Plasma amyloid-β (Aβ) peptides, alone or in ratio with p-tau217, show strong potential as Alzheimer’s disease biomarkers. While immunoprecipitation-mass spectrometry (IP-MS) is the preferred method for plasma Aβ quantification, current assays are resource- and time-intensive. Here, we developed a streamlined IP-MS method using a cost-effective instrument that significantly improved the efficiency of an original assay by incorporating a single immunoprecipitation step, an optimized buffer system, and approximately 75

The detection and monitoring of Alzheimer's disease (AD) biomarkers in plasma are crucial for early diagnosis and prognosis. However, the stability of plasma AD biomarkers can be compromised by the degradation caused by endogenous proteases present in blood. The efficacy of protease inhibitors in mitigating this degradation is yet to be established. This study evaluated the stability of five plasma AD biomarkers (GFAP, NFL, Aβ1-40, Aβ1-42, and pTau181) in plasma over time under room temperature (RT) conditions. Samples were collected concurrently into either standard K2EDTA or BD-P100 tubes, with the latter being coated with protease inhibitors. Our objective was to assess the effectiveness of protease inhibitors in preserving the stability of plasma AD biomarkers in plasma. We evaluated two experimental approaches. In Approach 1, whole blood samples (n=16) from the same draw were collected into K2EDTA and BD-P100 tubes and stored at RT for 0h and 24h. In Approach 2, plasma samples (n=19) were isolated from whole blood collected into BD-P100 and K2EDTA tubes and immediately stored at RT for 0h, and 24h. The levels of plasma AD biomarkers were measured using immunoprecipitation with mass spectrometry (IP-MS) and Single Molecule Array (SIMOA). The IP-MS assay replicated a protocol (Nakamura et al., 2018) for Aβ1-40 and Aβ1-42 measurement. The IP-MS assay indicated that the use of BD-P100 tubes significantly reduced Aβ peptide degradation compared with the K2EDTA tube. In Approach 1, the IP-MS Aβ signal decreased by 23-34% for samples collected in K2EDTA tubes relative to 17-18% for samples collected in BD-P100 tubes. In Approach 2, the IP-MS Aβ peptide signal decreased by 14-16% for K2EDTA tubes, with a lower decrease of 3-7% for BD-P100 tubes. The SIMOA assays produced similar results for Aβ peptides. No significant variations were detected in time-dependent pTau181, GFAP, and NFL levels for samples collected using both tubes. The study showed that tubes supplemented with protease inhibitors significantly improve the preanalytical stability of Aβ1-40 and Aβ1-42 in plasma. These results have important implications for preanalytical procedures that are needed to enable widespread access and utilization of plasma biomarkers in resource-limited settings.

Amyloid β (Aβ) deposition in the brain is a pathological hallmark of Alzheimer's disease (AD). While immunoprecipitation-mass spectrometry (IP-MS) stands out as an accurate method for quantifying blood-based Aβ peptides, its major limitations such as prolonged sample preparation, extensive analysis time, large specimen volume, and high costs, present opportunities for improvement. Consequently, we aimed to develop a novel plasma IP-MS Aβ assay that employs simplified and significantly shorter analytical procedures, along with much-reduced sample volumes. We evaluated effects of reducing the sample volume, changing the buffers, implementing heavy labeled internal standards, and switching to a more cost-effective MALDI-Tof mass spectrometer versus the original Shimadzu IP-MS Aβ assay (Nakamura et al., 2018 Nature). This led to an improved assay, referred to as the Pittsburgh IP-MS Aβ assay. Analytical validation covered linearity, accuracy, precision, and recovery. In clinical validation, we compared the new and original Aβ assays for identifying Aβ-PET positivity and cognitive impairment in two independent cohorts. The Pittsburgh assay streamlined the immuno-enrichment step with a novel binding buffer, cutting background noise and reducing processing time by 50%. Additionally, it allowed for potential sample volumes as low as 50 µl and decreased antibody and paramagnetic bead usage by 75% each. The assay utilized the cost-friendly MALDI-Tof instrument from Bruker, a departure from the AXIMA Performance platform used in the original assay. The analytical validation showed consistent signal linearity, improved spike recovery with heavy labeled Aβ1-40 or Aβ1-42, but equivalent precision between the two IP-MS methods. In the AGUEDA cohort (n=148), the Pittsburgh assay demonstrated better AUCs than the older method for identifying Aβ-PET-positive individuals. This trend persisted in the University of Pittsburgh ADRC cohort (n=30) when comparing accuracy to identify patients with clinical assessed AD positive. We have developed a more efficient and cost-effective IP-MS plasma Aβ assay, outperforming the original Shimadzu assay in clinical utility, while upholding consistently high analytical performance. The cost, time, and reagent savings along with the utilization of a more affordable and widely available instrument will empower more research laboratories to conduct IP-MS analysis of Aβ in blood more effectively.

