Shotgun Proteomics Research Articles

Semiquantitative analysis of protein expression in heated rat lens using shotgun proteomics.

Previous studies have reported that a strong correlation between the estimated cumulative thermal exposure in the crystalline lens and the incidence of nuclear cataracts; however, the precise relationship between temperature and cataracts remains to be fully elucidated. In the present study, the shotgun liquid chromatography/mass spectroscopy‑based global proteomic approach was applied to investigate cataract‑inducing factors in lens cultured at normal (35.0˚C) and slightly warmer (37.5˚C) conditions. In the rat lens, 190 proteins (total) were identified. Of these, 48 proteins (25.3%) were found in lenses cultured at both 35.0˚C and 37.5˚C. Moreover, 85 proteins (44.7%) were unique to lenses cultured at 35.0˚C, while 57 proteins (30.0%) were unique to lenses cultured at 37.5˚C. Protein expression changes in rat lenses cultured at 37.5˚C were examined using a label‑free semiquantitative approach that uses spectral counting and Gene Ontology analysis. Filensin and vimentin protein expression, key factors in maintaining lens structure, were decreased. These findings may serve as a valuable indicator for elucidating the relationship between temperature and the onset of nuclear cataracts.

BIOM-08. LEVERAGING CSF PROTEOMICS TO OPTIMIZE CNS LYMPHOMA MANAGEMENT

Abstract INTRODUCTION Despite exquisite responses to initial therapy for CNS lymphoma (CNSL), relapse is common and overall responses are disappointing compared to other non-CNS diffuse large B-cell lymphomas. Incorporation of multi-agent intraventricular chemotherapy (MAIVC) alongside systemic therapy is novel. Identification of minimally-invasive biomarkers of response to MAIVC are necessary but lacking. The objectives of this study were to 1) identify baseline cerebrospinal fluid (CSF)-based proteomic predictive biomarkers of MAIVC treatment response and 2) identify tumor burden markers for longitudinal monitoring of MAIVC treatment response in CNSL. METHODS A cohort of patients with primary or secondary CNSL that were treated with MAIVC at Penn State Health (2015-2021) was included. Each MAIVC cycle was administered via Ommaya reservoir and included a 2-drug combination comprised of methotrexate, rituximab, cytarabine, etoposide, topotecan, gemcitabine, or thiotepa. Patient-matched CSF was sampled at pre- and post-therapeutic endpoints and profiled using shotgun proteomics. Patients were grouped by treatment response status. Early responders were defined as patients that achieved malignant cell-free CSF within 6 MAIVC cycles, whereas never responders were those patients that i) did not achieve malignant cell-free CSF by endpoint or ii) required salvage surgery or radiation during MAIVC. RESULTS 59 patients were included (21 primary; 38 secondary CNSL) with a median age of 66 years (range 25-90) and median number of MAIVC cycles of 8 (range 2-23). A proteomic-based classifier, comprised of SGCE, LCP1 and AGRN, discriminated between early and never responders with an AUROC of 0.95. Comparison of CSF at baseline (<3 cycles MAIVC) vs. post-treatment (>12 cycles MAIVC) identified H3F3A, YWHAE, LCP1, CA1 and GAPDH as tumor burden biomarkers. CONCLUSION Here we developed a proteomic signature capable of predicting CNSL patient response to MAIVC, with potential to improve clinical decision making. Furthermore, the biomarkers associated with tumor burden represent important indicators of treatment response. Ongoing efforts are underway to evaluate these candidates as actionable therapeutic targets.

