Identify Candidate Proteins Research Articles

Discovery of plasma proteome markers associated with clinical outcome and immunological stress after cardiac surgery.

Molecular mechanisms underlying perioperative acute phase reactions in cardiac surgery are largely unknown. We aimed to characterise perioperative alterations of the acute phase plasma proteome in a cohort of adult patients undergoing on-pump cardiac surgery using high-throughput mass spectrometry and to identify candidate proteins potentially relevant to postoperative clinical outcome through a novel, multi-step approach. This study is an analysis of the Bern Perioperative Biobank, a prospective cohort of adults who underwent cardiac surgery with the use of cardiopulmonary bypass (CPB) at Bern University Hospital between January and December 2019. Blood samples were taken before induction of anaesthesia and on postoperative day one. Proteomic analyses were performed by mass spectrometry. Through a multi-step, exploratory approach, hit-proteins were first identified according to their perioperative prevalence and dynamics. The set of hit-proteins were associated with predefined clinical outcome measures (all-cause one-year mortality, length of hospital stay, postoperative myocardial infarction and stroke until hospital discharge). 192 patients [75.5% male, median age 67.0 (IQR 60.0-73.0)] undergoing cardiac surgery with the use of CPB were included in this analysis. In total, we identified and quantified 402 proteins across all samples, whereof 30/402 (7%) proteins were identified as hit-proteins. Three hit-proteins-LDHB, VCAM1 and IGFBP2-demonstrated the strongest associations with clinical outcomes. After adjustment both for age, sex, BMI and for multiple comparisons, the scaled preoperative levels of IGFBP2 were associated with 1-year all-cause mortality (OR 10.63; 95% CI: 2.93-64.00; p = 0.046). Additionally, scaled preoperative levels of LDHB (OR 5.58; 95% CI: 2.58-8.57; p = 0.009) and VCAM1 (OR 2.32; 95% CI: 0.88-3.77; p = 0.05) were found to be associated with length of hospital stay. We identified a subset of promising candidate plasma proteins relevant to outcome after on-pump cardiac surgery. IGFBP2 showed a strong association with clinical outcome measures and a significant association of preoperative levels with 1-year all-cause mortality. Other proteins strongly associated with outcome were LDHB and VCAM1, reflecting the dynamics in the acute phase response, inflammation and myocardial injury. We recommend further investigation of these proteins as potential outcome markers after cardiac surgery. ClinicalTrials.gov; NCT04767685, data are available via ProteomeXchange with identifier PXD046496.

Mitophagy biomarkers are changed in the continuum of Alzheimer’s disease

AbstractBackgroundAutophagy is a cellular self “garbage clearance ”system through which cells eliminate and recycle dysfunctional cytoplasmatic components, such as defective organelles and misfolded protein aggregates. Mitophagy is the sub‐type of autophagy that recognizes and degrades damaged or superfluous mitochondria to maintain cellular homeostasis. Emerging evidence demonstrates that autophagy and mitophagy are impaired in Alzheimer’s disease (AD); its upregulation ameliorates Aß pathology and slows down the cognitive decline in animal models of AD, thus representing a potentially treatable target. The change in the mitophagy biomarkers in individuals with AD needs to be clarified. We aimed to explore the changes of mitophagy biomarkers in biomarkers‐defined individuals in various stages of AD and compare them to cognitively unimpaired individuals (CU).MethodWe included 228 biomarker‐defined individuals from the Czech Brain Aging Study:98 AD dementia (ADD), 88 mild cognitive impairment due to AD (MCI‐AD), and 42 CU individuals. They underwent clinical examination, complex neuropsychological assessment, and brain MRI. Commercial ELISA kits were used to measure standard AD biomarkers in CSF (Aß42, Aß40, pTau, tTau), a biomarker of neurodegeneration (neurofilament light chain – NfL), synaptic dysfunction (neurogranin, Ng), and mitophagy biomarkers in CSF (PINK1‐ mitochondrial kinase crucial for mitophagy) and in serum (BNIP3L‐ mitophagy receptor, TFEB‐ transcription factor critical for lysosomal degradation).ResultWe found a significant increase in PINK1 CSF and BNIP3L serum levels in ADD compared to MCI‐AD and CU (ps<.001). TFEB serum levels were decreased in ADD compared to MCI‐AD and CU (ps<.05). Additionally, in ADD, PINK1 correlated with NfL (r = 0.27) and Ng (r = 0.47). In MCI‐AD, there was a correlation between PINK1 and Ng (r = 0.51), but not with NfL. There was no correlation in CU individuals with NfL or Ng.ConclusionWe identified candidate proteins of mitophagy that are changed in various cognitive stages of AD. In the ADD stage, there is an upregulation of the mitophagy inducers, PINK1 and BNIP3L, together with decreased levels of TFEB, a master regulator of lysosomal biogenesis. One of the possible explanations is that these findings reflect impairment in the final stage of autophagy–lysosomal degradation. Further research is needed to clarify its pathophysiological importance in the AD cascade.

