Pattern Of Protein Phosphorylation Research Articles (Page 1)

Protein restriction reprograms the multi-organ proteomic landscape of mouse aging.

The emergence of beta-lactamase producing multidrug-resistant (MDR) gram-negative bacteria presents a significant challenge to effective treatment of infections. This study focuses on the isolation, amplification, and molecular characterization of β-lactamase genes from clinical strains of Escherichia coli and Klebsiella pneumoniae. Seven new partial gene sequences, including novel variants of blaOXA and blaNDM, were identified after screening 108 clinical samples and submitted to NCBI GenBank. In silico analysis revealed considerable diversity and distribution of these resistance genes among different strains of bacteria. Gene structure predictions using GENSCAN showed that blaOXA genes typically contain single exons with moderate GC content, whereas blaNDM genes feature longer exons with higher GC content. Multiple sequence alignment showed that NDM and OXA β-lactamases were highly similar, with only slight differences in a few amino acids. The study also analyzed the physico-chemical properties, functional domains, and phosphorylation patterns of the β-lactamase proteins. Secondary structure prediction indicated a dominance of beta sheets, contributing to protein stability, while tertiary modeling provided insights into their 3D structure. Overall, these findings provide critical insights into the genetic diversity and potential mechanisms of β-lactamase-mediated resistance, offering valuable information for the development of novel therapeutic strategies and surveillance programs.

Introduction: Heart failure (HF) is a leading cause of mortality. Protein kinase G1 (PKG1) activation improves HF outcomes but often causes hypotension. Identifying blood pressure (BP)-independent PKG1 effectors could offer new therapeutic targets. Mixed lineage kinase 3 (MLK3) is a novel PKG1a substrate, and whole-body MLK3 knockout mice develop more severe left ventricular (LV) dysfunction after transaortic constriction but also have hypertension. The cardiac myocyte (CM) and BP-independent effects of MLK3 remain unclear. Hypothesis: MLK3 kinase function in the CM opposes basal pathological LV remodeling and dysfunction independent of BP. Methods: Male and female 3- and 6-month-old CM-specific MLK3 deletion (CMKO, n=12) and control MLK3 intact (MLK3 fl/fl Cre-, n=11) littermate mice were studied. Wild type (WT) mice were injected with MLK3 kinase inhibitor URMC-099 or vehicle for 2 weeks (10mg/kg 2x/day, n=4 per group). Echocardiography, organ masses, and LV hemodynamics were measured. We performed phosphoproteomics from the above groups to examine MLK3 kinase-dependent and CM-specific signaling. Results: At 3 months, LV dP/dt was reduced in CMKO males, while dP/dt and dP/dt min were reduced in females, compared with MLK3 intact. At 6 months, CMKO mice exhibited increased LV/tibia length, heart weight/tibia length , and end-diastolic dimension, along with reduced ejection fraction, dP/dt, dP/dt min, and contractile index in females. Notably, BP was not increased in MLK3 CMKO mice. MLK3 kinase inhibition decreased ejection fraction in females, compared with vehicle. MLK3 protein expression was unchanged between WT males and females at 3 and 6 months. Principal component analysis of phosphoproteomic data revealed distinct protein expression and phosphorylation patterns between MLK3 CMKO and MLK3-inhibited groups. Conclusion: CM-specific MLK3 deletion and systemic MLK3 kinase inhibition lead to pathological LV remodeling and dysfunction independent of BP effects, particularly in females, supporting a requirement of intact MLK3 kinase function for normal cardiac function. These findings, combined with the observed phosphoproteomic changes suggest that modulating MLK3 or MLK3-dependent signaling could improve LV function in HF.

