Mapping dynamic molecular changes in hippocampal subregions after traumatic brain injury through spatial proteomics.

BACKGROUND: Traumatic brain injury (TBI) often results in diverse molecular responses, challenging traditional proteomic studies that measure average changes at tissue levels and fail to capture the complexity and heterogeneity of the affected tissues. Spatial proteomics offers a solution by providing insights into sub-region-specific alterations within tissues. This study focuses on the hippocampal sub-regions, analyzing proteomic expression profiles in mice at the acute (1 day) and subacute (7 days) phases of post-TBI to understand subregion-specific vulnerabilities and long-term consequences. METHODS: Three mice brains were collected from each group, including Sham, 1-day post-TBI and 7-day post-TBI. Hippocampal subregions were extracted using Laser Microdissection (LMD) and subsequently analyzed by label-free quantitative proteomics. RESULTS: The spatial analysis reveals region-specific protein abundance changes, highlighting the elevation of FN1, LGALS3BP, HP, and MUG-1 in the stratum moleculare (SM), suggesting potential immune cell enrichment post-TBI. Notably, established markers of chronic traumatic encephalopathy, IGHM and B2M, exhibit specific upregulation in the dentate gyrus bottom (DG2) independent of direct mechanical injury. Metabolic pathway analysis identifies disturbances in glucose and lipid metabolism, coupled with activated cholesterol synthesis pathways enriched in SM at 7-Day post-TBI and subsequently in deeper DG1 and DG2 suggesting a role in neurogenesis and the onset of recovery. Coordinated activation of neuroglia and microtubule dynamics in DG2 suggest recovery mechanisms in less affected regions. Cluster analysis revealed spatial variations post-TBI, indicative of dysregulated neuronal plasticity and neurogenesis and further predisposition to neurological disorders. TBI-induced protein upregulation (MUG-1, PZP, GFAP, TJP, STAT-1, and CD44) across hippocampal sub-regions indicates shared molecular responses and links to neurological disorders. Spatial variations were demonstrated by proteins dysregulated in both or either of the time-points exclusively in each subregion (ELAVL2, CLIC1 in PL, CD44 and MUG-1 in SM, and SHOC2, LGALS3 in DG). CONCLUSIONS: Utilizing advanced spatial proteomics techniques, the study unveils the dynamic molecular responses in distinct hippocampal subregions post-TBI. It uncovers region-specific vulnerabilities and dysregulated neuronal processes, and potential recovery-related pathways that contribute to our understanding of TBI's neurological consequences and provides valuable insights for biomarker discovery and therapeutic targets.

Integrated proteomic profiling identifies amino acids selectively cytotoxic to pancreatic cancer cells.

Pancreatic adenocarcinoma (PDAC) is one of the most deadly cancers, characterized by extremely limited therapeutic options and a poor prognosis, as it is often diagnosed during late disease stages. Innovative and selective treatments are urgently needed, since current therapies have limited efficacy and significant side effects. Through proteomics analysis of extracellular vesicles, we discovered an imbalanced distribution of amino acids secreted by PDAC tumor cells. Our findings revealed that PDAC cells preferentially excrete proteins with certain preferential amino acids, including isoleucine and histidine, via extracellular vesicles. These amino acids are associated with disease progression and can be targeted to elicit selective toxicity to PDAC tumor cells. Both in vitro and in vivo experiments demonstrated that supplementation with these specific amino acids effectively eradicated PDAC cells. Mechanistically, we also identified XRN1 as a potential target for these amino acids. The high selectivity of this treatment method allows for specific targeting of tumor metabolism with very low toxicity to normal tissues. Furthermore, we found this treatment approach is easy-to-administer and with sustained tumor-killing effects. Together, our findings reveal that exocytosed amino acids may serve as therapeutic targets for designing treatments of intractable PDAC and potentially offer alternative treatments for other types of cancers.