Genetic mechanisms and biological processes underlying host response to ophidiomycosis (snake fungal disease) inferred from tissue-specific transcriptome analyses.

Emerging infectious diseases in wildlife species caused by pathogenic fungi are of growing concern, yet crucial knowledge gaps remain for diseases with potentially large impacts. For example, there is detailed knowledge about host pathology and mechanisms underlying response for chytridiomycosis in amphibians and white-nose syndrome in bats, but such information is lacking for other more recently described fungal infections. One such disease is ophidiomycosis, caused by the fungus Ophidiomyces ophidiicola, which has been identified in many species of snakes, yet the biological mechanisms and molecular changes occurring during infection are unknown. To gain this information, we performed a controlled experimental infection in captive Prairie rattlesnakes (Crotalus viridis) with O. ophidiicola at two different temperatures: 20 and 26°C. We then compared liver, kidney, and skin transcriptomes to assess tissue-specific genetic responses to O. ophidiicola infection. Given previous histopathological studies and the fact that snakes are ectotherms, we expected highest fungal activity on skin and a significant impact of temperature on host response. Although we found fungal activity to be localized on skin, most of the differential gene expression occurred in internal tissues. Infected snakes at the lower temperature had the highest host mortality whereas two-thirds of the infected snakes at the higher temperature survived. Our results suggest that ophidiomycosis is likely a systemic disease with long-term effects on host response. Our analysis also identified candidate protein coding genes that are potentially involved in host response, providing genetic tools for studies of host response to ophidiomycosis in natural populations.

Interception Proximity Labeling for Interrogating Cell Efflux Microenvironment.

The difficulty in elucidating the microenvironment of extracellular H2O2 efflux has led to the lack of a critical extracellular link in studies of the mechanisms of redox signaling pathways. Herein, we mounted horseradish peroxidase (HRP) to glycans expressed globally on the living cell surface and constructed an interception proximity labeling (IPL) platform for H2O2 efflux. The release of endogenous H2O2 is used as a "physiological switch" for HRP to enable proximity labeling. Using this platform, we visualize the oxidative stress state of tumor cells under the condition of nutrient withdrawal, as well as that of macrophages exposed to nonparticulate stimuli. Furthermore, in combination with a proteomics technique, we identify candidate proteins at the invasion interface between fungal mimics (zymosan) and macrophages by interception labeling of locally accumulated H2O2 and confirm that Toll-like receptor 2 binds zymosan in a glycan-dependent manner. The IPL platform has great potential to elucidate the mechanisms underlying biological processes involving redox pathways.

Identification of plasma proteomic signatures associated with the progression of cardia gastric cancer and precancerous lesions

ObjectiveConsidering that there are no effective biomarkers for the screening of cardia gastric cancer (CGC), we developed a noninvasive diagnostic approach, employing data-independent acquisition (DIA) proteomics to identify candidate protein markers. MethodsPlasma samples were obtained from 40 subjects, 10 each for CGC, cardia high-grade dysplasia (CHGD), cardia low-grade dysplasia (CLGD), and healthy controls. Proteomic profiles were obtained through liquid chromatography-mass spectrometry (LC-MS/MS-based DIA proteomics. Candidate plasma proteins were identified by weighted gene co-expression network analysis (WGCNA) combined with machine learning and further validated by the Human Protein Atlas (HPA) database. The area under the receiver operating characteristic curve (AUC) was used to evaluate the performance of the biomarker panel. ResultsThere was a clear distinction in proteomic features among CGC, CHGD, CLGD, and the healthy controls. According to the WGCNA, we found 42 positively associated and 164 inversely associated proteins related to CGC progression and demonstrated several canonical cancer-associated pathways. Combined with the results from random forests, LASSO regression, and immunohistochemical results from the HPA database, we identified three candidate proteins (GSTP1, CSRP1, and LY6G6F) that could together distinguish CLGD (AUC = 0.91), CHGD (AUC = 0.99) and CGC (AUC = 0.98) from healthy controls with excellent accuracy. ConclusionsThe panel of protein biomarkers showed promising diagnostic potential for CGC and precancerous lesions. Further validation and a larger-scale study are warranted to assess its potential clinical applications, suggesting a potential avenue for CGC prevention in the future.