The successful establishment of infection relies on an ability to sense and adapt to the host signaling state. One key mechanism of virus-host sensing is host-mediated post-translational modifications of viral proteins. While viral protein phosphorylation by host kinases is known to modulate viral functions, the global prevalence of kinase motifs across diverse viruses, and the signaling pathways they reflect, remains to be systematically explored. Here, we annotated human kinase motifs in 1,505 viral proteomes and uncovered enriched motifs in viral proteins that diverged from patterns observed in human proteins. Integration of our findings with 21,606 viral protein structures and deep mass spectrometry phosphoproteomics of infected human cells revealed that surface-accessible residues were preferentially phosphorylated and exhibited greater kinase specificity compared to buried sites. Virus-enriched motifs mapped predominantly to stress, inflammation, and cell cycle pathways—key signaling hubs dysregulated during infection that are central to the virus-host arms race—most strikingly for Flaviviridae, Togaviridae, Herpesviridae, and Retroviridae families. Temporal phosphoproteomic profiling of host kinase activity during alphavirus infection revealed dynamic patterns of stress kinase activation and viral protein phosphorylation, and the inhibition of MAP kinases reduced viral replication and phosphorylation at viral motifs with specificity for ERK and JNK kinases. Our findings suggest that viruses have evolved as biosensors of the host signaling state to optimize their life cycles, revealing new antiviral opportunities aimed at disrupting virus decision-making by manipulating host signaling cues.

Quantitative phosphoproteomic reveals that the induction of competence modulates protein phosphorylation in Streptococcus pneumonaie.

Unravelling the intricate patterns of site-specific protein phosphorylation during Epimorphic regeneration holds the key to unlocking the secrets of tissue complexity. Understanding these precise modifications and their impact on protein function could shed light on the remarkable regenerative capacity of tissues, with potential implications for therapeutic interventions. In this study we have systematically mapped the global phosphorylation modifications within regenerating tissue of zebrafish caudal fins, elucidating the intricate landscape of signalling pathway associate with the regeneration process. Based on mass spectrometry analysis, we identified 440 phosphorylated proteins using the immunoprecipitation method with phosphoserine, phosphothreonine, and phosphotyrosine antibodies, and 74 phosphorylated proteins using the TiO₂ column enrichment method were found differentially phosphorylated during the regeneration process from 12 hpa to 7 dpa compared to the control. Interestingly 95% of the proteins identified from TiO2 enrichment method were also found to be identified through the phosphoprotein antibody pull down method impacting the high accuracy and significance of the methods and greater association of the 70 proteins undergoing differential phosphorylation during the process of regeneration. Whole mount immunohistochemistry analysis reveals high association of phosphorylation at 1dpa, 2dpa and 3dpa regeneration time points. Network pathway analysis revealed that cancer-related diseases, organismal injuries and abnormalities as the most strongly associated canonical network pathways with the differentially expressed phosphoproteome in the mechanism of regeneration. This research enhances our comprehension on protein post-translational modification in the context of zebrafish caudal fin tissue regeneration, shedding light on its prospective application in the field of regenerative medicine.

Malignant Pleural Mesothelioma (MPM) is the most prevalent type of Mesothelioma and currently has no effective treatment options. This underscores the urgent need to explore new therapeutic agents and innovative strategies. Phenolic acids are significant natural compounds recognised for their effectiveness in treating various diseases, including cancer. This study evaluates the anti-carcinogenic properties of Cinnamic acid (CINN) and its derivative Caffeic acid (CA) in both MPM and non-cancerous mesothelial cells. Results show that CA exhibited greater efficiency than CINN in reducing cancer cell survival. This enhanced efficacy is primarily attributed to CA’s higher selectivity index and its ability to inhibit proliferation at lower concentrations. Consequently, further analysis was conducted using CA. The subsequent findings revealed that CA suppressed proliferative markers, Ki67 and PCNA, inhibited colony formation and wound healing in MM cells. Experiments also exposed that it suppresses the phosphorylation of ERK1/2 and AKT proteins in a concentration-dependent manner, while the phosphorylation of STAT3 remains unaffected. The pattern of protein phosphorylation and expression suppression by CA in 3D cells resembles that in 2D cells, although it occurred at higher concentrations. Additionally, CA significantly enhanced the expression of p53-regulated proteins p21 and p27, resulting in G2/M arrest in both SPC111 and SPC212 cell lines. Moreover, elevated concentrations of CA were associated with an increased number of dead cells, as demonstrated by DAPI/PI and AO/EtBr fluorescence staining. The increased Bax/Bcl-2 protein ratio, and BH3-only proteins (Bik and PUMA) and the cleavage of caspase-3 indicated that CA induces mitochondrial apoptosis. Our research with MM cells and three-dimensional micro-tumours suggests that CA may be a promising alternative for future MM therapies. However, it is vital to conduct high-throughput in vivo studies to elucidate further the potential importance of CA in treating this devastating disease.