Cerebrospinal Fluid Protein Markers Indicate Neuro-Damage in SARS-CoV-2-Infected Nonhuman Primates.

Neurologic manifestations are among the most frequently reported complications of COVID-19. However, given the paucity of tissue samples and the highly infectious nature of the etiologic agent of COVID-19, we have limited information to understand the neuropathogenesis of COVID-19. Therefore, to better understand the impact of COVID-19 on the brain, we used mass-spectrometry-based proteomics with a data-independent acquisition mode to investigate cerebrospinal fluid (CSF) proteins collected from two different nonhuman primates, Rhesus Macaque and African Green Monkeys, for the neurologic effects of the infection. These monkeys exhibited minimal to mild pulmonary pathology but moderate to severe central nervous system (CNS) pathology. Our results indicated that CSF proteome changes after infection resolution corresponded with bronchial virus abundance during early infection and revealed substantial differences between the infected nonhuman primates and their age-matched uninfected controls, suggesting these differences could reflect altered secretion of CNS factors in response to SARS-CoV-2-induced neuropathology. We also observed the infected animals exhibited highly scattered data distributions compared to their corresponding controls indicating the heterogeneity of the CSF proteome change and the host response to the viral infection. Dysregulated CSF proteins were preferentially enriched in functional pathways associated with progressive neurodegenerative disorders, hemostasis, and innate immune responses that could influence neuroinflammatory responses following COVID-19. Mapping these dysregulated proteins to the Human Brain Protein Atlas found that they tended to be enriched in brain regions that exhibit more frequent injury following COVID-19. It, therefore, appears reasonable to speculate that such CSF protein changes could serve as signatures for neurologic injury, identify important regulatory pathways in this process, and potentially reveal therapeutic targets to prevent or attenuate the development of neurologic injuries following COVID-19.

Circulating Plasma Exosomal Proteins of Either SHIV-Infected Rhesus Macaque or HIV-Infected Patient Indicates a Link to Neuropathogenesis.

Despite the suppression of human immunodeficiency virus (HIV) replication by combined antiretroviral therapy (cART), 50-60% of HIV-infected patients suffer from HIV-associated neurocognitive disorders (HAND). Studies are uncovering the role of extracellular vesicles (EVs), especially exosomes, in the central nervous system (CNS) due to HIV infection. We investigated links among circulating plasma exosomal (crExo) proteins and neuropathogenesis in simian/human immunodeficiency virus (SHIV)-infected rhesus macaques (RM) and HIV-infected and cART treated patients (Patient-Exo). Isolated EVs from SHIV-infected (SHIV-Exo) and uninfected (CTL-Exo) RM were predominantly exosomes (particle size < 150 nm). Proteomic analysis quantified 5654 proteins, of which 236 proteins (~4%) were significantly, differentially expressed (DE) between SHIV-/CTL-Exo. Interestingly, different CNS cell specific markers were abundantly expressed in crExo. Proteins involved in latent viral reactivation, neuroinflammation, neuropathology-associated interactive as well as signaling molecules were expressed at significantly higher levels in SHIV-Exo than CTL-Exo. However, proteins involved in mitochondrial biogenesis, ATP production, autophagy, endocytosis, exocytosis, and cytoskeleton organization were significantly less expressed in SHIV-Exo than CTL-Exo. Interestingly, proteins involved in oxidative stress, mitochondrial biogenesis, ATP production, and autophagy were significantly downregulated in primary human brain microvascular endothelial cells exposed with HIV+/cART+ Patient-Exo. We showed that Patient-Exo significantly increased blood-brain barrier permeability, possibly due to loss of platelet endothelial cell adhesion molecule-1 protein and actin cytoskeleton structure. Our novel findings suggest that circulating exosomal proteins expressed CNS cell markers-possibly associated with viral reactivation and neuropathogenesis-that may elucidate the etiology of HAND.

BoMiProt 2.0: An update of the bovine milk protein database.