A microglial activity state biomarker panel differentiates FTD-granulin and Alzheimer’s disease patients from controls

BackgroundWith the emergence of microglia-modulating therapies there is an urgent need for reliable biomarkers to evaluate microglial activation states.MethodsUsing mouse models and human induced pluripotent stem cell-derived microglia (hiMGL), genetically modified to yield the most opposite homeostatic (TREM2-knockout) and disease-associated (GRN-knockout) states, we identified microglia activity-dependent markers. Non-targeted mass spectrometry was used to identify proteomic changes in microglia and cerebrospinal fluid (CSF) of Grn- and Trem2-knockout mice. Additionally, we analyzed the proteome of GRN- and TREM2-knockout hiMGL and their conditioned media. Candidate marker proteins were tested in two independent patient cohorts, the ALLFTD cohort (GRN mutation carriers versus non-carriers), as well as the proteomic data set available from the EMIF-AD MBD study.ResultsWe identified proteomic changes between the opposite activation states in mouse microglia and CSF, as well as in hiMGL cell lysates and conditioned media. For further verification, we analyzed the CSF proteome of heterozygous GRN mutation carriers suffering from frontotemporal dementia (FTD). We identified a panel of six proteins (FABP3, MDH1, GDI1, CAPG, CD44, GPNMB) as potential indicators for microglial activation. Moreover, we confirmed three of these proteins (FABP3, GDI1, MDH1) to be significantly elevated in the CSF of Alzheimer’s (AD) patients. Remarkably, each of these markers differentiated amyloid-positive cases with mild cognitive impairment (MCI) from amyloid-negative individuals.ConclusionsThe identified candidate proteins reflect microglia activity and may be relevant for monitoring the microglial response in clinical practice and clinical trials modulating microglial activity and amyloid deposition. Moreover, the finding that three of these markers differentiate amyloid-positive from amyloid-negative MCI cases in the AD cohort suggests that these proteins associate with a very early immune response to seeded amyloid. This is consistent with our previous findings in the Dominantly Inherited Alzheimer’s Disease Network (DIAN) cohort, where soluble TREM2 increases as early as 21 years before symptom onset. Moreover, in mouse models for amyloidogenesis, seeding of amyloid is limited by physiologically active microglia further supporting their early protective role. The biological functions of some of our main candidates (FABP3, CD44, GPNMB) also further emphasize that lipid dysmetabolism may be a common feature of neurodegenerative disorders.

Identification of novel protein biomarkers and drug targets for colorectal cancer by integrating human plasma proteome with genome

BackgroundThe proteome is a major source of therapeutic targets. We conducted a proteome-wide Mendelian randomization (MR) study to identify candidate protein markers and therapeutic targets for colorectal cancer (CRC).MethodsProtein quantitative trait loci (pQTLs) were derived from seven published genome-wide association studies (GWASs) on plasma proteome, and summary-level data were extracted for 4853 circulating protein markers. Genetic associations with CRC were obtained from a large-scale GWAS meta-analysis (16,871 cases and 26,328 controls), the FinnGen cohort (4957 cases and 304,197 controls), and the UK Biobank (9276 cases and 477,069 controls). Colocalization and summary-data-based MR (SMR) analyses were performed sequentially to verify the causal role of candidate proteins. Single cell-type expression analysis, protein-protein interaction (PPI), and druggability evaluation were further conducted to detect the specific cell type with enrichment expression and prioritize potential therapeutic targets.ResultsCollectively, genetically predicted levels of 13 proteins were associated with CRC risk. Elevated levels of two proteins (GREM1, CHRDL2) and decreased levels of 11 proteins were associated with an increased risk of CRC, among which four (GREM1, CLSTN3, CSF2RA, CD86) were prioritized with the most convincing evidence. These protein-coding genes are mainly expressed in tissue stem cells, epithelial cells, and monocytes in colon tumor tissue. Two interactive pairs of proteins (GREM1 and CHRDL2; MMP2 and TIMP2) were identified to be involved in osteoclast differentiation and tumorigenesis pathways; four proteins (POLR2F, CSF2RA, CD86, MMP2) have been targeted for drug development on autoimmune diseases and other cancers, with the potentials of being repurposed as therapeutic targets for CRC.ConclusionsThis study identified several protein biomarkers to be associated with CRC risk and provided new insights into the etiology and promising targets for the development of screening biomarkers and therapeutic drugs for CRC.

Plant adenylate cyclases have come full circle.

In bacteria, fungi and animals, 3'-5'-cyclic adenosine monophosphate (cAMP) and adenylate cyclases (ACs), enzymes that catalyse the formation of 3',5'-cAMP from ATP, are recognized as key signalling components. In contrast, the presence of cAMP and its biological roles in higher plants have long been a matter of controversy due to the generally lower amounts in plant tissues compared with that in animal and bacterial cells, and a lack of clarity on the molecular nature of the generating and degrading enzymes, as well as downstream effectors. While treatment with 3',5'-cAMP elicited many plant responses, ACs were, however, somewhat elusive. This changed when systematic searches with amino acid motifs deduced from the conserved catalytic centres of annotated ACs from animals and bacteria identified candidate proteins in higher plants that were subsequently shown to have AC activities in vitro and in vivo. The identification of active ACs moonlighting within complex multifunctional proteins is consistent with their roles as molecular tuners and regulators of cellular and physiological functions. Furthermore, the increasing number of ACs identified as part of proteins with different domain architectures suggests that there are many more hidden ACs in plant proteomes and they may affect a multitude of mechanisms and processes at the molecular and systems levels.