The multitude of DNA lesion types, and the nuclear dynamic context in which they occur, presents a challenge for genome integrity maintenance as this requires the engagement of different DNA repair pathways. Specific "repair controllers" that facilitate DNA repair pathway crosstalk between double-strand break (DSB) repair and base excision repair (BER) and that regulate BER protein engagement at lesion sites have yet to be identified. Here, we find that DNA polymerase β (Polβ), crucial for BER, is ubiquitylated in a BER complex-dependent manner by TRIP12, an E3 ligase that partners with UBR5 to restrain DSB repair signaling. Furthermore, we find that TRIP12, but not UBR5, controls cellular levels and chromatin loading of Polβ. Required for Polβ foci formation, TRIP12 influences Polβ involvement after radiation-induced DNA damage, a process regulated by TRIP12-mediated ubiquitylation of Polβ. Notably, excessive TRIP12-mediated engagement of Polβ affects DSB formation and radiation sensitivity, underscoring its role in promoting precedence for BER over DSB repair. The herein discovered function of TRIP12, in the governance of Polβ-directed BER, supports a role for TRIP12 in assuring BER lesion removal at complex DSB sites to optimize DSB repair at the nexus of DNA repair pathways.

OBJECTIVE:To characterize differences in vaginal proteomic profiles among women with mesh exposure, mesh-related pain, and controls. METHODS:10 well-integrated mesh samples (removed incidentally or normally incorporated distant from complication), 10 mesh exposure samples, 10 mesh-pain samples and 10 control vaginal biopsies of participants without mesh from a Mesh and Pelvic Floor Tissue Biorepository were analyzed. Differentially expressed protein analysis, gene ontology enrichment, clinical covariate modeling and a hypothesis-driven assessment of 100 a priori specified proteins were performed. RESULTS:Participant age of well-integrated mesh specimens (62 ± 13 years) was similar to complication samples (exposure 52 ± 7, pain 49 ± 14 years) but older than controls (42 ± 6 years). Mean BMI (28 ± 6 kg/m2) and median implantation duration (6 ± 5 years) were similar between groups. Extracellular remodeling proteins, including MMP9 and TIMP3 and neutrophil activation proteins showed the greatest difference between complications and controls. GO terms related to immunity, response to stress, cell proliferation, apoptosis, and inflammatory and fibrosis mediators were enriched in exposure. Mesh related pain group showed enrichment in collagen fibril organization and cartilage development compared to well-integrated mesh, while only morphogenesis of an epithelium and cornification were different between pain and exposure. Clinical covariate analysis revealed similar pathways. ELISA performed on a separate cohort supports elevation of TIMP3 in well-integrated mesh samples. CONCLUSIONS:Even well-integrated polypropylene mesh dramatically alters the local environment. Pain and exposure complications progress through epithelial remodeling, dysregulated extracellular matrix, and fibrosis pathways similarly regardless of clinical risk factors, supporting that constructive matrix remodeling therapies and neutrophil targeting may be useful therapeutics. STATEMENT OF SIGNIFICANCE:Polypropylene mesh is used in urogynecologic surgery to improve the durability and reduce invasiveness of surgical repairs, however it is associated with a high complication rate. In order to improve the response to polypropylene mesh implanted on the vagina and to design better materials, we must understand the pathways through which these complications develop. This proteomic analysis showed that inflammation, disordered extracellular and epithelial remodeling, apoptosis and fibrosis were the most different between complications and controls, both validating prior mechanistic studies and providing potential therapeutic targets, including matrix degradation protein inhibitors and neutrophil pathways.

