is summarized. The significance of proteomic approaches to improve salt tolerance in various crops is highlighted, and an integrated omics approach to achieve global food security is discussed. Novel proteins that respond to salt stress are potential candidates for future breeding of salt tolerance.     

Mycobacterial Membrane Protein Large 3 (MmpL3) Inhibitors: A Promising Approach to Combat Tuberculosis

Tuberculosis is a prominent aliment throughout the world and a leading cause of mortality among infectious diseases. Drug development for multi-drug resistance and reducing the current therapy time is the top priority. Mycobacterial membrane protein large 3 (MmpL3) is a promising target with high potential, however, it has not been explored to its greatest potential. It is a membrane transporter that translocates trehalose-monomycolate which is a precursor for the synthesis of mycolic acid that is essential for the synthesis of the bacterial cell wall and is pathogenic in nature. In this review, we have discussed the current development of MmpL3 inhibitors, different scaffolds, their derivatives, and their synthetic schemes and provide insight into the challenges in developing these inhibitors.     

Nanoencapsulation of Gla-Rich Protein (GRP) as a Novel Approach to Target Inflammation

Chronic inflammation is a major driver of chronic inflammatory diseases (CIDs), with a tremendous impact worldwide. Besides its function as a pathological calcification inhibitor, vitamin K-dependent protein Gla-rich protein (GRP) was shown to act as an anti-inflammatory agent independently of its gamma-carboxylation status. Although GRP’s therapeutic potential has been highlighted, its low solubility at physiological pH still constitutes a major challenge for its biomedical application. In this work, we produced fluorescein-labeled chitosan-tripolyphosphate nanoparticles containing non-carboxylated GRP (ucGRP) (FCNG) via ionotropic gelation, increasing its bioavailability, stability, and anti-inflammatory potential. The results indicate the nanosized nature of FCNG with PDI and a zeta potential suitable for biomedical applications. FCNG’s anti-inflammatory activity was studied in macrophage-differentiated THP1 cells, and in primary vascular smooth muscle cells and chondrocytes, inflamed with LPS, TNFα and IL-1β, respectively. In all these in vitro human cell systems, FCNG treatments resulted in increased intra and extracellular GRP levels, and decreased pro-inflammatory responses of target cells, by decreasing pro-inflammatory cytokines and inflammation mediators. These results suggest the retained anti-inflammatory bioactivity of ucGRP in FCNG, strengthening the potential use of ucGRP as an anti-inflammatory agent with a wide spectrum of application, and opening up perspectives for its therapeutic application in CIDs.