1,2-Unsaturated pyrrolizidine alkaloids (PA) induce severe acute and chronic hepatotoxicity. Effects include an impairment of the cell cycle. To elucidate this effect, cell cycle progression was analyzed by flow cytometry, changes in cell and nucleus size, differential gene expression and protein phosphorylation patterns of regulatory key proteins in the Chinese hamster lung fibroblast cell line V79 and/or the human hepatoblastoma cell line HepG2, both overexpressing human CYP3A4 (V793A4/HepG23A4). Lasiocarpine, a potent PA representative, reduced the cell viability of human CYP3A4-overexpressing cell lines concentration-dependently. Microscopic observation showed a marked increase in cell and nucleus size of V793A4 cells after exposure to 10 µM lasiocarpine. In the human CYP3A4-overexpressing cell lines, cells accumulate in G2/M phase after lasiocarpine treatment. Based on these findings, the gene expression pattern of cell cycle-related genes was investigated in HepG23A4 cells showing a decrease of e. g. WEE1, and CHEK1 and an increase of PAK1 and ATM. While results on cell cycle regulation at the level of gene expression are of limited relevance, protein phosphorylation plays an important role. Therefore, we also elucidated the protein phosphorylation status of regulatory key proteins. The results clearly indicate an induction of the DNA damage response and a late G2 arrest. In conclusion, an impairment of the cell cycle was observed. It correlates with the metabolic activation of lasiocarpine and is most likely mediated by adduct formation of the reactive pyrrole esters with DNA, leading to a disruption of cellular homeostasis and genomic instability.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00204-025-04185-y.

Zinc is known to influence chromatin stability, motility and protection against oxidative stress. While swim-up remains the preferred method for selecting sperm in Assisted Reproductive Technologies (ART), concerns arise regarding sperm DNA fragmentation associated with this procedure. Given zinc's significant role in protecting sperm DNA integrity and motility, we aimed to investigate the impact of zinc supplementation during the swim-up process on sperm quality. Semen samples from 203 normozoospermic men were used. Samples were divided into fractions and swim-up procedure was applied using human tubal fluid (mHTF) supplemented with three different concentrations of zinc or medium without supplementation as control. DNA fragmentation, chromatin maturity, reactive oxygen species (ROS) levels, motility and protein phosphorylation levels analyses were addressed to each fraction. The sperm DNA fragmentation was reduced in sperm recovered by swim-up in media with all concentrations of zinc assayed with respect to the control (p < 0.0001). Aniline blue staining showed better chromatin maturity in sperm recovered with 2.5- and 3.5-mM zinc (p = 0.045; p = 0.021). Kinematic parameters such as curvilinear velocity and beat-cross frequency showed improvement with 2.5mM zinc (p = 0.0080 and p = 0.0400), whereas straightness, linearity, and hypermotility showed improvement with 5mM zinc (p = 0.0075, p = 0.0069, and p = 0.0244). Protein phosphorylation patterns showed changes associated with treatment with zinc, and only 5mM zinc treatment showed a decrease in ROS levels. The addition of zinc to mHTF provided optimal physiological conditions for sperm recovered through swim-up. This supplementation should be considered for selecting sperm for use in ART.

Cronobacter sakazakii is a notorious foodborne opportunistic pathogen, particularly affecting vulnerable populations such as premature infants, and poses significant public health challenges. This study aimed to elucidate the role of the envZ/ompR genes in environmental tolerance, pathogenicity, and protein regulation of C. sakazakii. An envZ/ompR knockout mutant was constructed and assessed for its impact on bacterial growth, virulence, environmental tolerance, and protein regulation. Results demonstrate that deletion of envZ/ompR genes leads to reduced growth rate and attenuated virulence in animal models. Additionally, the knockout strain exhibited compromised environmental tolerance, particularly in desiccation and oxidative stress conditions, along with impaired adhesion and invasion abilities in epithelial cells. Proteomic analysis revealed significant alterations in protein expression and phosphorylation patterns, highlighting potential compensatory mechanisms triggered by gene deletion. Furthermore, investigation into protein deamidation and glucose metabolism uncovered a link between envZ/ompR deletion and energy metabolism dysregulation. Interestingly, the downregulation of MalK and GrxC proteins was identified as contributing factors to altered desiccation tolerance and disrupted redox homeostasis, respectively, providing mechanistic insights into the phenotypic changes observed. Overall, this study enhances understanding of the multifaceted roles of envZ/ompR in C. sakazakii physiology and pathogenesis, shedding light on potential targets for therapeutic intervention and food safety strategies.