Milk is a biofluid with various functions, containing carbohydrates, lipids, proteins, vitamins, and minerals. Owing to its importance and availability of vast proteomics information, our research group designed a database for bovine milk proteins (N = 3159) containing the primary and secondary information called BoMiProt. Due to the gaining interest and intensively published literature in the last three years, BoMiProt has been upgraded with newer identified proteins (N = 7459) from peer-reviewed journals, significantly expanding the database from different milk fractions (e.g., whey, fat globule membranes, and exosomes). Additionally, class, architecture, topology, and homology, structural classification of proteins, known and predicted disorder, predicted transmembrane helices, and structures have been included. Each protein entry in the database is thoroughly cross-referenced, including 1392 BoMiProt defined proteins provided with secondary information, such as protein function, biochemical properties, post-translational modifications, significance in milk, domains, fold, AlphaFold predicted models and crystal structures. The proteome data in the database can be retrieved using several search parameters using protein name, accession IDs, and FASTA sequence. Overall, BoMiProt represents an extensive compilation of newer proteins, including structural, functional, and hierarchical information, to help researchers better understand mammary gland pathophysiology, including their potential application in improving the nutritional quality of dairy products.

Targeted proteomics using parallel reaction monitoring confirms salivary proteins indicative of metastatic triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype due to the absence of hormonal receptors. Our study aimed to identify and determine the effectiveness of salivary proteins as candidate markers for metastatic TNBC subtype using parallel reaction monitoring mass spectrometry (PRM-MS). Three salivary proteins (lipocalin-1, SMR3B, and plastin-2) that showed significant differential expression in label-free quantitation (LFQ) between TNBC (N = 6) and health subjects (HS; N = 6) were selected for further validation. The developed PRM assay was used to quantify peptides GLST and NNLE (lipocalin-1), VYAL and MINL (Plastin-2) and GPYP, and IPPP (SMR3B) on a different cohort of TNBC patients (N = 20) and HS (N = 20) for evaluating their discriminating performances. Quantitative validation using PRM correlated well with the LFQ results, and 5 peptides from three proteins showed a similar up-or down-regulation. Subsequently, these proteins were validated by Western blot analysis. Compared to one protein's performance as an individual marker, the five-signature panel with salivary GLST, VYAL, MINL, GPYP, and IPPP achieved better performance in differentiating aggressive TNBC and HS with sensitivity (80%) and specificity (95%). Targeted proteomic analysis of the prioritized proteins highlights a peptide-based signature in saliva as the potential predictor to distinguish between TNBC and HS. SIGNIFICANCE OF THE STUDY: This study was designed to identify and quantify potential markers in saliva from the triple-negative breast cancer (TNBC) patients using parallel reaction monitoring assay. Three salivary proteins, Lipocalin-1 (LCN-1), Submaxillary androgen-regulated protein 3B (SMR3B), and Plastin-2 (LCP-1) selected in the discovery-phase were further quantified by targeted proteomics and Western blots. The salivary proteins successfully differentiated TNBC patients from healthy subjects with a sensitivity (80%) and specificity (95%).

PD-L1 interacts with Frizzled 6 to activate ß-catenin and form a positive feedback loop to promote cancer stem cell expansion.

Cancer stem cells (CSCs) drive tumor initiation, progression, metastasis, and drug resistance. We report here that programmed cell death ligand 1 (PD-L1) is constitutively expressed in cancer cells to maintain and expand CSC through a novel mechanism in addition to promoting cancer cell immune evasion. We discovered that PD-L1 interacts with receptor Frizzled 6 to activate ß-catenin signaling and increase ß-catenin-targeted gene expression, such as a putative stem cell marker leucine-rich-repeat-containing G-protein-coupled receptor 5. Blockage of PD-L1 function, using a specific small hairpin RNA or a specific antibody, inhibits disease progression by reducing the CSC population in both colorectal and breast tumors. Moreover, ß-catenin conversely regulates PD-L1 expression through a ß-catenin complex binding site in the PD-L1 promoter. Our discoveries reveal that besides assistant tumor cell immune escaping, PD-L1 and ß-catenin signaling form a positive feedback loop to promote cancer progression through CSC maintenance and expansion.