Predicting Protein Corona Formation on Carbon Nanotubes

Engineered nanoparticles are poised to transform sensing, imaging, and delivery in biological systems. In particular, single-walled carbon nanotubes (SWCNTs) are uniquely suited for biological sensing and imaging due to their tissue-transparent and photostable near-infrared fluorescence. SWCNTs have been functionalized with various biomolecules, including synthetic peptides and proteins, to construct fluorescence-based nanosensors. Optimizing these -nanoparticle interactions is key in enhancing nanotechnology function. Yet, functionalized SWCNTs and other nanotechnologies more broadly suffer from as-of-yet unpredictable interactions with the biological environments in which they are applied: when nanoparticles are introduced into biological systems, endogenous proteins rapidly bind and often lead to decreased ability of the nanoparticles to perform their intended functions. As such, testing compatibility and function of nanotechnologies in biological systems requires a heuristic approach, where this unpredictable protein corona formation often prevents effective implementation.In this work, we develop a classifier that identifies proteins binding to nanotubes based solely on the protein’s amino acid sequence. We build and validate our classifier using mass spectrometry-based quantitative proteomic data that characterizes proteins bound to SWCNT-based nanosensors. Distribution changes among protein properties allow us to determine protein features associated with increased likelihood of SWCNT binding, including high content of solvent-exposed glycines and non-secondary structure-associated amino acids. Understanding these key protein features that drive protein-nanotube binding informs the design of physics-based modeling approaches of such interfacial nano-bio interactions. Lastly, we apply the classifier to rapidly identify candidate proteins with high binding affinity to SWCNTs and experimentally validate adsorption of these proteins with a kinetic exchange assay to evaluate the predictive power of our model. In sum, this classifier provides a step toward undertaking the otherwise intractable problem of predicting protein-nanoparticle interactions.

The Screening of the Protective Antigens of Aeromonas hydrophila Using the Reverse Vaccinology Approach: Potential Candidates for Subunit Vaccine Development.

The threat of bacterial septicemia caused by Aeromonas hydrophila infection to aquaculture growth can be prevented through vaccination, but differences among A. hydrophila strains may affect the effectiveness of non-conserved subunit vaccines or non-inactivated A. hydrophila vaccines, making the identification and development of conserved antigens crucial. In this study, a bioinformatics analysis of 4268 protein sequences encoded by the A. hydrophila J-1 strain whole genome was performed based on reverse vaccinology. The specific analysis included signal peptide prediction, transmembrane helical structure prediction, subcellular localization prediction, and antigenicity and adhesion evaluation, as well as interspecific and intraspecific homology comparison, thereby screening the 39 conserved proteins as candidate antigens for A. hydrophila vaccine. The 9 isolated A. hydrophila strains from diseased fish were categorized into 6 different molecular subtypes via enterobacterial repetitive intergenic consensus (ERIC)-PCR technology, and the coding regions of 39 identified candidate proteins were amplified via PCR and sequenced to verify their conservation in different subtypes of A. hydrophila and other Aeromonas species. In this way, conserved proteins were screened out according to the comparison results. Briefly, 16 proteins were highly conserved in different A. hydrophila subtypes, of which 2 proteins were highly conserved in Aeromonas species, which could be selected as candidate antigens for vaccines development, including type IV pilus secretin PilQ (AJE35401.1) and TolC family outer membrane protein (AJE35877.1). The present study screened the conserved antigens of A. hydrophila by using reverse vaccinology, which provided basic foundations for developing broad-spectrum protective vaccines of A. hydrophila.

Cerebrospinal Fluid Protein Biomarker Discovery in CLN3.

Syndromic CLN3-Batten is a fatal, pediatric, neurodegenerative disease caused by variants in CLN3, which encodes the endolysosomal transmembrane CLN3 protein. No approved treatment for CLN3 is currently available. The protracted and asynchronous disease presentation complicates the evaluation of potential therapies using clinical disease progression parameters. Biomarkers as surrogates to measure the progression and effect of potential therapeutics are needed. We performed proteomic discovery studies using cerebrospinal fluid (CSF) samples from 28 CLN3-affected and 32 age-similar non-CLN3 individuals. Proximal extension assay (PEA) of 1467 proteins and untargeted data-dependent mass spectrometry [MS; MassIVE FTP server (ftp://MSV000090147@massive.ucsd.edu)] were used to generate orthogonal lists of protein marker candidates. At an adjusted p-value of <0.1 and threshold CLN3/non-CLN3 fold-change ratio of 1.5, PEA identified 54 and MS identified 233 candidate biomarkers. Some of these (NEFL, CHIT1) have been previously linked with other neurologic conditions. Others (CLPS, FAM217B, QRICH2, KRT16, ZNF333) appear to be novel. Both methods identified 25 candidate biomarkers, including CHIT1, NELL1, and ISLR2 which had absolute fold-change ratios >2. NELL1 and ISLR2 regulate axonal development in neurons and are intriguing new candidates for further investigation in CLN3. In addition to identifying candidate proteins for CLN3 research, this study provides a comparison of two large-scale proteomic discovery methods in CSF.