Exercise training effectively relieves anxiety disorders via modulating specific brain networks. The role of post-translational modification of proteins in this process, however, has been underappreciated. Here we performed a mouse study in which chronic restraint stress-induced anxiety-like behaviors can be attenuated by 14-day persistent treadmill exercise, in association with dramatic changes of protein phosphorylation patterns in the medial prefrontal cortex (mPFC). In particular, exercise was proposed to modulate the phosphorylation of Nogo-A protein, which drives the ras homolog family member A (RhoA)/ Rho-associated coiled-coil-containing protein kinases 1(ROCK1) signaling cascade. Further mechanistic studies found that liver-derived kynurenic acid (KYNA) can affect the kynurenine metabolism within the mPFC, to modulate this RhoA/ROCK1 pathway for conferring stress resilience. In sum, we proposed that circulating KYNA might mediate stress-induced anxiety-like behaviors via protein phosphorylation modification within the mPFC, and these findings shed more insights for the liver-brain communications in responding to both stress and physical exercise.

DUSP4 is a member of the DUSP (dual-specificity phosphatase) subfamily that is selective to the mitogen-activated protein kinases (MAPK) and has been implicated in a range of biological processes and functions in Alzheimer's disease (AD). In this study, we utilized the stereotactic delivery of adeno-associated virus (AAV)-DUSP4 to overexpress DUSP4 in the dorsal hippocampus of 5xFAD and wildtype (WT) mice, then used mass spectrometry (MS)-based proteomics along with the label-free quantification to profile the proteome and phosphoproteome in the hippocampus. We identified protein expression and phosphorylation patterns modulated in 5xFAD mice and examined the sex-specific impact of DUSP4 overexpression on the 5xFAD proteome/phosphoproteome. In 5xFAD mice, a substantial number of proteins were up- or down-regulated in both male and female mice in comparison to age and sex-matched WT mice, many of which are involved in AD-related biological processes, such as activated immune response or suppressed synaptic activities. Many proteins in pathways, such as immune response were found to be suppressed in response to DUSP4 overexpression in male 5xFAD mice. In contrast, such a shift was absent in female mice. For the phosphoproteome, we detected an array of phosphorylation sites regulated in 5xFAD compared to WT and modulated via DUSP4 overexpression in each sex. Interestingly, 5xFAD- and DUSP4-associated phosphorylation changes occurred in opposite directions. Strikingly, both the 5xFAD- and DUSP4-associated phosphorylation changes were found to be mostly in neurons and play key roles in neuronal processes and synaptic functions. Site-centric pathway analysis revealed that both the 5xFAD- and DUSP4-associated phosphorylation sites were enriched for a number of kinase sets in females but only a limited number of sets of kinases in male mice. Taken together, our results suggest that male and female 5xFAD mice responded to DUSP4 overexpression via shared and sex-specific molecular mechanisms, which might underly similar reductions in amyloid pathology in both sexes while learning deficits were reduced in only females with DUSP4 overexpression. Finally, we validated our findings with the sex-specific AD-associated proteomes in human cohorts and further developed DUSP4-centric proteomic network models and signaling maps for each sex.

