Comprehensive characterization of the Hsp70 interactome reveals novel client proteins and interactions mediated by posttranslational modifications.

Hsp70 interactions are critical for cellular viability and the response to stress. Previous attempts to characterize Hsp70 interactions have been limited by their transient nature and the inability of current technologies to distinguish direct versus bridged interactions. We report the novel use of cross-linking mass spectrometry (XL-MS) to comprehensively characterize the Saccharomyces cerevisiae (budding yeast) Hsp70 protein interactome. Using this approach, we have gained fundamental new insights into Hsp70 function, including definitive evidence of Hsp70 self-association as well as multipoint interaction with its client proteins. In addition to identifying a novel set of direct Hsp70 interactors that can be used to probe chaperone function in cells, we have also identified a suite of posttranslational modification (PTM)-associated Hsp70 interactions. The majority of these PTMs have not been previously reported and appear to be critical in the regulation of client protein function. These data indicate that one of the mechanisms by which PTMs contribute to protein function is by facilitating interaction with chaperones. Taken together, we propose that XL-MS analysis of chaperone complexes may be used as a unique way to identify biologically important PTMs on client proteins.

Managed Care after Acute Myocardial Infarction (MC-AMI) improves prognosis in AMI survivors with pre-existing heart failure: A propensity score matching analysis of Polish nationwide program of comprehensive post-MI care.

BACKGROUND: Despite improvement in acute myocardial infarction (AMI) treatment, post-discharge mortality remains high. The outcomes are supposed to be even worse in patients with post-MI heart failure (HF), as only a half of patients with newly diagnosed HF survive four years. AIMS: The study aimed to analyze whether managed care after acute myocardial infarction (MC-AMI) is associated with better survival in AMI survivors with a pre-existing diagnosis of HF. RESULTS: The study included 7228 patients with a pre-existing diagnosis of HF who survived the hospitalization for AMI in Poland between November 2017 and December 2020, of whom 2268 (31.4%) were referred for the MC-AMI program. The median follow-up was 1.5 (0.7-2.3) years. In the unmatched analysis, patients without MC-AMI had more than twice higher 12-month mortality (21.8% vs. 9.9%; P <0.01) than MC-AMI participants. The difference remained significant after propensity score matching (16,8% vs. 10.0%; P <0.01). In multivariable analysis, participation in MC-AMI was an independent factor of 12-month survival. MC-AMI participants had a lower stroke rate (1.5% vs. 3.0%; P <0.01) and fewer hospital admissions due to HF (22.9% vs. 27.6%; P <0.01). CONCLUSIONS: After propensity score matching, participation in MC-AMI was associated with lower rates of stroke, HF hospitalizations, and all-cause mortality in the 12-month follow-up and was an independent factor of 12-month survival in AMI survivors with pre-existing HF.

The Blood Proteoform Atlas: A reference map of proteoforms in human hematopoietic cells.

Human biology is tightly linked to proteins, yet most measurements do not precisely determine alternatively spliced sequences or posttranslational modifications. Here, we present the primary structures of ~30,000 unique proteoforms, nearly 10 times more than in previous studies, expressed from 1690 human genes across 21 cell types and plasma from human blood and bone marrow. The results, compiled in the Blood Proteoform Atlas (BPA), indicate that proteoforms better describe protein-level biology and are more specific indicators of differentiation than their corresponding proteins, which are more broadly expressed across cell types. We demonstrate the potential for clinical application, by interrogating the BPA in the context of liver transplantation and identifying cell and proteoform signatures that distinguish normal graft function from acute rejection and other causes of graft dysfunction.

Meat Quality of the Native Carpathian Goat Breed in Comparison with the Saanen Breed.