Assessing the applicability of protein residues in combination with lipid residues to reconstruct Indus foodways from Gujarat

When extracted and analysed in tandem, proteomics and lipid residue analysis can provide high resolution identification of ancient foodstuff. Here, we apply lipid residue and shotgun proteomic analyses to 11 ceramic vessel sherds from the Gujarat, India-based Indus Valley Civilization site of Kotada Bhadli. Our results demonstrate variable success. Lipids were successfully recovered from each ceramic vessel and suggest the presence of dairy and meat from cattle/buffalo, and meat from sheep/goat and monogastric animals, such as pigs and birds. Additionally, we were also able to identify the presence of plant products such as leafy vegetables, oils and broomcorn millets. In contrast, none of the extracted proteins could be confidently traced to specific foods or ingredients and were thus unable to contribute to broader interpretations of foodways at Kotada Bhadli. Nevertheless, our results present an opportunity to discuss pathways for improving proteomic methods, and advocate for the need to report negative results as well as positive ones. We support continued efforts to apply multi-proxy approaches to the study of ancient ceramics and consider future applications of shotgun proteomics in this rapidly evolving field.

Essential protocols for decoding the composition and the functional effects of the nanoparticle protein corona

Identifying the function and composition of the protein corona (i.e., the set of host proteins interacting with nanoparticles) is considered a crucial step in the development of nanoparticles for medical and pharmacological applications. Evidence suggests that host proteins can alter NP stability, biocompatibility, and pharmacokinetics features. Therefore, in this review, we provide an updated conceptual, methodological, and experimental guideline for the study of the NP protein corona. We surveyed recent literature (2009–2024) focusing on in vitro and in vivo studies. We show that several methods, including shot-gun proteomics, protein identification after in-gel digestion, and TMT proteomics, must be carefully applied and integrated to shed light on this complex phenomenon. Hence, we discuss in detail the relative protocols, highlighting the importance of the experimental conditions, ranging from the administration route to basic, but determinant, parameters like the kind of biological host fluids, the incubation times and the NP concentrations. Additionally, we propose a series of protocols that involve studying the protein corona using purified serum or plasma proteins, as well as sera depleted of specific complement proteins, to investigate the role of their deposition on the nanoparticle surface. We also explore how the role of the protein corona in inducing uptake by phagocytic cells can be examined; finally, we discuss several methodological approaches to study the effects of different coatings on the composition of the protein corona. Available data indicated that it is possible to characterize and punctually study the differential adsorption of specific proteins onto the nanoparticle surface. This allows designing NP chemical coatings features to actively guide the protein corona formation, thus improving nanotheranostic development.

The Toxin Diversity, Cytotoxicity, and Enzymatic Activity of Cape Cobra (Naja nivea) Venom.

"True" cobras (genus Naja) are among the venomous snakes most frequently involved in snakebite accidents in Africa and Asia. The Cape cobra (Naja nivea) is one of the African cobras of highest medical importance, but much remains to be learned about its venom. Here, we used a shotgun proteomics approach to better understand the qualitative composition of N. nivea venom and tested its cytotoxicity and protease activity as well as its effect on intracellular Ca2+ release and NO synthesis. We identified 156 venom components representing 17 protein families, with the dominant ones being three-finger toxins, mostly of the short-chain type. Two-thirds of the three-finger toxin entries identified were assigned as cytotoxins, while the remainder were categorized as neurotoxins, including short-chain, long-chain, and ancestral three-finger toxins. We also identified snake venom metalloproteinases and members of CRISP, l-amino acid oxidase, and other families. Protease activity and its effect on intracellular Ca2+ release and NO synthesis were low. Phospholipase A2 activity was surprisingly high, despite this toxin family being marginally recovered in the analyzed venom. Cytotoxicity was relevant only at higher venom concentrations, with macrophage and neuroblastoma cell lines showing the lowest viability. These results are in line with the predominantly neurotoxic envenomation symptoms caused by Cape cobra bites. The present overview of the qualitatively complex and functionally intriguing venom of N. nivea may provide insights into the pathobiochemistry of this species' venom.