Acetyl-CoA Acetyltransferase 2 Confers Radioresistance by Inhibiting Ferroptosis in Esophageal Squamous Cell Carcinoma

The overall survival of patients with esophageal squamous cell carcinoma (ESCC) is not high due to the lack of markers to evaluate concurrent chemoradiation therapy (CCRT) resistance. The aim of this study is to use proteomics to identify a protein related to radiation therapy resistance and explore its molecular mechanisms. Proteomic data for pretreatment biopsy tissues from 18 patients with ESCC who underwent CCRT (complete response [CR] group, n=8; incomplete response [<CR] group, n=10) were collected and combined with ESCC proteomic data from iProx (n=124) to identify candidate proteins that confer resistance to CCRT. Subsequently, 125 paraffin-embedded biopsies were used for immunohistochemical validation. Colony formation assays, after ionizing radiation (IR), of acetyl-CoA acetyltransferase 2 (ACAT2)-overexpressing, -knockdown or knockout ESCC cells were used to determine the effects of ACAT2 on radioresistance. reactive oxygen species, C11-BODIPY, and Western blotting were employed to reveal the potential mechanism of ACAT2-mediated radioresistance after IR. Enrichment analysis of differentially expressed proteins (<CR vs CR) showed that the pathways of molecules conferring CCRT resistance in ESCC were related to lipid metabolism, whereas molecules conferring CCRT sensitivity were mainly related to immunity pathways. ACAT2 was selected from proteomics and validated by immunohistochemistry as a risk factor for reduced overall survival and CCRT or radiation therapy resistance among ESCC patients. ACAT2 overexpression conferred resistance to IR treatment, whereas ACAT2 knockdown or knockout conferred IR sensitivity. ACAT2 knockout cells were prone to have elevated reactive oxygen species production, enhanced lipid peroxidation, and reduced levels of glutathione peroxidase 4 after IR compared with irradiated wild-type cells. ACAT2 knockout cells could be rescued from IR-mediated toxicity by ferrostatin-1 and liproxstatin. ACAT2 overexpression confers radioresistance by inhibiting ferroptosis in ESCC, suggesting ACAT2 could be a potential biomarker of poor radiotherapeutic response and a therapeutic target for enhancing the radiosensitivity of ESCC.

In-Depth Serum Proteomics Reveals the Trajectory of Hallmarks of Cancer in Hepatitis B Virus–Related Liver Diseases

Hepatocellular carcinoma (HCC) is a prevalent cancer in China, with chronic hepatitis B (CHB) and liver cirrhosis (LC) being high-risk factors for developing HCC. Here, we determined the serum proteomes (762 proteins) of 125 healthy controls and Hepatitis B virus–infected CHB, LC, and HCC patients and constructed the first cancerous trajectory of liver diseases. The results not only reveal that the majority of altered biological processes were involved in the hallmarks of cancer (inflammation, metastasis, metabolism, vasculature, and coagulation) but also identify potential therapeutic targets in cancerous pathways (i.e., IL17 signaling pathway). Notably, the biomarker panels for detecting HCC in CHB and LC high-risk populations were further developed using machine learning in two cohorts comprised of 200 samples (discovery cohort = 125 and validation cohort = 75). The protein signatures significantly improved the area under the receiver operating characteristic curve of HCC (CHB discovery and validation cohort = 0.953 and 0.891, respectively; LC discovery and validation cohort = 0.966 and 0.818, respectively) compared to using the traditional biomarker, alpha-fetoprotein, alone. Finally, selected biomarkers were validated with parallel reaction monitoring mass spectrometry in an additional cohort (n = 120). Altogether, our results provide fundamental insights into the continuous changes of cancer biology processes in liver diseases and identify candidate protein targets for early detection and intervention.