Goats provide valuable products that are appreciated by consumers who are looking for food that is not only tasty but also healthy, and, probably, one of them is goat meat. Breeding of local breeds such as the native Carpathian goat has been gaining importance in recent years, which creates an opportunity for the development of the goat meat market. The aim of this study was to investigate the influence of goat breed on the basic chemical, fatty and amino acid composition, colour and sensory evaluation of meat. The research material consisted of Carpathian goats from the NRIAP experimental plant located in the southern part of Poland, and goats from a farm keeping Saanen goats in south-eastern Poland. Ten male goat kids from each breed were taken to the NRIAP farm. The quality of meat obtained from the leg (m. biceps femoris) of male goat kids about 150 days old at slaughter was analysed. The meat of the Carpathian goat was characterised by a lower content of protein and cholesterol (p < 0.01), and a higher content of fat and general collagen compared to the meat from Saanen goats (p < 0.05). Cholesterol content in goat meat of both breeds was similar and ranged from 55.08 mg/100 g (Carpathian) to 56.79 mg/100 g (Saanen). Despite the higher collagen content, the goat meat of Carpathian breeds was characterised by lower shear force, less hardness (p < 0.05) and chewiness, being a more delicate meat. The fat of Carpathian goat breeds was characterised by a higher content of monounsaturated acids, mainly C 18:1n:9, and a more favourable (lower) saturation index, S/P (p < 0.05). The meat of Carpathian goats was characterised by a higher health-promoting quality compared to the meat from Saanen goats. In the goat meat of both breeds, there were no differences between the total content of exogenous and endogenous amino acids. The essential/nonessential amino acids (EAA/NEAA) ratio in the meat of the analysed breeds was 0.88:0.89. However, the meat of the Carpathian goats was statistically significantly higher concerning the content of phenylalanine, histidine, proline, alanine and tyrosine, as compared to the meat of the Saanen goats. The obtained results confirm the high quality of the meat of the local Carpathian breed in comparison to the Saanen breed.

A Comparison of Oxidative Stress Biomarkers in the Serum of Healthy Polish Dairy Goats with Those Naturally Infected with Small Ruminant Lentivirus in the Course of Lactation.

The present study examines the effects of natural infection by small ruminant lentivirus (SRLV) in the two most common goat breeds in Poland, i.e., Polish white improved and polish fawn improved. It focuses on biomarkers of oxidative stress, oxidatively modified proteins and antioxidant defenses, ceruloplasmin level as an acute phase protein, and the activities of antioxidant enzymes in the goat serum. It was conducted on 24 goats divided into two equal groups: one SRLV-seropositive (SRLV-SP) and another SRLV-seronegative (SRLV-SN). Both groups were identical in terms of breed and parity. Despite infection, the SRLV-SP goats demonstrated no symptoms of caprine arthritis-encephalitis. In addition, the SRLV-SP goats did not reveal pronounced dysfunctions in oxidative stress biomarkers in the serum compared to the SRLV-SN animals. However, both groups demonstrated elevated levels of the aldehydic and ketonic derivatives of oxidatively modified proteins during the lactation period. In addition, both groups retained a high total antioxidant capacity in serum despite the decrease of enzyme antioxidant defenses throughout the 200-day lactation period.

Managed Care after Acute Myocardial Infarction (MC-AMI) Reduces Total Mortality in 12-Month Follow-Up-Results from a Poland's National Health Fund Program of Comprehensive Post-MI Care-A Population-Wide Analysis.

INTRODUCTION: Advances in the acute treatment of myocardial infarction (AMI) substantially reduced in-hospital mortality, but the post-discharge prognosis is still unacceptable. The Managed Care in Acute Myocardial Infarction (MC-AMI) is a program of Poland's National Health Fund that aims at comprehensive post-AMI care to improve long-term prognosis. The aim of the study was to assess the effect of MC-AMI on all-cause mortality in one-year follow-up. METHODS: MC-AMI includes acute MI treatment, complex revascularization, cardiac rehabilitation (CR), scheduled one-year outpatient follow-up, and prevention of sudden cardiac death. In this retrospective observational study performed in a province of Silesia, Poland, we analyzed 3893 MC-AMI participants, and compared them to 6946 patients in the control group. After propensity score matching, we compared two groups of 3551 subjects each. To assess the effect of MC-AMI and other variables on mortality, we preformed a Cox regression. RESULTS: MC-AMI was related with mortality reduction by 38% in a 12-month observation period and the effect persisted even after. Multivariable Cox regression analysis revealed MC-AMI participation to be inversely associated with 1-year mortality (HR 0.52, 95%CI 0.42-0.65, p < 0.001). Besides that, older age (HR 1.47/10 y), ST-elevation AMI (HR 1.41), heart failure (HR 2.08), diabetes (HR 1.52), and dialysis (HR 2.38) were significantly associated with the primary endpoint. Among MC-AMI components, cardiac rehabilitation (HR 0.34) and strict outpatient care (HR 0.42) are the crucial factors affecting mortality reduction. CONCLUSIONS: Participation in MC-AMI reduced 1-year mortality by 38% and the effect persisted after the program had been completed.