Changes in the proteomics and metabolomics profiles of Cormus Domestica (L.) fruits during the ripening process

BackgroundCormus domestica (L.) is a monophyletic wild fruit tree belonging to the Rosaceae family, with well-documented use in the Mediterranean region. Traditionally, these fruits are harvested and stored for at least 2 weeks before consumption. During this period, the fruit reaches its well-known and peculiar organoleptic and texture characteristics. However, the spread of more profitable fruit tree species, resulted in its progressive erosion. In this work we performed proteomic and metabolomic fruit analyses at three times after harvesting, to characterise postharvest physiological and molecular changes, it related to nutritional and organoleptic properties at consumption.ResultsProteomics and metabolomics analysis were performed on fruits harvested at different time points: freshly harvested fruit (T0), fruit two weeks after harvest (T1) and fruit four weeks after harvest (T2). Proteomic analysis (Shotgun Proteomic in LC-MS/MS) resulted in 643 proteins identified. Most of the differentially abundant proteins between the three phases observed were involved in the softening process, carbohydrate metabolism and stress responses. Enzymes, such as xyloglucan endotransglucosylase/hydrolase, pectin acetylesterase, beta-galactosidase and pectinesterase, accumulated during fruit ripening and could explain the pulp breakdown observed in C. domestica. At the same time, enzymes abundant in the early stages (T0), such as sucrose synthase and malic enzyme, explain the accumulation of sugars and the lowering of acidity during the process. The metabolites extraction from C. domestica fruits enabled the identification of 606 statistically significant differentially abundant metabolites. Some compounds such as piptamine and resorcinol, well-known for their antimicrobial and antifungal properties, and several bioactive compounds such as endocannabinoids, usually described in the leaves, accumulate in C. domestica fruit during the post-harvest process.ConclusionsThe metabolomic and proteomic profiling of the C. domestica fruit during the postharvest process, evaluated in the study, provides a considerable contribution to filling the existing information gap, enabling the molecular and phytochemical characterisation of this erosion-endangered fruit. Data show biochemical changes that transform the harvested fruit into palatable consumable product.

Carrier-Guided Proteome Analysis in a High Protein Background: An Improved Approach to Host Cell Protein Identification.

Many shotgun proteomics experiments are negatively influenced by highly abundant proteins, such as those measuring residual host cell proteins (HCP) amidst highly abundant recombinant biotherapeutic or plasma proteins amidst albumin and immunoglobulins. While western blotting and ELISAs can reveal the presence of specific low abundance proteins from highly abundant background proteins, mass spectrometry approaches are required to define the low abundance protein composition in these scenarios. The challenge in detecting low abundance proteins in a high protein background by standard shotgun approaches is that spectra are often not triggered on their peptides in data dependent acquisition methods but rather on the highly abundant background peptides. Here, we use tandem mass tags (TMT) to introduce a carrier proteome approach to enhance the detection of proteins, such as from residual host cell proteomes amidst a highly abundant background. Using a mixture of bovine serum albumin (BSA) and E. coli as a mock high background/low abundance target protein formulation, we demonstrate proof-of-principle experiments allowing the improved detection of target proteins amidst a high protein background. While we observed significant coisolation interference, we mitigated it by using a spike-in interference detection TMT channel. Finally, we use the approach to identify 300 residual E. coli proteins from a protein A pulldown of a human IgG antibody, demonstrating that it may be applicable to analysis of HCPs in biotherapeutic protein formulations.

Native Digestion and Shotgun Proteomics for Host Cell Protein Profiling of Adeno-Associated Viruses.

Host cell proteins (HCPs) are contaminants of biotherapeutics produced from engineered living systems; they can influence the product's quality, efficacy, and toxicity. Liquid chromatography coupled to mass spectrometry can detect HCPs thereby mitigating their risks. However, highly abundant biotherapeutics hamper the detection of low-level HCPs. Sample preparation termed native digestion has proven effective to preferentially digest and draw out HCPs from intact antibodies. Here, we adapted native digestion to adeno-associated viruses (AAV), which is a vector gaining popularity for gene therapy. We leveraged quantitative proteomics using capillary-flow liquid chromatography-mass spectrometry (LC-MS) and demonstrated that native digestion was more effective than applying denaturing conditions to extract the HCPs associated with different AAV serotypes.

Plasma Proteomics of Type 2 Diabetes, Hypertension, and Co-Existing Diabetes/Hypertension in Thai Adults.