Abstract 624: The role of draining lymph nodes in the post-transplant recurrence of liver cancer: Examining immune response using spatial proteomics

Abstract Hepatocellular Carcinoma (HCC) and Cholangiocarcinoma (CCA) are the predominant forms of liver cancer, with a devastating combined incidence of 40,000 cases per year in the United States. Despite many differences between the cancers, liver transplant has found great success in treating both over traditional methods. However, up to 35% of patients’ cancer still recur within five years. Given the risk and resource intensiveness of this treatment, it is critical to identify factors that influence recurrence in these cancers. Previous studies have found the micro-environments of HCC tumors tend to be immune-active while those of CCA tumors tend to be immunosuppressive. Increased immune activity at the tumor level is associated with better patient outcomes, but the role and impact of the regional immune response in liver cancer is not well understood. Liver draining lymph node tissue was collected from patients undergoing orthotopic liver transplant (OLT) for HCC, CCA, or other reasons (control). Nanostring GeoMx Digital Spatial Profiler (DSP) was used to measure immune marker levelsWe found increased immune activity in the LNs of patients with HCC compared to CCA, showing significantly higher levels of lymphocytes and immune activity markers, matching previous tumor microenvironment findings. Additionally, comparing recurrent and non-recurrent HCC or CCA samples, we see generally increased immune activity in non-recurrent lymph nodes, also identifying specific immune markers in both cancer types that correlate with cancer recurrence or non-recurrence. These also reveal patterns of activity specific to both cancer type and recurrence status. Overall, this study provides novel insights into the regional immune response in liver cancers and identifies candidate proteins to serve as predictors of recurrence in liver cancer. We suggest CD163, CD66b, CD8, and CD56 as markers to predict post-OLT HCC recurrence and CD163, CD66b, ID01, and OX40L as candidate markers to predict post-OLT CCA recurrence using LN samples. Further spatial analysis of this data may further characterize immune activity in these lymph nodes, and future studies focusing on identified differences in immune activity may reveal factors affecting liver cancer aggressiveness and recurrence. Citation Format: Peyton Hickman, Aubrey E. Thompson, Justin Nguyen. The role of draining lymph nodes in the post-transplant recurrence of liver cancer: Examining immune response using spatial proteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 624.

Downregulation of Heat Shock Protein 72 Contributes to Fibrostenosis in Crohn's Disease.

Crohn's disease (CD) with recurrent inflammation can cause intestinal fibrostenosis due to dysregulated deposition of extracellular matrix. However, little is known about the pathogenesis of fibrostenosis. Here, we performed a differential proteomic analysis between normal, inflamed, and fibrostenotic specimens of patients with CD and investigated the roles of the candidate proteins in myofibroblast activation and fibrosis. We performed two-dimensional difference gel electrophoresis and identified candidate proteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and orbitrap liquid chromatography-mass spectrometry. We also verified the levels of candidate proteins in clinical specimens and examined their effects on 18Co myofibroblasts and Caco-2 intestinal epithelial cells. We identified five of 30 proteins (HSP72, HSPA5, KRT8, PEPCK-M, and FABP6) differentially expressed in fibrostenotic CD. Among these proteins, the knockdown of heat shock protein 72 (HSP72) promoted the activation and wound healing of myofibroblasts. Moreover, knockdown of HSP72 induced the epithelial-mesenchymal transition of intestinal epithelial cells by reducing E-cadherin and inducing fibronectin and α-smooth muscle actin, which contribute to fibrosis. HSP72 is an important mediator that regulates myofibroblasts and epithelial-mesenchymal transition in fibrosis of CD, suggesting that HSP72 can serve as a target for antifibrotic therapy.

TMT-based comparative proteomic analysis of Dezhou donkey spermatozoa related to freezability

The freezability difference between donkey ejaculates is a limiting factor of sperm cryopreservation. Our recent study shows that the freezability of donkey semen is related to the seminal plasma proteome. In this study, we aimed to identify the different abundance sperm proteins in good freezability ejaculates (GFEs) and poor freezability ejaculates (PFEs) using a Tandem Mass Tag (TMT) peptide labeling coupled with LC-MS/MS approach. A total of 2682 proteins were identified, among which 58 were significantly up-regulated in GFEs and 16 were down-regulated compared with PFEs. Bioinformatic analysis results revealed that the majority of different abundance proteins (DAPs) participated in copper and calcium binding, regulation of RNA biosynthetic process, positive regulation of innate immune response, and negative regulation of programmed cell death. KEGG pathway enrichment analysis showed the up-regulated proteins in GF group were mainly involved in N-Glycan biosynthesis and protein processing in endoplasmic reticulum. Our study was the first to analyze the proteome of sperm from donkey ejaculates with different freezabilities. The identified candidate proteins might be used to explore the molecular mechanism related to donkey sperm cryotolerance and might improve the screening of jacks with good sperm freezability. SignificanceCryopreserved semen has been widely used in assisted reproductive technology. However, semen cryopreservation is a damaging process, which can cause oxidative stress, reduce sperm motility and motility. There are differences in sperm freezability reported to exist between or within breeds, and even between fractions coming from the same ejaculate. The freezability difference between donkey ejaculates is a limiting factor of sperm cryopreservation.The mechanisms that affect the freezing difference in sperm quality remain to be investigated, and freezability differences was found to be related to protein composition of spermatozoa. Some protein markers that can indicate good freezability or poor freezability semen have been identified in mammals. Until now, there is no information about the relationship between donkey spermatozoa proteome and freezability. Additional novel biomarkers of semen freezability in donkey spermatozoa are also needed. The identified candidate proteins might be used to explore the molecular mechanism related to donkey sperm cryotolerance and might improve the screening of jacks with good sperm freezability.

