Biological Implications of a Stroke Therapy Based in Neuroglobin Hyaluronate Nanoparticles. Neuroprotective Role and Molecular Bases

Exogenous neuroprotective protein neuroglobin (Ngb) cannot cross the blood–brain barrier. To overcome this difficulty, we synthesized hyaluronate nanoparticles (NPs), able to deliver Ngb into the brain in an animal model of stroke (MCAO). These NPs effectively reached neurons, and were microscopically identified after 24 h of reperfusion. Compared to MCAO non-treated animals, those treated with Ngb-NPs showed survival rates up to 50% higher, and better neurological scores. Tissue damage improved with the treatment, but no changes in the infarct volume or in the oxidative/nitrosative values were detected. A proteomics approach (p-value < 0.02; fold change = 0.05) in the infarcted areas showed a total of 219 proteins that significantly changed their expression after stroke and treatment with Ngb-NPs. Of special interest, are proteins such as FBXO7 and NTRK2, which were downexpressed in stroke, but overexpressed after treatment with Ngb-NPs; and ATX2L, which was overexpressed only under the effect of Ngb. Interestingly, the proteins affected by the treatment with Ngb were involved in mitochondrial function and cell death, endocytosis, protein metabolism, cytoskeletal remodeling, or synaptic function, and in regenerative processes, such as dendritogenesis, neuritogenesis, or sinaptogenesis. Consequently, our pharmaceutical preparation may open new therapeutic scopes for stroke and possibly for other neurodegenerative pathologies.

Comparative Proteomics of Mulberry Leaves at Different Developmental Stages Identify Novel Proteins Function Related to Photosynthesis

Mulberry leaves at different positions are different in photosynthetic rate, nutrient substance and feeding impact to silkworms. Here, we investigated the proteomic differences of the first (L1), sixth (L6), and twentieth (L20) mulberry leaves at different stem positions (from top to the base) using a label-free quantitative proteomics approach. L1 contained less developed photosynthetic apparatus but was more active in protein synthesis. L20 has more channel proteins and oxidoreductases relative to L6. Proteins that detected in all measured leaves were classified into three groups according to their expression patterns in L1, L6, and L20. The protein group that displayed the maximum amount in L6 has the highest possibility that function related to photosynthesis. Nine function unknown proteins belong to this group were further analyzed in the light responsive expression, evolutionary tree and sub-cellular localization analysis. Based on the results, five proteins were suggested to be involved in photosynthesis. Taken together, these results reveal the molecular details of different roles of mulberry leaves at different developmental stages and contribute to the identification of five proteins that might function related to photosynthesis.

Comprehensive Profiling of Mammalian Tribbles Interactomes Implicates TRIB3 in Gene Repression

The three human Tribbles (TRIB) pseudokinases have been implicated in a plethora of signaling and metabolic processes linked to cancer initiation and progression and can potentially be used as biomarkers of disease and prognosis. While their modes of action reported so far center around protein–protein interactions, the comprehensive profiling of TRIB interactomes has not been reported yet. Here, we have developed a robust mass spectrometry (MS)-based proteomics approach to characterize Tribbles’ interactomes and report a comprehensive assessment and comparison of the TRIB1, -2 and -3 interactomes, as well as domain-specific interactions for TRIB3. Interestingly, TRIB3, which is predominantly localized in the nucleus, interacts with multiple transcriptional regulators, including proteins involved in gene repression. Indeed, we found that TRIB3 repressed gene transcription when tethered to DNA in breast cancer cells. Taken together, our comprehensive proteomic assessment reveals previously unknown interacting partners and functions of Tribbles proteins that expand our understanding of this family of proteins. In addition, our findings show that MS-based proteomics provides a powerful tool to unravel novel pseudokinase biology.

Photoaffinity capture compounds to profile the Magic Spot Nucleotide interactomes

Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival when confronted with adverse external challenges. In times of antibiotic crisis, a detailed understanding of the stringent response is of critical importance, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium cell lysates applying a family of trifunctional photoaffinity capture compounds. We introduce different MSN probes covering diverse phosphorylation patterns, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of diverse putative MSN receptors both from cytosolic and membrane fractions that, upon validation, unveil new MSN targets. We find, for example, that the dinucleoside polyphosphate hydrolase activity of the non-Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The photoaffinity capture compounds described herein will be useful to identify MSN interactomes under varying conditions and across bacterial species.

Protein Diversity and Immune Specificity of Hemocyanin From Shrimp Litopenaeus vannamei

Hemocyanin is an important non-specific innate immune defense molecule with phenoloxidase, antiviral, antibacterial, hemolytic, and antitumor activities. To better understand the mechanism of functional diversity, proteomics approach was applied to characterize hemocyanin (HMC) expression profiles from Litopenaeus vannamei. At first, hemocyanin was purified by Sephadex G-100 and DEAE-cellulose (DE-52) columns from shrimp serum, and 34 protein spots were identified as HMC on the 2-DE gels. Furthermore, we found that 9 HMC spots about 75 or 77 kDa were regulated by Streptococcus agalactiae and Vibrio parahaemolyticus infection at 6, 12, and 24 h. In addition, 6 different pathogen-binding HMC fractions, viz., HMC-Mix, HMC-Vp, HMC-Va, HMC-Vf, HMC-Ec, and HMC-Sa, showed different agglutinative and antibacterial activities. Moreover, lectin-blotting analysis showed significant differences in glycosylation level among HMC isomers and bacteria-binding HMC fractions. Particularly, the agglutinative activities of the HMC fractions were almost completely abolished when HMC was deglycosylated by O-glycosidase, which suggest that O-linked sugar chains of HMC played important roles in the innate immune recognition. Our findings demonstrated for the first time that L. vannamei HMC had molecular diversity in protein level, which is closely associated with its ability to recognize diverse pathogens, whereas glycan modification probably contributed to HMC’s diversity and multiple immune activities.