The study explored proteomics to better understand the relationship between type 2 diabetes (T2DM) and hypertension (HT) in Thai adults, using shotgun proteomics and bioinformatics analysis. Plasma samples were taken from 61 subjects: 14 healthy subjects (mean age = 40.85 ± 7.12), 13 with T2DM (mean age = 57.38 ± 6.03), 16 with HT (mean age = 66.87 ± 10.09), and 18 with coexisting T2DM/HT (mean age = 58.22 ± 10.65). Proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Protein-protein interactions were analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) version 11.5. We identified six unique proteins in T2DM patients, including translationally controlled 1 (TPT1) and nibrin (NBN), which are associated with the DNA damage response. In HT patients, seven unique proteins were identified, among them long-chain fatty acid-CoA ligase (ASCL), which functions in the stimulation of triacylglycerol and cholesterol synthesis, and NADPH oxidase activator 1 (NOXA1), which is involved in high blood pressure via angiotensin II-induced reactive oxygen species (ROS)-generating systems. In coexisting T2DM/HT patients, six unique proteins were identified, of which two-microtubule-associated protein 1A (MAP1A)-might be involved in dementia via RhoB-p53 and diacylglycerol kinase beta (DGKB), associated with lipid metabolism. This study identified new candidate proteins that are possibly involved in the pathology of these diseases.

Impact of a teat disinfectant based on Lactococcus cremoris on the cow milk proteome

BackgroundDairy cow milking practices require cleaning and disinfection of the teat skin before and after milking to ensure the safety and quality of milk and prevent intramammary infections. Antimicrobial proteins of natural origin can be valuable alternatives to traditional disinfectants. In a recent field trial, we demonstrated that a teat dip based on a nisin A-producing Lactococcus cremoris (L) had comparable efficacy to conventional iodophor dip (C) in preventing dairy cow mastitis. Here, we present the differential shotgun proteomics investigation of the milk collected during the trial.MethodsFour groups of quarter milk samples with low (LSCC) and high somatic cell count (HSCC) collected at the beginning (T0) and end (TF) of the trial were analyzed for a total of 28 LSCC (14 LSCC T0 and 14 LSCC TF) and 12 HSCC (6 HSCC T0 and 6 HSCC TF) samples. Milk proteins were digested into peptides, separated by nanoHPLC, and analyzed by tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Tribrid mass spectrometer. The proteins were identified with MaxQuant and interaction networks of the differential proteins were investigated with STRING. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045030.ResultsIn healthy milk (LSCC), we detected 90 and 80 differential proteins at T0 and TF, respectively. At TF, the Lactococcus group showed higher levels of antimicrobial proteins. In mastitis milk (HSCC), we detected 88 and 106 differential proteins at T0 and TF, respectively. In the Lactococcus group, 14 proteins with antimicrobial and immune defense functions were enriched at TF vs. 4 proteins at T0. Cathelicidins were among the most relevant enriched proteins. Western immunoblotting validation confirmed the differential abundance.ConclusionsAt T0, the proteomic differences observed in healthy milk of the two groups were most likely dependent on physiological variation. On the other hand, antimicrobial and immune defense functions were higher in the milk of cows with mammary gland inflammation of the Lactococcus-treated group. Among other factors, the immunostimulatory action of nisin A might be considered as a contributor.

Intraspecific venom variation in the Iberian asp viper (Vipera aspis zinnikeri) across natural and intensive agricultural habitats

Snake venom composition varies at different levels. To date, comparative venom studies have seldom taken into account the role of habitat type in the occurrence of snake venom variation. Here we investigated the presence of venom variation across different populations of the Iberian asp viper (Vipera aspis zinnikeri) inhabiting two contrasting habitats: natural vs. intensive agricultural. We used shotgun proteomics to describe the protein composition of the venoms of six adults from two distinct localities. Furthermore, to test whether local conditions and habitat can alter venom composition in this taxon, we compared the SDS-PAGE profiles of 40 adult venoms from six populations, three in natural habitats and three in intensive agricultural environments. The venoms were composed of 21 toxin families, of which five (CTL, PLA2, VEGF, svSP, and svMP) comprised 69–82 % of each proteome. The relative abundances of toxin families varied considerably at inter- and intra-population levels. Linear regression performed on non-metric multidimensional scaling values showed a significant effect of locality of origin and habitat type on the differences detected between individual SDS-PAGE venom profiles. Our results suggest the presence of regional variation in V. a. zinnikeri venom, potentially reinforcing the role of local pressures in shaping snake venom composition. SignificanceThis work provides the first proteomic characterization of the venom of the Iberian asp viper, Vipera aspis zinnikeri, obtained by means of shotgun proteomics. The statistical analysis of 40 individual SDS-PAGE venom profiles highlights that venom variation in this taxon can be associated with geographical origin and habitat type of the area where each viper was collected. Our results suggest the presence of regional variation in V. a. zinnikeri venom, reinforcing the role that local pressures may play as drivers of snake venom variation.