Using Proteomics Analysis to Identify Cerebrospinal Fluid Biomarkers in Normal Pressure Hydrocephalus

AbstractBackgroundNormal Pressure Hydrocephalus (NPH) is a neurological condition where there is enlarged ventricular size with normal opening pressure at lumbar puncture. Etiology is uncertain but it is presumed to occur from an imbalance in the production and/or absorption of cerebrospinal fluid (CSF) leading to expansion of the ventricles. Clinical features include gait dysfunction, cognitive impairments, bladder incontinence. Diagnosis is challenging, given a large overlap in clinical and neuroradiological features and co‐morbidity with other more common diseases of aging, especially Alzheimer’s disease (AD) and Lewy body diseases. CSF amyloid and tau biomarkers can be helpful to identify AD pathology but do not exclude concurrent NPH. Currently, there are no biofluid biomarkers to assist in diagnosis of NPH. Here we describe a proteomic discovery pilot to identify candidate protein biomarkers associated with NPH.Method176 banked CSF samples from the MassGeneral Institute for Neurodegenerative Disease biorepository were separated into three groups: NPH (n=30), AD (n=65), and cognitively unimpaired neurological controls (CU‐N; n=81). Diagnoses were established based on comprehensive chart review and CSF AD biomarkers. For proteomic analysis, samples were prepared in batches and analyzed by LC‐MS/MS on an Orbitrap Fusion using a data‐independent acquisition method with non‐overlapping windows from 400‐1000 m/z. Batches were run sequentially, and raw files were searched per batch using Scaffold‐DIA and a custom data‐dependent generated library.ResultFive proteins present in hemoglobulin (HBD and HBB), immunoglobulins (LV39 and KV105), and fibrinogen (FIBA) were increased in NPH compared to AD (Benjamini‐Hochberg adjusted p‐value pAdj &lt;0.05). CSF erythrocyte counts were undetectable in all but one NPH subjects suggesting that a traumatic LP was not the cause of the increased levels. Two neuron associated proteins (VGF and NPTXR) were detected at lower levels in NPH compared to CU‐N subjects (pAdj&lt;0.0001). Alpha‐1‐antichymotrypsin (AACT), was detected at high concentrations both in NPH and AD compared to CU‐N subjects (pAdj&lt;0.02).ConclusionProteomic analysis in NPH may identify novel pathological processes involved in the disease process and aid in the development of biomarkers in NPH.

A Methylotrophic Bacterium Growing with the Antidiabetic Drug Metformin as Its Sole Carbon, Nitrogen and Energy Source.

Metformin is one of the most prescribed antidiabetic agents worldwide and is also considered for other therapeutic applications including cancer and endocrine disorders. It is largely unmetabolized by human enzymes and its presence in the environment has raised concern, with reported toxic effects on aquatic life and potentially also on humans. We report on the isolation and characterisation of strain MD1, an aerobic methylotrophic bacterium growing with metformin as its sole carbon, nitrogen and energy source. Strain MD1 degrades metformin into dimethylamine used for growth, and guanylurea as a side-product. Sequence analysis of its fully assembled genome showed its affiliation to Aminobacter niigataensis. Differential proteomics and transcriptomics, as well as mini-transposon mutagenesis of the strain, point to genes and proteins essential for growth with metformin and potentially associated with hydrolytic C-N cleavage of metformin or with cellular transport of metformin and guanylurea. The obtained results suggest the recent evolution of the growth-supporting capacity of strain MD1 to degrade metformin. Our results identify candidate proteins of the enzymatic system for metformin transformation in strain MD1 and will inform future research on the fate of metformin and its degradation products in the environment and in humans.

Plasma Proteomic Profiling Illustrates Endothelial and Complement Signature in Patients with Alcohol-Associated Cirrhosis