Oxidative stress-induced FABP5 S-glutathionylation protects against acute lung injury by suppressing inflammation in macrophages

Oxidative stress contributes to the pathogenesis of acute lung injury. Protein S-glutathionylation plays an important role in cellular antioxidant defense. Here we report that the expression of deglutathionylation enzyme Grx1 is decreased in the lungs of acute lung injury mice. The acute lung injury induced by hyperoxia or LPS is significantly relieved in Grx1 KO and Grx1fl/flLysMcre mice, confirming the protective role of Grx1-regulated S-glutathionylation in macrophages. Using a quantitative redox proteomics approach, we show that FABP5 is susceptible to S-glutathionylation under oxidative conditions. S-glutathionylation of Cys127 in FABP5 promotes its fatty acid binding ability and nuclear translocation. Further results indicate S-glutathionylation promotes the interaction of FABP5 and PPARβ/δ, activates PPARβ/δ target genes and suppresses the LPS-induced inflammation in macrophages. Our study reveals a molecular mechanism through which FABP5 S-glutathionylation regulates macrophage inflammation in the pathogenesis of acute lung injury.

A proteomics study identifying interactors of the FSHD2 gene product SMCHD1 reveals RUVBL1-dependent DUX4 repression

Structural Maintenance of Chromosomes Hinge Domain Containing 1 (SMCHD1) is a chromatin repressor, which is mutated in > 95% of Facioscapulohumeral dystrophy (FSHD) type 2 cases. In FSHD2, SMCHD1 mutations ultimately result in the presence of the cleavage stage transcription factor DUX4 in muscle cells due to a failure in epigenetic repression of the D4Z4 macrosatellite repeat on chromosome 4q, which contains the DUX4 locus. While binding of SMCHD1 to D4Z4 and its necessity to maintain a repressive D4Z4 chromatin structure in somatic cells are well documented, it is unclear how SMCHD1 is recruited to D4Z4, and how it exerts its repressive properties on chromatin. Here, we employ a quantitative proteomics approach to identify and characterize novel SMCHD1 interacting proteins, and assess their functionality in D4Z4 repression. We identify 28 robust SMCHD1 nuclear interactors, of which 12 are present in D4Z4 chromatin of myocytes. We demonstrate that loss of one of these SMCHD1 interacting proteins, RuvB-like 1 (RUVBL1), further derepresses DUX4 in FSHD myocytes. We also confirm the interaction of SMCHD1 with EZH inhibitory protein (EZHIP), a protein which prevents global H3K27me3 deposition by the Polycomb repressive complex PRC2, providing novel insights into the potential function of SMCHD1 in the repression of DUX4 in the early stages of embryogenesis. The SMCHD1 interactome outlined herein can thus provide further direction into research on the potential function of SMCHD1 at genomic loci where SMCHD1 is known to act, such as D4Z4 repeats, the inactive X chromosome, autosomal gene clusters, imprinted loci and telomeres.

Identification of antibiotic resistance proteins via MiCId's augmented workflow. A mass spectrometry-based proteomics approach

Fast and accurate identifications of pathogenic bacteria along with their associated antibiotic resistance proteins are of paramount importance for patient treatments and public health. While mass spectrometry has become an important, technique for diagnostics of infectious disease, there is a need for mass spectrometry workflows offering this capability. To meet this need, we have augmented the previously published Microorganism Classification and Identification (MiCId) workflow for this capability. To evaluate the performance of the newly augmented MiCId workflow, we have used MS/MS datafiles from samples of 10 antibiotic resistance bacterial strains belonging to three different species: Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The evaluation results show that MiCId's workflow has a sensitivity value around 85% (with a lower bound at about 72%) and a precision greater than 95% in the identification of antibiotic resistance proteins. Using MS/MS datasets from samples of two bacterial clonal isolates, one being antibiotic-sensitive while the other (obtained from the same patient at different times) being multidrug-resistant, we applied MiCId's workflow to investigate possible mechanisms of antibiotic resistance in these pathogenic bacteria; the results showed that MiCId's conclusions are in agreement with the published study. Furthermore, we show that MiCId's workflow is fast. It provides microorganismal identifications, protein identifications, sample biomass estimates, and antibiotic resistance protein identifications in 6-17 minutes per MS/MS sample using computing resources that are available in most desktop and laptop computers, making it a highly portable workflow. This study demonstrated that MiCId's workflow is fast, portable, and with high sensitivity and high precision, making it a valuable tool for rapid identifications of bacteria as well as detection of their antibiotic resistance proteins. The new version of MiCId (v.07.01.2021) is freely available for download at https://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html.

On-tissue spatial proteomics integrating MALDI-MS imaging with shotgun proteomics reveals soy consumption-induced biomarkers in a fragile X syndrome mouse model

Soy-based diets are associated with increased seizures and autism. Thus, there is an acute need for unbiased protein biomarker identification in Fragile X syndrome (FXS) in response to soy consumption. Herein, we present a spatial proteomics approach integrating mass spectrometry imaging (MSI) with label-free proteomics in a mouse model of FXS to map the spatial distribution and quantify the levels of proteins in the hippocampus and hypothalamus brain regions. In total, 1,004 unique peptides were spatially resolved, demonstrating the diverse array of peptidomes present in the tissue slices and the broad coverage of the strategy. A group of proteins that are known to be involved in the GABAergic system, synaptic transmission, and co-expression network analysis indicated that protein in soy group was significantly associated with metabolism and synapse modules in the Fmr1KO brain. Ultimately, this spatial proteomics work laid the ground for identifying novel therapeutic targets and biomarkers for FXS.