High-performance, resource-efficient methods for plasma amyloid-β (Aβ) quantification in Alzheimer's disease are lacking; existing mass spectrometry-based assays are resource- and time-intensive. We developed a streamlined mass spectrometry method with a single immunoprecipitation step, an optimized buffer system, and ≤75% less antibody requirement. Analytical and clinical performances were compared with an in-house reproduced version of a well-known two-step assay. The streamlined assay showed high dilution linearity (r2>0.99) and precision (< 10% coefficient of variation), low quantification limits (Aβ1-40: 12.5 pg/ml; Aβ1-42: 3.125 pg/ml), and high signal correlation (r2~0.7) with the two-step immunoprecipitation assay. The novel single-step assay showed more efficient recovery of Aβ peptides via fewer immunoprecipitation steps, with significantly higher signal-to-noise ratios, even at plasma sample volumes down to 50 pl. Both assays had equivalent performances in distinguishing non-elevated vs. elevated brain Aβ-PET individuals. The new method enables simplified yet robust evaluation of plasma Aβ biomarkers in Alzheimer's disease.

The multitude of DNA lesion types, and the nuclear dynamic context in which they occur, present a challenge for genome integrity maintenance as this requires the engagement of different DNA repair pathways. Specific 'repair controllers' that facilitate DNA repair pathway crosstalk between double strand break (DSB) repair and base excision repair (BER), and regulate BER protein trafficking at lesion sites, have yet to be identified. We find that DNA polymerase β (Polβ), crucial for BER, is ubiquitylated in a BER complex-dependent manner by TRIP12, an E3 ligase that partners with UBR5 and restrains DSB repair signaling. Here we find that, TRIP12, but not UBR5, controls cellular levels and chromatin loading of Polβ. Required for Polβ foci formation, TRIP12 regulates Polβ involvement after DNA damage. Notably, excessive TRIP12-mediated shuttling of Polβ affects DSB formation and radiation sensitivity, underscoring its precedence for BER. We conclude that the herein discovered trafficking function at the nexus of DNA repair signaling pathways, towards Polβ-directed BER, optimizes DNA repair pathway choice at complex lesion sites.

ABSTRACTINTRODUCTIONThe reliability of plasma Alzheimer’s disease (AD) biomarkers can be compromised by protease-induced degradation. This limits the feasibility of conducting plasma biomarker studies in environments that lack the capacity for immediate processing and appropriate storage of blood samples. We hypothesized that blood collection tube supplementation with protease inhibitors can improve the stability of plasma biomarkers at room temperatures (RT). This study conducted a comparative analysis of blood biomarker stability in traditional ethylenediaminetetraacetic acid (EDTA) tubes versus BD™ P100 collection tubes, the latter being coated with a protease inhibitor cocktail. The stability of six plasma AD biomarkers was evaluated over time under RT conditions.METHODSWe evaluated three experimental approaches. In Approach 1, pooled plasma samples underwent storage at RT for up to 96 hours. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0h or 24h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA or P100 tubes, followed by storage at RT for 0h or 24h before isolating the plasma for analyses. Biomarkers were measured with Single Molecule Array (Simoa) and immunoprecipitation-mass spectrometry (IP-MS) assays.RESULTSBoth the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improved the stability of Aβ42 and Aβ40 across all approaches. Additionally, the Aβ42/Aβ40 ratio levels were significantly stabilized only in the IP-MS assay in Approach 3. No significant differences were observed in the levels of plasma p-tau181, GFAP, and NfL for samples collected using either tube type in any of the approaches.CONCLUSIONSupplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aβ42 and Aβ40, and the Aβ ratio for IP-MS assay. This has crucial implications for preanalytical procedures, particularly in resource-limited settings.