INTRODUCTION Cirrhosis related coagulation derangements result in altered hemostasis. Endothelial markers such as von Willebrand factor (VWF) antigen and the factor VIII-to-protein C ratio (FVIII/PC) have prognostic significance in patients with cirrhosis. Additional endothelial markers have not been well described in patients with alcohol-associated cirrhosis. We performed plasma proteomic profiling in patients with alcohol-associated cirrhosis to gain insight into the potential roles of endothelial and hemostatic proteins associated with severity of cirrhosis. METHODS Two independent cohorts of patients with Child-Pugh class A (CPA) and C (CPC) alcohol-associated cirrhosis were recruited. Controls were patients without underlying liver disease. For plasma proteomic profiling, plasma supernatant was frozen, then sent to Eve Technologies (Calgary, Alberta, Canada). Linear discrimination analysis (LDA) was applied to the first cohort to identify a set of proteins that clustered patients based on the severity of liver disease (CPA vs CPC) and controls. Identified proteins were then validated using a second independent cohort of patients with alcohol-associated cirrhosis to determine if the protein signature was replicable. Boruta feature selection was performed on cohort 2 to identify candidate proteins to explain the differences in FVIII/PC ratio. A p-value of < 0.05 was considered statistically significant. RESULTS Cohort 1 included 13 patients: 4 CPA, 9 CPC (median age was 57; 4 women; 9 had dyslipidemia, 2 diabetes mellitus, 2 hypertension, and 2 chronic kidney disease). Cohort 2 included 12 patients: 7 CPA, 5 CPC (median age 50; 5 women; 7 had dyslipidemia, 1 diabetes mellitus, 1 hypertension and 0 chronic kidney disease). For cohort 1 LDA clustered patients based on CPA vs CPC with an endothelial, inflammatory and complement signature (fig.1). The proteins set that achieved robust LDA clustering of CPA, CPC, and control patients in cohort 1 included: sVCAM-1 and PAI-1 (endothelial markers); C3, C4, and CFH (complement cascade factors); IL-8 (inflammatory chemokine), NCAM (neural adhesion maker) and BDNF (neurotrophin); MPO (marker of neutrophilic activation); Eotaxin (eosinophil associated chemokine); MIP-1d (activator of T-cells and monocytes). The same protein profile applied to the validation cohort 2 was also able to cluster CPA, CPC, and control patients (fig. 2). Boruta feature selection for proteins of importance for the FVIII/PC ratio included sVCAM-1; CFI, C4b, C4, C5, CFB (complement factors); IL-6 (inflammatory cytokine), and VWF. CONCLUSIONS Using plasma proteomic profiling we describe a signature of endothelial, complement, hemostatic, and inflammatory activation that distinguishes the severity of liver disease in patients with alcohol-associated cirrhosis. Exploratory analyses suggest that endothelial and complement proteins may also contribute to the differences in the FVIII/PC ratio which has prognostic significance in patients with cirrhosis. Future work should continue to highlight the role of endothelium in the progression of liver disease and describe alternative prognostic markers to help guide clinicians. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Novel plasma and brain proteins that are implicated in multiple sclerosis.

Understanding how variations in the plasma and brain proteome contribute to multiple sclerosis susceptibility can provide important insights to guide drug repurposing and therapeutic development for the disease. However, the role of genetically predicted protein abundance in multiple sclerosis remains largely unknown. Integrating plasma proteomics (n = 3301) and brain proteomics (n = 376 discovery; n = 152 replication) into multiple sclerosis genome-wide association studies (n = 14 802 cases and 26 703 controls), we employed summary-based methods to identify candidate proteins involved in multiple sclerosis susceptibility. Next, we evaluated associations of the corresponding genes with multiple sclerosis at tissue-level using large gene expression quantitative trait data from whole-blood (n = 31 684) and brain (n = 1194) tissue. Further, to assess transcriptional profiles for candidate proteins at cell-level, we examined gene expression patterns in immune cell types (Dataset 1: n = 73 cases and 97 controls; Dataset 2: n = 31 cases and 31 controls) for identified plasma proteins, and in brain cell types (Dataset 1: n = 4 cases and 5 controls; Dataset 2: n = 5 cases and 3 controls) for identified brain proteins. In a longitudinal multiple sclerosis cohort (n = 203 cases followed up to 15 years), we also assessed the corresponding gene-level associations with the outcome of disability worsening. We identified 39 novel proteins associated with multiple sclerosis risk. Based on five identified plasma proteins, four available corresponding gene candidates showed consistent associations with multiple sclerosis risk in whole-blood, and we found TAPBPL upregulation in multiple sclerosis B cells, CD8+ T cells and natural killer cells compared with controls. Among the 34 candidate brain proteins, 18 were replicated in a smaller cohort and 14 of 21 available corresponding gene candidates also showed consistent associations with multiple sclerosis risk in brain tissue. In cell-specific analysis, six identified brain candidates showed consistent differential gene expression in neuron and oligodendrocyte cell clusters. Based on the 39 protein-coding genes, we found 23 genes that were associated with disability worsening in multiple sclerosis cases. The findings present a set of candidate protein biomarkers for multiple sclerosis, reinforced by high concordance in downstream transcriptomics findings at tissue-level. This study also highlights the heterogeneity of cell-specific transcriptional profiles for the identified proteins and that numerous candidates were also implicated in disease progression. Together, these findings can serve as an important anchor for future studies of disease mechanisms and therapeutic development.