Integrated physiological and comparative proteomics analysis of contrasting genotypes of pearl millet reveals underlying salt-responsive mechanisms.

Salinity stress poses a significant risk to plant development and agricultural yield. Therefore, elucidation of stress-response mechanisms has become essential to identify salt-tolerance genes in plants. In the present study, two genotypes of pearl millet (Pennisetum glaucum L.) with contrasting tolerance for salinity exhibited differential morpho-physiological and proteomic responses under 150 mM NaCl. The genotype IC 325825 was shown to withstand the stress better than IP 17224. The salt-tolerance potential of IC 325825 was associated with its ability to maintain intracellular osmotic, ionic, and redox homeostasis and membrane integrity under stress. The IC 325825 genotype exhibited a higher abundance of C4 photosynthesis enzymes, efficient enzymatic and non-enzymatic antioxidant system, and lower Na+ /K+ ratio compared with IP 17224. Comparative proteomics analysis revealed greater metabolic perturbation in IP 17224 under salinity, in contrast to IC 325825 that harbored pro-active stress-responsive machinery, allowing its survival and better adaptability under salt stress. The differentially abundant proteins were in silico characterized for their functions, subcellular-localization, associated pathways, and protein-protein interaction. These proteins were mainly involved in photosynthesis/response to light stimulus, carbohydrate and energy metabolism, and stress responses. Proteomics data were validated through expression profiling of the selected genes, revealing a poor correlation between protein abundance and their relative transcript levels. This study has provided novel insights into salt adaptive mechanisms in P. glaucum, demonstrating the power of proteomics-based approaches. The critical proteins identified in the present study could be further explored as potential objects for engineering stress tolerance in salt-sensitive major crops.

Integrated Metabolomics and Proteomics Dynamics of Serum Samples Reveals Dietary Zeolite Clinoptilolite Supplementation Restores Energy Balance in High Yielding Dairy Cows.

Dairy cows can suffer from a negative energy balance (NEB) during their transition from the dry period to early lactation, which can increase the risk of postpartum diseases such as clinical ketosis, mastitis, and fatty liver. Zeolite clinoptilolite (CPL), due to its ion-exchange property, has often been used to treat NEB in animals. However, limited information is available on the dynamics of global metabolomics and proteomic profiles in serum that could provide a better understanding of the associated altered biological pathways in response to CPL. Thus, in the present study, a total 64 serum samples were collected from 8 control and 8 CPL-treated cows at different time points in the prepartum and postpartum stages. Labelled proteomics and untargeted metabolomics resulted in identification of 64 and 21 differentially expressed proteins and metabolites, respectively, which appear to play key roles in restoring energy balance (EB) after CPL supplementation. Joint pathway and interaction analysis revealed cross-talks among valproic acid, leucic acid, glycerol, fibronectin, and kinninogen-1, which could be responsible for restoring NEB. By using a global proteomics and metabolomics strategy, the present study concluded that CPL supplementation could lower NEB in just a few weeks, and explained the possible underlying pathways employed by CPL.

Deciphering the enigma of missing DNA binding domain of LacI family transcription factors.