Leg muscles of migratory locust (Locusta migratoria) as a protein source: Extraction, protein composition and foaming properties

Migratory locust (Locusta migratoria) is an edible insect species that is usually consumed at the adult developmental stage and represents a valuable source of proteins. Locusts can be processed into food ingredients or directly consumed after removal of legs and wings. Legs represent a unique matrix, which comprises a chitin-based exoskeleton typical for insects and muscle tissues commonly occurring also in other animals. Muscle proteins can be easily extracted from traditional animal sources, but the efficiency of these methods for locust legs as well as the functional characteristics of the resulting protein extracts have not been previously explored. This study first compared protein extraction methods at acid, alkaline, and salt-assisted conditions. All three methods resulted in a significantly higher extraction yield from locust leg muscles (0.465–0.595 g/g) than from whole locusts (0.061–0.125 g/g). Shotgun proteomics of the protein fraction extracted at alkaline conditions assigned 310 muscle proteins, comprising components assigned to energy and carbon metabolism, as well as to skeletal, protein folding, membrane trafficking, and cell adhesion functions. The techno-functional potential of locust leg muscles was assessed by a foamability assay. Foam stability of locust leg extracts varied as a function of pH extraction and re-solubilization conditions and was significantly higher (58.3–70.8 %) than for whey proteins taken as a benchmark (38.3–51.7 %); foam capacity at t = 0 h was in the range of 48.6–68.8 %. This study demonstrates that locust legs, which can be a by-product of locust consumption or processing, should be considered a rich source of muscle proteins with promising technological functionality.

Analysis of Antibiotic Response in Clinical Wound Pseudomonas aeruginosa Isolates: Unveiling Proteome Dynamics of Tobramycin-Tolerant Phenotype

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic human pathogen, causing serious chronic infections. P. aeruginosa can adapt efficiently to antibiotic stressors via different genotypic or phenotypic strategies such as resistance and tolerance. The adaptation regulatory system is not always very well understood. In this study, we use shotgun proteomics to investigate the system-level response to tobramycin in two clinical wound P. aeruginosa isolates and PAO1. We profiled each strain for its antibiotic drug-tolerant phenotype using supra-minimum inhibitory concentrations (supra-MIC) of tobramycin and applied proteomics to investigate the protein expression profiles. The MIC revealed that all isolates were susceptible to tobramycin but at supra-MIC concentrations at stationary growth, a degree of tolerance was observed for the isolates. We identified around 40 % of the total proteins encoded by the P. aeruginosa genome and highlighted shared and unique protein signatures for all isolates. Comparative proteome profiling in the absence of antibiotic treatment showed divergent fingerprints, despite similarities in the growth behavior of the isolates. In the presence of tobramycin, the isolates shared a common response in the downregulation of proteins involved in the two-component system, whereas stress response proteins were present at higher levels. Our findings provide insight into the use of proteomic tools to dissect the system-level response in clinical isolates in the absence and presence of antibiotic stress.