Lemnaceae are small freshwater plants with extraordinary high growth rates. We aimed to test whether this correlates with a more efficient photosynthesis, the primary energy source for growth. To this end, we compared photosynthesis properties of the duckweed Lemna minor and the terrestrial model plant Arabidopsis thaliana. Chlorophyll fluorescence analyses revealed high similarity in principle photosynthesis characteristics; however, Lemna exhibited a more effective light energy transfer into photochemistry and more stable photosynthesis parameters especially under high light intensities. Western immunoblot analyses of representative photosynthesis proteins suggested potential post-translational modifications in Lemna proteins that are possibly connected to this. Phospho-threonine phosphorylation patterns of thylakoid membrane proteins displayed a few differences between the two species. However, phosphorylation-dependent processes in Lemna such as photosystem II antenna association and the recovery from high-light-induced photoinhibition were not different from responses known from terrestrial plants. We thus hypothesize that molecular differences in Lemna photosynthesis proteins are associated with yet unidentified mechanisms that improve photosynthesis and growth efficiencies. We also developed a high-magnification video imaging approach for Lemna multiplication which is useful to assess the impact of external factors on Lemna photosynthesis and growth.

Sarin is a highly toxic organophosphorus nerve agent that irreversibly inhibits neuronal enzyme acetylcholinesterase. In the prevailing scenario, it is of paramount importance to develop early diagnosis and medical countermeasures for sarin exposure. A deeper understanding of the molecular mechanism of sarin intoxication and perturbations in the associated cellular processes is likely to provide valuable clues for the elucidation of diagnostic markers and therapeutic targets for sarin exposure. Present study, uncovered the changes in phosphorylation patterns of multiple proteins in different rat brain regions after sarin intoxication using 2-DE/MS approach. It provided a holistic view of the phosphorylation-mediated changes in the cellular proteome and highlighted various signaling and response pathways affected at an early time point of sarin intoxication. We found total 22 proteins in the cortex, 25 proteins in the corpus striatum, and 17 proteins in the hippocampus, showed ≥1.5 fold changes (hyper- or hypo- phosphorylated) with respect to control, either at 2.5 h or 1 d after sarin exposure. These results indicated the differential expression of phosphoproteins involved in protein folding in the endoplasmic reticulum, carbon metabolism, metabolic function, and energy metabolism. Four candidates (protein disulfide-isomerase A3, heat shock cognate 71 kDa protein, alpha-enolase, and creatine kinase B-type), hyperphosphorylated in all three brain regions, can be further studied to understand the molecular mechanism behind neurodegenerative changes mediated by sarin exposure. The study sheds light on major pathogenic processes initiated during sarin intoxication and provides putative diagnostic markers/therapeutic targets for further validation.

The use of immunohistochemical techniques to study the patterns of protein phosphorylation has revolutionized the study of signaling pathways. This technique allows detecting the phosphorylated state of signaling proteins in formalin-fixed and paraffin-embedded tissue sections by using phosphospecific antibodies. This chapter describes in detail the immunohistocshemical protocols from which the study of phosphoproteins in tissue sections can be approached.

Triple-negative breast cancers (TNBCs) are characterized by a lack of approved targeted therapies and remain a challenge in the clinic. Several overexpressed proteins, including epidermal growth factor receptor (EGFR), have been associated with TNBCs and are considered potential therapeutic targets. However, EGFR inhibitors alone failed to demonstrate a cutting-edge advantage for treating TNBCs over conventional chemotherapies. Studies have shown that selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene also affect TNBC cell viability. The combination of gefitinib and raloxifene was assessed against TNBC cell lines in vitro. Two TNBC cell lines, MDA-MB-231 and MDA-MB-468, were used to investigate the combination of gefitinib and raloxifene on cell viability, DNA synthesis, and apoptosis. The combination was assessed on intracellular signaling pathways, colony formation, migration, and angiogenesis. In the present study, raloxifene, in combination with gefitinib, decreased cell viability. The combination potentiates apoptosis and affects the expression and phosphorylation pattern of proteins involved in cell proliferation, such as NFκB, β-catenin, and EGFR. Furthermore, evidence of apoptosis activation was also observed, along with a decreased cell migration and tumorigenicity of TNBC cells. Moreover, the combined treatment decreased the ability of neovascularization as assessed by tube formation of endothelial cells. These results suggested the potential of the combination of raloxifene and gefitinib for the prevention of TNBC growth and the appearance of metastatic events. Our findings provide the basis for future studies on the mechanism involved in raloxifene-gefitinib inhibition of ER-negative tumor growth.