Conclusions We demonstrate for the first time, that AT-derived ceramides are causally related with dysregulated myocardial redox signalling and adverse cardiovascular disease outcomes in patients with advanced atherosclerosis. As such, GlcC16 may be an important therapeutic target for the prevention and treatment of cardiovascular complications in obesity and diabetes.

The “block and lock” strategy is one approach that might elicit a sterilizing cure for HIV-1 infection. The “block” refers to a compound’s ability to inhibit latent HIV-1 proviral transcription, while the “lock” refers to its capacity to induce permanent proviral silencing. We previously identified PF-3758309, a pan-isoform inhibitor of p21-activated kinases (PAKs), as a potent inhibitor of HIV-1 latency reversal. The goal of this study was to define the mechanism(s) involved. We found that both 24ST1NLESG cells (a cell line model of HIV-1 latency) and purified CD4+ naïve and central memory T cells express high levels of PAK2 and lower levels of PAK1 and PAK4. Knockdown of PAK1 or PAK2, but not PAK4, in 24ST1NLESG cells resulted in a modest, but statistically significant, decrease in the magnitude of HIV-1 latency reversal. Overexpression of PAK1 significantly increased the magnitude of latency reversal. A phospho-protein array analysis revealed that PF-3758309 down-regulates the NF-κB signaling pathway, which provides the most likely mechanism by which PF-3758309 inhibits latency reversal. Finally, we used cellular thermal shift assays combined with liquid chromatography and mass spectrometry to ascertain whether PF-3758309 off-target binding contributed to its activity. In 24ST1NLESG cells and in peripheral blood mononuclear cells, PF-3758309 bound to mitogen-activated protein kinase 1 and protein kinase A; however, knockdown of either of these kinases did not impact HIV-1 latency reversal. Collectively, our study suggests that PAK1 and PAK2 play a key role in the maintenance of HIV-1 latency.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the United States. Tyrosine sulfation, catalyzed by the tyrosylprotein sulfotransferase 2 (TPST2), is a post-translational modification essential for protein-protein interactions and cellular functions. Solute carrier family 35 member B (SLC35B2) is a key transporter that transports the universal sulfate donor 3'phosphoadenosine 5'-phosphosulfate into the Golgi apparatus where the protein sulfation occurs. The goal of this study was to determine whether and how the SLC35B2-TPST2 axis of tyrosine sulfation plays a role in PDAC. METHODS: Gene expression was analyzed in PDAC patients and mice. Human PDAC MIA PaCa-2 and PANC-1 cells were used for in vitro studies. TPST2-deficient MIA PaCa-2 cells were generated to assess xenograft tumor growth in vivo. Mouse PDAC cells derived from the Kras(LSL-G12D/+)Tp53(L/+)Pdx1-Cre (KPC) mice were used to generate Tpst2 knockout KPC cells to evaluate tumor growth and metastasis in vivo. RESULTS: High expressions of SLC35B2 and TPST2 were correlated with poor PDAC patient survival. Knocking down SLC35B2 or TPST2, or pharmacologicically inhibiting sulfation, resulted in the inhibition of PDAC cell proliferation and migration in vitro. TPST2-deficient MIA PaCa-2 cells showed inhibited xenograft tumor growth. Orthotopic inoculation of Tpst2 knockout KPC cells in mice showed inhibition of primary tumor growth, local invasion, and metastasis. Mechanistically, the integrin beta 4 was found to be a novel substrate of TPST2. Inhibition of sulfation destabilizes integrin beta 4 protein, which may have accounted for the suppression of metastasis. CONCLUSIONS: Targeting the SLC35B2-TPST2 axis of tyrosine sulfation may represent a novel approach for therapeutic intervention of PDAC.