Catabolite repressor activator (Cra) is a member of the LacI family transcriptional regulator distributed across a wide range of bacteria and regulates the carbon metabolism and virulence gene expression. In numerous studies to crystallize the apo form of the LacI family transcription factor, the N-terminal domain (NTD), which functions as a DNA-binding domain, has been enigmatically missing from the final resolved structures. It was speculated that the NTD is disordered or unstable and gets cleaved during crystallization. Here, we have determined the crystal structure of Cra from Escherichia coli (EcCra). The structure revealed a well-defined electron density for the C-terminal domain (CTD). However, electron density was missing for the first 56 amino acids (NTD). Our data reveal for the first time that EcCra undergoes a spontaneous cleavage at the conserved Asn 50 (N50) site, which separates the N-terminal DNA binding domain from the C-terminal effector molecule binding domain. With the site-directed mutagenesis, we confirm the involvement of residue N50 in the spontaneous cleavage phenomenon. Furthermore, the Isothermal titration calorimetry (ITC) assay of the EcCra-NTD with DNA showed EcCra-NTD is in a functional conformation state and retains its DNA binding activity.

Mammary microbial dysbiosis leads to the zoonosis of bovine mastitis: a One-Health perspective.

Bovine mastitis is a prototypic emerging and reemerging bacterial disease that results in cut-by-cut torture to animals, public health and the global economy. Pathogenic microbes causing mastitis have overcome a series of hierarchical barriers resulting in the zoonotic transmission from bovines to humans either by proximity or remotely through milk and meat. The disease control is challenging and has been attributed to faulty surveillance systems to monitor their emergence at the human-animal interface. The complex interaction between the pathogens, the hidden pathobionts and commensals of the bovine mammary gland that create a menace during mastitis remains unexplored. Here, we review the zoonotic potential of these pathogens with a primary focus on understanding the interplay between the host immunity, mammary ecology and the shift from symbiosis to dysbiosis. We also address the pros and cons of the current management strategies and the extent of the success in implementing the One-Health approach to keep these pathogens at bay.

Quantitative alterations in bovine milk proteome from healthy, subclinical and clinical mastitis during S. aureus infection.

Bovine mastitis, caused by Staphylococcus aureus, is a major impediment to milk production and lacks markers to indicate disease progression in cows and buffaloes. Thus, the focus of this study was to identify proteins marking the transition from subclinical to clinical mastitis. Whey proteins were isolated from 6 group's i.e. healthy, subclinical and clinical mastitis of Holstein Friesian cow and Murrah buffalo. Mass spectrometry and statistical analysis (ANOVA and t-tests) were performed on 12 biological samples each from cow and buffalo (4 per healthy, subclinical and clinical mastitis) resulting in a total of 24 proteome datasets. Collectively, 1479 proteins were identified of which significant proteins were shortlisted by a combination of fold change (≤ 0.5 or ≥ 2) and q < 0.05. Of these proteins, 128 and 163 indicated disease progression in cow and buffalo, respectively. Change in expression of haptoglobin and fibronectin from Holstein Friesian while spermadhesin and osteopontin from Murrah correlated with disease progression. Similarly, angiogenin and cofilin-1 were upregulated while ubiquitin family members were downregulated during disease transition. Subsequently, selected proteins (e.g. osteopontin and fibrinogen-α) were validated by Western blots. The results of this study provide deeper insights into whey proteome dynamics and signature patterns indicative of disease progression. BIOLOGICAL SIGNIFICANCE: Bovine mastitis is the most lethal infectious disease causing a huge economic loss in the dairy industry. In an attempt, to understand the dynamics of whey proteome in response to S. aureus infection, whey protein collected from healthy, subclinical and clinical mastitic HF and Mu were investigated. A total of 1479 proteins were identified, of which 128 and 163 had signature pattern in each stage indicative of the progression of the disease. The results of the present study provide a foundation to better understand the complexity of mastitis that will ultimately help facilitate early therapeutic and husbandry-based intervention to improve animal health and milk quality.

BoMiProt: A database of bovine milk proteins.