Targeted detection and quantitation of histone modifications from 1,000 cells.

Histone post-translational modifications (PTMs) create a powerful regulatory mechanism for maintaining chromosomal integrity in cells. Histone acetylation and methylation, the most widely studied histone PTMs, act in concert with chromatin-associated proteins to control access to genetic information during transcription. Alterations in cellular histone PTMs have been linked to disease states and have crucial biomarker and therapeutic potential. Traditional bottom-up mass spectrometry of histones requires large numbers of cells, typically one million or more. However, for some cell subtype-specific studies, it is difficult or impossible to obtain such large numbers of cells and quantification of rare histone PTMs is often unachievable. An established targeted LC-MS/MS method was used to quantify the abundance of histone PTMs from cell lines and primary human specimens. Sample preparation was modified by omitting nuclear isolation and reducing the rounds of histone derivatization to improve detection of histone peptides down to 1,000 cells. In the current study, we developed and validated a quantitative LC-MS/MS approach tailored for a targeted histone assay of 75 histone peptides with as few as 10,000 cells. Furthermore, we were able to detect and quantify 61 histone peptides from just 1,000 primary human stem cells. Detection of 37 histone peptides was possible from 1,000 acute myeloid leukemia patient cells. We anticipate that this revised method can be used in many applications where achieving large cell numbers is challenging, including rare human cell populations.

Use of the HRM Method in Quick Identification of FecXO Mutation in Highly Prolific Olkuska Sheep.

Olkuska is a highly prolific sheep breed in Poland. Thanks to earlier identification of the genetic basis of its prolificacy, a mutation in the BMP-15 gene, we can use molecular biology tools to identify this causative mutation affecting prolificacy. In our research, we used the High-Resolution Melting (HRM) and Sanger sequencing methods to identify the genotypes of the studied animals. The result obtained by the HRM method is identical to those obtained by the sequencing method, which confirms the effectiveness of the HRM method and the possibility of quick and cheap identification of individuals with a FecXO mutation.

Chromatin occupancy and epigenetic analysis reveal new insights into the function of the GATA1 N terminus in erythropoiesis.

Mutations in GATA1, which lead to expression of the GATA1s isoform that lacks the GATA1 N terminus, are seen in patients with Diamond-Blackfan anemia (DBA). In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in Gata1s mice. Defects in yolks sac and fetal liver hematopoiesis included impaired terminal maturation and reduced numbers of erythroid progenitors. RNA-sequencing revealed that both erythroid and megakaryocytic gene expression patterns were altered by the loss of the N terminus, including aberrant upregulation of Gata2 and Runx1. Dysregulation of global H3K27 methylation was found in the erythroid progenitors upon loss of N terminus of GATA1. Chromatin-binding assays revealed that, despite similar occupancy of GATA1 and GATA1s, there was a striking reduction of H3K27me3 at regulatory elements of the Gata2 and Runx1 genes. Consistent with the observation that overexpression of GATA2 has been reported to impair erythropoiesis, we found that haploinsufficiency of Gata2 rescued the erythroid defects of Gata1s fetuses. Together, our integrated genomic analysis of transcriptomic and epigenetic signatures reveals that, Gata1 mice provide novel insights into the role of the N terminus of GATA1 in transcriptional regulation and red blood cell maturation which may potentially be useful for DBA patients.

Coupling Fluorescence-Activated Cell Sorting and Targeted Analysis of Histone Modification Profiles in Primary Human Leukocytes.