Abstract Background NFPTs are the most common operated pituitary tumours and can present with visual field defects, hormone deficiencies and headache. Surgery is treatment of choice but recurrence rate is high ranging from 10-50%, depending on the extent of tumour removal. No confirmed predictive biomarkers for NFPT recurrence have been identified, apart from Ki-67. We applied high-throughput mass spectrometry-based phosphoproteomic approach to explore the phosphorylation pattern of proteins in NFPTs in order to identify predictive markers of invasion and recurrence. Methods Based on radiological, histopathological, and surgical features, NFPTs were sub-grouped into three groups: non-invasive (n=5), invasive (n=10) and recurrent (n=5) subtypes. Invasiveness was determined by radiology (Knosp classification 3&amp;4), histopathological invasion (bone, dura and mucosa) and intraoperative findings. Tumour recurrence was based on radiological data for a mean±SD follow-up of112±39 months. Fresh-frozen pituitary tumour tissues were used for protein extraction and phosphopeptides were enriched using TiO 2 and labelled with tandem mass tags and subjected tomass spectrometry (Orbitrap)for quantification. Candidate hyper-phosphorylated proteins were validated by immunohistochemistry in 200 additional tumour samples by immunoblotting (n=36). Results In total, we identified 3185 phosphopeptides and observed significant difference in phosphorylation levels of invasive and recurrent groups. Compared to non-invasive cases, in invasive group we found, 452 hyper and 93 hypo phosphorylated proteins, while in the recurrent group there were 790 hyper and 307 hypo phosphorylated proteins. Phospho-serine showed the highest level of difference (90.3%) among the groups, followed by threonine (8.9%) and tyrosine (0.8%). One of the top differentially phosphorylated proteins was Ser552 of β-catenin showing significant hyper-phosphorylation in recurrent (p&amp;lt;0. 001) and invasive (p&amp;lt;0. 001) NFPTs. We also observed hyper-phosphorylation in tumours with suprasellar (p&amp;lt;0. 05) and cavernous sinus extension (p&amp;lt;0. 01). There was no correlation with tumour diameter and volume. Receiver operating characteristics curve analysis was performed to find the optimal cut-off value of β-catenin pSer552 immunohistochemical H-score in patients who had recurrence (n=44) or non-recurrence (n=156)and observed an area under curve of 0.717 (95% CI: 0.61-0.80),indicating a good prognostic ability for theβ-catenin pSer552H-score. A cut-off value of 160 for theβ-catenin pSer552H-score gives a sensitivity of 69% and a specificity of 73% for tumour recurrence. Kaplan-Meier survival curve analysis shows strong statistical correlation in the recurrence free survival (p&amp;lt;0. 0001) and the nuclear positive staining of β-catenin pSer552 with a hazard ratio of 3.1 (95% CI 1.5-6.3). Conclusions our study has identified hyper-phosphorylation of β-catenin at the Ser552 in recurrent and invasive NFPT subgroups. The H-score of β-catenin p552 correlates with tumour recurrence free survival in a large cohort of NFPT patients, which supports that β-catenin pSer552 could be used as predictive biomarker for NFPT recurrence. Presentation: No date and time listed