Identification of Novel Biomarkers for Post-Kasai Portoenterostomy in Biliary Atresia through Shotgun Proteomics Analysis

Introduction: Biliary Atresia (BA) causes neonatal cholestasis jaundice. The primary therapeutic treatment for BA is the Kasai portoenterostomy. Current diagnostic approaches for BA are imprecise and time-consuming, making early diagnosis crucial for successful treatment outcomes. Objective: This study aims to analyze proteins from Peripheral Blood Mononuclear Cells (PBMCs) obtained from children with BA compared with healthy children Methods and Study Design: We employed a large-scale, total shotgun quantitative serum proteomics approach to analyze the protein from PBMC samples from a discovery cohort. This approach allowed for the simultaneous identification and quantification of multiple proteins, enabling the detection of disease-specific protein expression patterns. The study is proteomic-based study. Results: We identified 24 proteins, by Liquid Chromatography-Mass Spectrometry (LC-MS) analysis that exhibited high discriminatory power for five subjects with BA post-Kasai operation compared to ten healthy controls. ATP2A3, LIN28B, SLC25A3, ITGB3, COX5A, and HLA-B identified proteins of upregulation were predicted to associate with BA post-Kasai operation. Discussion: Our findings highlight the utility of proteomic techniques in BA research. The identified proteomic markers offer promise for improving BA diagnostic accuracy and timeliness, leading to enhanced treatment outcomes for affected children. Conclusion: Proteomic analysis revealed a set of potential biomarkers for early and accurate diagnosis of biliary atresia. These biomarkers hold significant clinical value and have the potential to transform the management of biliary atresia by facilitating timely intervention and improving patient outcomes.

Research biopsies in kidney transplantation: an evaluation of surgical techniques and optimal tissue mass allowing molecular and histological analyses

BackgroundResearch biopsies have great potential to advance scientific knowledge by helping to establish predictors of favourable or unfavourable outcomes in kidney transplantation. We evaluated punch and core biopsies of different sizes to determine the optimal size for clinical use.MethodsA total of 54 punch biopsies and 18 core needle biopsies were retrieved by three transplant surgeons. Each surgeon obtained three separate 2 mm, 3 mm and 4 mm punch biopsy samples and three 23 mm (length) core needle biopsies from two pig kidneys.Results4 mm punch biopsies yielded the greatest amount of protein (2.11 ± 0.41 mg) with good reproducibility between surgeons and biopsy types (Coefficient of Variation ∼ 22.13%). All surgeons found 2 mm biopsies technically challenging to obtain and sample processing was difficult due to the sample size. Shotgun proteomics identified 3853 gene products with no significant difference in the quantitative proteome of 2 mm and 3 mm punch biopsies. However, the expression of 158 Kidney enriched genes, was higher in bigger and deeper 4 mm punch and core needle biopsies compared to 2 mm biopsy. Only 80% of 2 mm biopsies demonstrated the presence of glomeruli, whereas glomeruli were present in 100% of all other biopsy sizes.ConclusionsThe 2 mm punch biopsy has been shown to be challenging to use and frequently provides inadequate tissue for histology and proteomics while 3 mm research biopsies were the smallest size that were technically obtainable with adequate tissue for molecular studies.

Integrated Chemometrics and Data-Independent Acquisition Proteomics for the Discovery of Meat Authenticity Biomarkers: A Study on Early Post-Mortem Pectoralis major Muscle Proteomes of Ross 308 and Ranger Classic Chicken Produced by Organic versus Antibiotic-Free Farming Systems.

Many factors, such as the farming systems and preslaughter rearing practices, can influence the physiological and metabolic functions of poultry with consequent effects on poultry meat quality. In this trial, label-free shotgun proteomics was used to analyze the early post-mortem Pectoralis major muscle proteomes of Ross 308 and Ranger Classic chicken strains raised under two divergent farming systems these being organic and antibiotic-free. The combination of chemometrics using partial-least-square discriminant analysis (PLS-DA) and shotgun proteomics allowed clear discrimination between the different groups. Chicken strains were discriminated by differences in the abundance of 73 and 62 proteins within the antibiotic-free and organic farming systems, respectively. The abundances of 71 and 52 proteins were impacted by the farming system within the Ross 308 and Ranger Classic chicken strains, respectively. The analyses allowed for the proposal of several putative biomarkers of meat authenticity, which were found to be related to muscle structure and energy metabolism pathways. This study is a significant step forward in elucidating the potential of proteomics profiling and chemometrics in chicken meat, which may provide opportunities for the efficient assessment of chicken authenticity.