Histone posttranslational modifications (PTMs) are essential for regulating chromatin and maintaining gene expression throughout cell differentiation. Despite the deep level of understanding of immunophenotypic differentiation pathways in hematopoietic cells, few studies have investigated global levels of histone PTMs required for differentiation and maintenance of these distinct cell types. Here, we describe an approach to couple fluorescence-activated cell sorting (FACS) with targeted mass spectrometry to define global "epi-proteomic" signatures for primary leukocytes. FACS was used to sort closely and distantly related leukocytes from normal human peripheral blood for quantitation of histone PTMs with a multiple reaction monitoring LC-MS/MS method measuring histone PTMs on histones H3 and H4. We validate cell sorting directly into H2SO4 for immediate histone extraction to decrease time and number of steps after FACS to analyze histone PTMs. Relative histone PTM levels vary in T cells across healthy donors, and the majority of PTMs remain stable up to 2 days following initial blood draw. Large differences in the levels of histone PTMs are observed across the mature lymphoid and myeloid lineages, as well as between different types within the same lineage, though no differences are observed in closely related T cell subtypes. The results show a streamlined approach for quantifying global changes in histone PTMs in cell types separated by FACS that is poised for clinical deployment.

The vermiform appendix impacts the risk of developing Parkinson's disease.

The pathogenesis of Parkinson's disease (PD) involves the accumulation of aggregated α-synuclein, which has been suggested to begin in the gastrointestinal tract. Here, we determined the capacity of the appendix to modify PD risk and influence pathogenesis. In two independent epidemiological datasets, involving more than 1.6 million individuals and over 91 million person-years, we observed that removal of the appendix decades before PD onset was associated with a lower risk for PD, particularly for individuals living in rural areas, and delayed the age of PD onset. We also found that the healthy human appendix contained intraneuronal α-synuclein aggregates and an abundance of PD pathology-associated α-synuclein truncation products that are known to accumulate in Lewy bodies, the pathological hallmark of PD. Lysates of human appendix tissue induced the rapid cleavage and oligomerization of full-length recombinant α-synuclein. Together, we propose that the normal human appendix contains pathogenic forms of α-synuclein that affect the risk of developing PD.

Stability of histone post-translational modifications in samples derived from liver tissue and primary hepatic cells.

Chromatin structure, a key contributor to the regulation of gene expression, is modulated by a broad array of histone post-translational modifications (PTMs). Taken together, these "histone marks" comprise what is often referred to as the "histone code". The quantitative analysis of histone PTMs by mass spectrometry (MS) offers the ability to examine the response of the histone code to physiological signals. However, few studies have examined the stability of histone PTMs through the process of isolating and culturing primary cells. To address this, we used bottom-up, MS-based analysis of histone PTMs in liver, freshly isolated hepatocytes, and cultured hepatocytes from adult male Fisher F344 rats. Correlations between liver, freshly isolated cells, and primary cultures were generally high, with R2 values exceeding 0.9. However, a number of acetylation marks, including those on H2A K9, H2A1 K13, H3 K4, H3 K14, H4 K8, H4 K12 and H4 K16 differed significantly among the three sources. Inducing proliferation of primary adult hepatocytes in culture affected several marks on histones H3.1/3.2 and H4. We conclude that hepatocyte isolation, culturing and cell cycle status all contribute to steady-state changes in the levels of a number of histone PTMs, indicating changes in histone marks that are rapidly induced in response to alterations in the cellular milieu. This has implications for studies aimed at assigning biological significance to histone modifications in tumors versus cancer cells, the developmental behavior of stem cells, and the attribution of changes in histone PTMs to altered cell metabolism.

The Value of Activated Ion Electron Transfer Dissociation for High-Throughput Top-Down Characterization of Intact Proteins.

High-throughput top-down proteomic experiments directly identify proteoforms in complex mixtures, making high quality tandem mass spectra necessary to deeply characterize proteins with many sources of variation. Collision-based dissociation methods offer expedient data acquisition but often fail to extensively fragment proteoforms for thorough analysis. Electron-driven dissociation methods are a popular alternative approach, especially for precursor ions with high charge density. Combining infrared photoactivation concurrent with electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), can significantly improve ETD characterization of intact proteins, but benefits of AI-ETD have yet to be quantified in high-throughput top-down proteomics. Here, we report the first application of AI-ETD to LC-MS/MS characterization of intact proteins (<20 kDa), highlighting improved proteoform identification the method offers over higher energy-collisional dissociation (HCD), standard ETD, and ETD followed by supplemental HCD activation (EThcD). We identified 935 proteoforms from 295 proteins from human colorectal cancer cell line HCT116 using AI-ETD compared to 1014 proteoforms, 915 proteoforms, and 871 proteoforms with HCD, ETD, and EThcD, respectively. Importantly, AI-ETD outperformed each of the three other methods in MS/MS success rates and spectral quality metrics (e.g., sequence coverage achieved and proteoform characterization scores). In all, this four-method analysis offers the most extensive comparisons to date and demonstrates that AI-ETD both increases identifications over other ETD methods and improves proteoform characterization via higher sequence coverage, positioning it as a premier method for high-throughput top-down proteomics.