Left ventricular diastolic dysfunction is a structural and functional condition that precedes the development of heart failure with preserved ejection fraction (HFpEF). The etiology of diastolic dysfunction includes alterations in fuel substrate metabolism that negatively impact cardiac bioenergetics, and may precipitate the eventual transition to heart failure. To date, the molecular mechanisms that regulate early changes in fuel metabolism leading to diastolic dysfunction remain unclear. In this report, we use a diet-induced obesity model in aged mice to show that inhibitory lysine acetylation of the pyruvate dehydrogenase (PDH) complex promotes energetic deficits that may contribute to the development of diastolic dysfunction in mouse hearts. Cardiomyocyte-specific deletion of the mitochondrial lysine acetylation regulatory protein GCN5L1 prevented hyperacetylation of the PDH complex subunit PDHA1, allowing aged obese mice to continue using pyruvate as a bioenergetic substrate in the heart. Our findings suggest that changes in mitochondrial protein lysine acetylation represent a key metabolic component of diastolic dysfunction that precedes the development of heart failure.

1 Vascular Medicine Institute, 2 Center for Metabolism and Mitochondrial Medicine, Department of 6 Medicine, University of Pittsburgh, Pittsburgh, PA 15261. 3 Biomedical Mass Spectrometry Center, 7 Schools of the Health Sciences, University of Pittsburgh, Pittsburgh, PA 15261. 4 Department of Cell 8 Biology, University of Pittsburgh, Pittsburgh, PA 15261. 5 Department of Chemistry, University of 9 Pittsburgh, Pittsburgh, PA 15261. 10

Introduction Left ventricular diastolic dysfunction is a structural and functional condition that precedes the development of heart failure with preserved ejection fraction (HFpEF). The etiology of diastolic dysfunction includes alterations in fuel substrate metabolism that negatively impact cardiac bioenergetics, and may precipitate the eventual transition to heart failure. To date, the molecular mechanisms that regulate early changes in fuel metabolism leading to diastolic dysfunction remain unclear. However, recent work has suggested that changes in mitochondrial lysine acetylation may regulate this process in mouse models of HFpEF. Methods We used a diet-induced obesity model and quantitative acetylproteomics in aged mice to examine the role played by mitochondrial lysine acetylation in the development of diastolic dysfunction. Wildtype and cardiac-specific GCN5L1 knockout mice (which are deficient in mitochondrial lysine acetylation) aged 5–7 months were placed on a low fat diet (10% fat) or high fat diet (60% fat) for 30 weeks. Echocardiography was performed after 30 weeks of diet to assess cardiac structure and function, followed by euthanasia. After rapid isolation, hearts were subject to quantitative acetylproteomics, respirometry measurements, and biochemical measurements of metabolic enzyme acetylation and activity. In addition, cell culture models of site-specific lysine acetylation were used to test the mechanism underlying bioenergetic changes in mouse hearts. Results Cardiomyocyte-specific deletion of the mitochondrial lysine acetylation regulatory protein GCN5L1 prevented the development of diastolic dysfunction (measured as a change in E/e’ ratio) in response to a high fat diet. Quantitative acetylproteomics demonstrated that enzymes in the mitochondrial fatty acid oxidation and pyruvate utilization pathways were most affected by GCN5L1-dependent acetylation. Deletion of GCN5L1 prevented hyperacetylation of the pyruvate dehydrogenase complex subunit PDHA1, which increased its enzymatic activity, and allowed increased pyruvate utilization in hearts from obese, aged mice. Using a cell culture model of variable PDHA1 acetylation status, we confirmed that site-specific acetylation of five PDHA1 lysine residues significantly reduced its enzymatic activity in cardiac cells in vitro. Conclusions Our findings suggest that changes in mitochondrial protein lysine acetylation represent a key metabolic component of diastolic dysfunction that precedes the development of heart failure. Our work suggests that manipulation of PDHA1 acetylation levels in vivo may represent a novel target for therapeutic intervention in the treatment of diastolic dysfunction.