Integrated multi-omics unveil the impact of H-phosphinic analogs of glutamate and α-ketoglutarate on Escherichia coli metabolism

Desmethylphosphinothricin (L-Glu-γ-PH) is the H-phosphinic analogue of glutamate with carbon-phosphorus-hydrogen (C-P-H) bonds. In L-Glu-γ-PH the phosphinic group acts as a bioisostere of glutamate γ-carboxyl group allowing the molecule to be a substrate of Escherichia coli glutamate decarboxylase, a pyridoxal 5’-phosphate-dependent α-decarboxylase. In addition, the L-Glu-γ-PH decarboxylation product, GABA-PH, is further metabolized by bacterial GABA-transaminase, another pyridoxal 5’-phosphate-dependent enzyme, and succinic semialdehyde dehydrogenase, a NADP+-dependent enzyme. The product of these consecutive reactions, the so-called GABA shunt, is succinate-PH, the H-phosphinic analogue of succinate, a tricarboxylic acid cycle intermediate. Notably, L-Glu-γ-PH displays an antibacterial activity in the same concentration range of well-established antibiotics in E. coli. The dipeptide L-Leu-Glu-γ-PH was shown to display an even higher efficacy, likely as a consequence of an improved penetration into the bacteria.Herein, with the aim of further understanding the intracellular effects of L-Glu-γ-PH, 1H NMR-based metabolomics and LC-MS-based shotgun proteomics were used. This study included also the keto-derivative of L-Glu-γ-PH, α-ketoglutarate-γ-PH (α-KG-γ-PH), which also exhibits antimicrobial activity. L-Glu-γ-PH and α-KG-γ-PH are found to similarly impact the bacterial metabolism, though the overall effect of α-KG-γ-PH is more pervasive. Notably α-KG-γ-PH is converted intracellularly into L-Glu-γ-PH, but the opposite was not found. In general, both molecules impact the pathways where aspartate, glutamate and glutamine are used as precursors for the biosynthesis of related metabolites, activate the acid stress response and deprive cells of nitrogen.This work highlights the multi-target drug potential of L-Glu-γ-PH and α-KG-γ-PH and paves the way for their exploitation as antimicrobials.

Plasma proteomic profiles of patients with HIV infection and coinfection with hepatitis B/C virus undergoing anti‑retroviral therapy.

Chronic liver disease is becoming a leading cause of illness and mortality in patients living with human immunodeficiency virus (HIV; PLWH) undergoing suppressive anti-retroviral therapy. Its primary etiology is coinfection with hepatitis B and C virus (HBV and HCV, respectively). Chronic liver inflammation and fibrosis can potentially lead to the development of hepatocellular carcinoma (HCC). Therefore, monitoring of the disease progression in PLWH is required. The present study aimed to explore plasma protein profiles of PLWH and those coinfected with HBV and HCV using shotgun proteomics. HIV-monoinfected, HIV/HBV-coinfected, HIV/HCV-coinfected and uninfected control individuals were recruited. Patients in the three virus-infected groups had significantly higher levels of liver fibrosis indices (fibrosis-4 score and aspartate aminotransferase to platelet ratio index) compared with the control group. Liquid chromatography-tandem mass spectrometry analysis of plasma samples identified 1,074 proteins that were differentially expressed, where subsequent partial least squares-discriminant analysis model demonstrated clear clustering of proteomes from the four sample groups; 18 proteins that were significantly differentially expressed. Heatmap analysis identified two main groups of proteins, six proteins being upregulated only in the HIV/HBV-coinfection group and 10 proteins downregulated in all three virally infected groups. STITCH 5.0 analysis predicted an interaction network containing two identified proteins in the latter group, specifically ubiquitin interaction motif-containing 1 (UIMC1) and haptoglobin (HP), which are part of the profibrogenic TGF-1β/SMAD, inflammatory TNF and tumor suppressor BRCA1 pathways. Expression levels of UIMC1 and HP were significantly lower in HIV-infected groups compared with those in uninfected controls. Altogether, these proteomics data provide protein expression profiles potentially associated with HIV infection and coinfection with HBV/HCV, which may be applied to predict progression to advanced liver disease or HCC in PLWH.