Hemolysis is a primary ATP-release mechanism in human erythrocytes.

The hypothesis that regulated ATP release from red blood cells (RBCs) contributes to nitric oxide-dependent control of local blood flow has sparked much interest in underlying release mechanisms. Several stimuli, including shear stress and hypoxia, have been found to induce significant RBC ATP release attributed to activation of ATP-conducting channels. In the present study, we first evaluated different experimental approaches investigating stimulated RBC ATP release and quantifying hemolysis. We then measured ATP and free hemoglobin in each and every RBC supernatant sample to directly assess the contribution of hemolysis to ATP release. Hypotonic shock, shear stress, and hypoxia, but not cyclic adenosine monophosphate agonists, significantly enhanced ATP release. It tightly correlated, however, with free hemoglobin in RBC supernatants, indicating that lysis was responsible for most, if not all, ATP release. Luminescence ATP imaging combined with simultaneous infrared cell imaging showed that ATP was released exclusively from lysing cells with no contribution from intact cells. In summary, with all stimuli tested, we found no evidence of regulated ATP release from intact RBCs other than by cell lysis. Such a release mechanism might be physiologically relevant in vivo, eg, during exercise and hypoxia where intravascular hemolysis, predominantly of senescent cells, is augmented.

ATP release from mast cells by physical stimulation: a putative early step in activation of acupuncture points.

In Chinese medicine acupuncture points are treated by physical stimuli to counteract various diseases. These stimuli include mechanical stress as applied during the needle manipulation or tuina, high temperatures as applied during moxibustion, and red laser light applied during laser acupuncture. This study aimed to investigate cellular responses to stimuli that might occur in the tissue of acupuncture points. Since they have a characteristically high density of mast cells that degranulate in response to acupuncture, we asked whether these processes lead to ATP release. We tested in in vitro experiments on mast cells of the human mast-cell line HMC-1 the effects of the physical stimuli; mechanical stress was applied by superfusion of the cells with hypotonic solution, heat was applied by incubation of the cells at 52°C, and red laser light of 657 nm was used for irradiation. We demonstrate that all the stimuli induce ATP release from model human mast HMC-1 cells, and this release is associated with an intracellular free Ca(2+) rise. We hypothesize that ATP released from mast cells supplements the already known release of ATP from keratinocytes and, by acting on P2X receptors, it may serve as initial mediator of acupuncture-induced analgesia.

A probabilistic framework for peptide and protein quantification from data-dependent and data-independent LC-MS proteomics experiments.

A probability-based quantification framework is presented for the calculation of relative peptide and protein abundance in label-free and label-dependent LC-MS proteomics data. The results are accompanied by credible intervals and regulation probabilities. The algorithm takes into account data uncertainties via Poisson statistics modified by a noise contribution that is determined automatically during an initial normalization stage. Protein quantification relies on assignments of component peptides to the acquired data. These assignments are generally of variable reliability and may not be present across all of the experiments comprising an analysis. It is also possible for a peptide to be identified to more than one protein in a given mixture. For these reasons the algorithm accepts a prior probability of peptide assignment for each intensity measurement. The model is constructed in such a way that outliers of any type can be automatically reweighted. Two discrete normalization methods can be employed. The first method is based on a user-defined subset of peptides, while the second method relies on the presence of a dominant background of endogenous peptides for which the concentration is assumed to be unaffected. Normalization is performed using the same computational and statistical procedures employed by the main quantification algorithm. The performance of the algorithm will be illustrated on example data sets, and its utility demonstrated for typical proteomics applications. The quantification algorithm supports relative protein quantification based on precursor and product ion intensities acquired by means of data-dependent methods, originating from all common isotopically-labeled approaches, as well as label-free ion intensity-based data-independent methods.