Abstract Background The lack of therapies for HFpEF patients is a major unmet need, thus identifying cardiac specific pathways in HFpEF is a priority. Purpose Identify molecular features of protein and phosphoprotein in a murine model of HFpEF Methods Studies followed the principles of laboratory animal care (NIH Pub no. 85–23 revised 1985). HFpEF (N=4) was induced by NaCl drinking water, unilateral nephrectomy, and chronic aldosterone infusion (SAUNA) or saline (Sham; N=4) for 4 wks. Mice were euthanized and LV tissue was collected. Samples were homogenized, proteins extracted and subjected to digestion followed by phosphopeptide enrichment for proteomics and phosphoproteomics profiling. Results HFpEF mice had moderate hypertension (137.8±7.0 vs. Sham; 115.4±6.0mmHg; P&amp;lt;0.05), lung congestion (4.5±0.1 vs. 4.0±0.1; P&amp;lt;0.01), and LVH (3.7±0.1 vs. 3.3±0.1mg/g; P&amp;lt;0.05). ECHO showed normal LVEF with evidence of diastolic dysfunction in HFpEF mice. Mitral E velocity was reduced (1347±154 vs. 1971±284mm/s; P&amp;lt;0.05) and IVRT increased (24.3±2.6 vs. 14.4±1.6ms; P&amp;lt;0.05) vs. Sham. Protein and protein phosphosites from the LV were quantified by mass spectrometry. Analysis of the proteomics datasets revealed marked changes in sarcomere proteins, such as skeletal alpha (α)-actin (ACTA1; P=0.000039), beta (β)-myosin heavy chain (MYH7; P=0.006963) and myosin heavy chain 9 (MYH9; P=0.000408); the mitochondria-related proteins mitofusin 1 (MFN1; P=0.001059), mitochondrial dynamin like GTPase (aka optic atrophy protein 1, OPA1; P=0.046441) and transcription factor A mitochondrial (TFAM; P=0.005837); and the NAD-dependent protein deacetylase sirtuin-3 (SIRT3; P=0.000914). There was also a reduction in proteins involved in the oxidation of free fatty acid, pyruvate, and ketone bodies in the LV from HFpEF vs. Sham. Phosphoproteomics analysis also showed aberrant protein phosphorylation patterns linked to disparate subcellular compartments, ranging from sarcomere proteins (LIM domain-binding protein 3, LDB3; myozenin 2, Myoz2; titin, TTN), to nuclear-localized proteins (BAG family molecular chaperone regulator 3, BAG3; high mobility group protein HMG-I/HMG-Y, HMGA1) with known links to contractile dysfunction, LVH and/or cardiomyopathy. Additional GSEA analysis revealed the most relevant and enriched biological annotations in LV from HFpEF, to be processes involving immune system modulation and muscle contraction. Downregulated pathways were mainly related to multitude of GO terms associated with mitochondrial metabolism. Conclusion(s) This study presents the systematic, quantitative proteomics and phosphoproteomic analysis of the LV from the SAUNA HFpEF mice. We observed profound changes in proteins related to mitochondrial metabolism and function and heart contractile dysfunction in HFpEF which may be mediated by Sirtuin 3. Additional studies are warranted to investigate the specific role of Sirtuin 3 in mitochondrial metabolism and heart contractile dysfunction in HFpEF. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): National Institute of Heath (NIH/ NHLBI)

The objective of the study was to characterize the temporal changes of phosphorylation patterns of mTOR signaling proteins in response to two dietary protein sources in insulin dysregulated (ID, n = 8) and non-ID (n = 8) horses. Horses were individually housed and fed timothy grass hay and 2 daily concentrate meals so that protein was the first limiting nutrient and the total diet provided 120% of daily DE requirements for maintenance. On sample days, horses randomly received 0.25 g CP/kg BW of a pelleted alfalfa (AP) or commercial protein supplement (PS). Blood samples were collected before and 30, 60, 90, 120, 150, 180, 210, 240, 300, 360, 420, and 480 min post feeding and analyzed for plasma glucose, insulin and amino acid (AA) concentrations. Gluteus Medius muscle samples were obtained before and 90, 180, and 300 min after feeding and analyzed for relative abundance of phosphorylated mTOR pathway components using western immunoblot analysis. There was no effect of protein source on postprandial glucose and insulin responses (P ≥ 0.14) but consumption of PS elicited a 2 times larger AUC for essential AA (EAA), greater peak concentrations of EAA and a shorter time to reach peak EAA concentrations compared to AP. Abundance of phosphorylated mTOR (P = 0.08) and rpS6 (P = 0.10) tended to be ~1.5-fold greater after consumption of PS at 90 min compared to AP. Dephosphorylation patterns differed between protein sources and was slower for AP compared to PS. ID horses had a 2 times greater (P = 0.009) AUC and 3 times higher postprandial peak concentrations (P < 0.0001) for insulin compared to non-ID horses after consumption of both treatment pellets, but EAA responses were similar between groups (P = 0.53). Insulin status did not affect rpS6 or mTOR phosphorylation after consumption of either protein source (P ≥ 0.35), but phosphorylated rpS6 abundance was twice as high in ID compared to non-ID horses (P = 0.007). These results suggest that the consumption of higher quality protein sources may result in greater postprandial activation of the mTOR pathway compared to equal amounts of a forage-based protein source. Moreover, ID does not impair postprandial activation of mTOR and rpS6 proteins in horses following a protein-rich meal.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.