ABSTRACTLeft ventricular diastolic dysfunction is a structural and functional condition that precedes the development of heart failure with preserved ejection fraction (HFpEF). The etiology of diastolic dysfunction includes alterations in fuel substrate metabolism that negatively impact cardiac bioenergetics, and may precipitate the eventual transition to heart failure. To date, the molecular mechanisms that regulate early changes in fuel metabolism leading to diastolic dysfunction remain unclear. In this report, we use a diet-induced obesity model and quantitative acetylproteomics in aged mice to show that inhibitory lysine acetylation of the pyruvate dehydrogenase (PDH) complex promotes energetic deficits and diastolic dysfunction in mouse hearts. Cardiomyocyte-specific deletion of the mitochondrial lysine acetylation regulatory protein GCN5L1 prevented hyperacetylation of the PDH complex subunit PDHA1, allowing aged obese mice to continue using pyruvate as a bioenergetic substrate in the heart. Our findings suggest that changes in mitochondrial protein lysine acetylation represent a key metabolic component of diastolic dysfunction that precedes the development of heart failure.

Schizophrenia (Sz) is a highly polygenic disorder, with common, rare, and structural variants each contributing only a small fraction of overall disease risk. Thus, there is a need to identify downstream points of convergence that can be targeted with therapeutics. Reduction of microtubule-associated protein 2 (MAP2) immunoreactivity (MAP2-IR) is present in individuals with Sz, despite no change in MAP2 protein levels. MAP2 is phosphorylated downstream of multiple receptors and kinases identified as Sz risk genes, altering its immunoreactivity and function. Using an unbiased phosphoproteomics approach, we quantified 18 MAP2 phosphopeptides, 9 of which were significantly altered in Sz subjects. Network analysis grouped MAP2 phosphopeptides into three modules, each with a distinct relationship to dendritic spine loss, synaptic protein levels, and clinical function in Sz subjects. We then investigated the most hyperphosphorylated site in Sz, phosphoserine1782 (pS1782). Computational modeling predicted phosphorylation of S1782 reduces binding of MAP2 to microtubules, which was confirmed experimentally. We generated a transgenic mouse containing a phosphomimetic mutation at S1782 (S1782E) and found reductions in basilar dendritic length and complexity along with reduced spine density. Because only a limited number of MAP2 interacting proteins have been previously identified, we combined co-immunoprecipitation with mass spectrometry to characterize the MAP2 interactome in mouse brain. The MAP2 interactome was enriched for proteins involved in protein translation. These associations were shown to be functional as overexpression of wild type and phosphomimetic MAP2 reduced protein synthesis in vitro. Finally, we found that Sz subjects with low MAP2-IR had reductions in the levels of synaptic proteins relative to nonpsychiatric control (NPC) subjects and to Sz subjects with normal and MAP2-IR, and this same pattern was recapitulated in S1782E mice. These findings suggest a new conceptual framework for Sz-that a large proportion of individuals have a "MAP2opathy"-in which MAP function is altered by phosphorylation, leading to impairments of neuronal structure, synaptic protein synthesis, and function.

Elevated aldehyde dehydrogenase (ALDH) activity correlates with poor outcome for many solid tumors as ALDHs may regulate cell proliferation and chemoresistance of cancer stem cells (CSCs). Accordingly, potent, and selective inhibitors of key ALDH enzymes may represent a novel CSC-directed treatment paradigm for ALDH+ cancer types. Of the many ALDH isoforms, we and others have implicated the elevated expression of ALDH1A3 in mesenchymal glioma stem cells (MES GSCs) as a target for the development of novel therapeutics. To this end, our structure of human ALDH1A3 combined with in silico modeling identifies a selective, active-site inhibitor of ALDH1A3. The lead compound, MCI-INI-3, is a selective competitive inhibitor of human ALDH1A3 and shows poor inhibitory effect on the structurally related isoform ALDH1A1. Mass spectrometry-based cellular thermal shift analysis reveals that ALDH1A3 is the primary binding protein for MCI-INI-3 in MES GSC lysates. The inhibitory effect of MCI-INI-3 on retinoic acid biosynthesis is comparable with that of ALDH1A3 knockout, suggesting that effective inhibition of ALDH1A3 is achieved with MCI-INI-3. Further development is warranted to characterize the role of ALDH1A3 and retinoic acid biosynthesis in glioma stem cell growth and differentiation.