Bovine milk has become an important biological fluid for proteomic research due to its nutritional and immunological benefits. To date, over 300 publications have reported changes in bovine milk protein composition based on seasons, lactation stages, breeds, health status and milk fractions while there are no reports on consolidation or overlap of data between studies. Thus, we have developed a literature-based, manually curated open online database of bovine milk proteome, BoMiProt (http://bomiprot.org), with over 3100 proteins from whey, fat globule membranes and exosomes. Each entry in the database is thoroughly cross-referenced including 397 proteins with well-defined information on protein function, biochemical properties, post-translational modifications and significance in milk from different publications. Of 397 proteins, over 199 have been reported with a structural gallery of homology models and crystal structures in the database. The proteome data can be retrieved using several search parameters such as protein name, accession IDs, FASTA sequence. Furthermore, the proteome data can be filtered based on milk fractions, post-translational modifications and/or structures. Taken together, BoMiProt represents an extensive compilation of bovine milk proteins from literature, providing a foundation for future studies to identify specific milk proteins which may be linked to mammary gland pathophysiology. BIOLOGICAL SIGNIFICANCE: Protein data identified from different previously published proteomic studies on bovine milk samples (21 publications) were gathered in the BoMiProt database. Unification of the identified proteins will give researchers an initial reference database on bovine milk proteome to understand the complexities of milk as a biological fluid. BoMiProt has a user-friendly interface with several useful features, including different search criteria for primary and secondary information of proteins along with cross-references to external databases. The database will provide insights into the existing literature and possible future directions to investigate further and improve the beneficial effects of bovine milk components and dairy products on human health.

Quantitative proteomics of milk whey reveals breed and season specific variation in protein abundance in Holstein Friesian cow and Murrah buffalo.

Bovine milk contains different components with nutritional and immunological benefits. It is easily accessible and a rich source of potential markers reflective of pathophysiological conditions; however, little is known about the changes in protein abundance associated with variation across breeds and seasons. In this study, we performed a comprehensive proteomic profiling of whey proteins from Holstein Friesian cow and Murrah buffalo across summer and winter seasons. Collectively, 490 proteins were identified with 113 and 144 differentially expressed proteins across seasons in cow and buffalo, respectively. Breed specific proteins like secretoglobin, e-cadherin and cathepsin-L were detected exclusively in HF, while basigin, conglutinin, and thrombomodulin were identified exclusively in Mu. Acute phase proteins (e.g. haptoglobin and α1AG) were more abundant in summer while antimicrobial proteins (e.g. conglutinin and osteopontin) were upregulated in winter. Similarly, proteins involved in lipid homeostasis (e.g. perilipin 2 and acyl CoA binding protein) showed breed specific variations. Selected representative mass spectrometric proteins (e.g. gelsolin and osteopontin) were validated by Western blot analysis. Results of this study indicate the dynamic nature of milk protein and provide a foundation for future studies of whey proteins which may be linked to diseases specific across breeds and seasons.

Protein glycosylation: Sweet or bitter for bacterial pathogens?

Protein glycosylation systems in many bacteria are often associated with crucial biological processes like pathogenicity, immune evasion and host-pathogen interactions, implying the significance of protein-glycan linkage. Similarly, host protein glycosylation has been implicated in antimicrobial activity as well as in promoting growth of beneficial strains. In fact, few pathogens notably modulate host glycosylation machineries to facilitate their survival. To date, diverse chemical and biological strategies have been developed for conjugate vaccine production for disease control. Bioconjugate vaccines, largely being produced by glycoengineering using PglB (the N-oligosaccharyltransferase from Campylobacter jejuni) in suitable bacterial hosts, have been highly promising with respect to their effectiveness in providing protective immunity and ease of production. Recently, a novel method of glycoconjugate vaccine production involving an O-oligosaccharyltransferase, PglL from Neisseria meningitidis, has been optimized. Nevertheless, many questions on defining antigenic determinants, glycosylation markers, species-specific differences in glycosylation machineries, etc. still remain unanswered, necessitating further exploration of the glycosylation systems of important pathogens. Hence, in this review, we will discuss the impact of bacterial protein glycosylation on its pathogenesis and the interaction of pathogens with host protein glycosylation, followed by a discussion on strategies used for bioconjugate vaccine development.