Impact of mineral and non-mineral sources of iron and sulfur on the metalloproteome of Methanosarcina barkeri.

Methanogens often inhabit sulfidic environments that favor the precipitation of transition metals such as iron (Fe) as metal sulfides, including mackinawite (FeS) and pyrite (FeS2). These metal sulfides have historically been considered biologically unavailable. Nonetheless, methanogens are commonly cultivated with sulfide (HS-) as a sulfur source, a condition that would be expected to favor metal precipitation and thus limit metal availability. Recent studies have shown that methanogens can access Fe and sulfur (S) from FeS and FeS2 to sustain growth. As such, medium supplied with FeS2 should lead to higher availability of transition metals when compared to medium supplied with HS-. Here, we examined how transition metal availability under sulfidic (i.e., cells provided with HS- as sole S source) versus non-sulfidic (cells provided with FeS2 as sole S source) conditions impact the metalloproteome of Methanosarcina barkeri Fusaro. To achieve this, we employed size exclusion chromatography coupled with inductively coupled plasma mass spectrometry and shotgun proteomics. Significant changes were observed in the composition and abundance of iron, cobalt, nickel, zinc, and molybdenum proteins. Among the differences were alterations in the stoichiometry and abundance of multisubunit protein complexes involved in methanogenesis and electron transport chains. Our data suggest that M. barkeri utilizes the minimal iron-sulfur cluster complex and canonical cysteine biosynthesis proteins when grown on FeS2 but uses the canonical Suf pathway in conjunction with the tRNA-Sep cysteine pathway for iron-sulfur cluster and cysteine biosynthesis under sulfidic growth conditions.IMPORTANCEProteins that catalyze biochemical reactions often require transition metals that can have a high affinity for sulfur, another required element for life. Thus, the availability of metals and sulfur are intertwined and can have large impacts on an organismismal biochemistry. Methanogens often occupy anoxic, sulfide-rich (euxinic) environments that favor the precipitation of transition metals as metal sulfides, thereby creating presumed metal limitation. Recently, several methanogens have been shown to acquire iron and sulfur from pyrite, an abundant iron-sulfide mineral that was traditionally considered to be unavailable to biology. The work presented here provides new insights into the distribution of metalloproteins, and metal uptake of Methanosarcina barkeri Fusaro grown under euxinic or pyritic growth conditions. Thorough characterizations of this methanogen under different metal and sulfur conditions increase our understanding of the influence of metal availability on methanogens, and presumably other anaerobes, that inhabit euxinic environments.

Nicotinamide riboside Induced Energy Stress and Metabolic Reprogramming in BEAS-2B Cells.

Nicotinamide riboside (NR), a NAD+ precursor, has received attention due to several health benefits it has induced in experimental models. Studies in cultured cells, animals, and humans consistently show increased NAD+ availability after NR supplementation, which is considered the only mode of NR action that leads to health benefits. In the present study, we show that a persistently low NR concentration (1 μM) in the growth medium of BEAS-2B human cells, grown in a monolayer, induces energy stress, which precedes a cellular NAD+ increase after 192 h. NR concentrations greater than 1 μM under the specified conditions were cytotoxic in the 2D cell culture model, while all concentrations tested in the 3D cell culture model (BEAS-2B cell spheroids exposed to 1, 5, 10, and 50 μM NR) induced apoptosis. Shotgun proteomics revealed that NR modulated the abundance of proteins, agreeing with the observed effects on cellular energy metabolism and cell growth or survival. Energy stress may activate pathways that lead to health benefits such as cancer prevention. Accordingly, the premalignant 1198 cell line was more sensitive to NR cytotoxicity than the phenotypically normal parent BEAS-2B cell line. The role of a mild energy stress induced by low concentrations of NR in its beneficial effects deserves further investigation. On the other hand, strategies to increase the bioavailability of NR require attention to toxic effects that may arise.