Immunoprecipitation Mass Spectrometry Research Articles

6-Phosphogluconate dehydrogenase promotes glycolysis and fatty acid synthesis by inhibiting the AMPK pathway in lung adenocarcinoma cells

Abnormal metabolism has emerged as a prominent hallmark of cancer and plays a pivotal role in carcinogenesis and progression of lung adenocarcinoma (LUAD). In this study, single-cell sequencing revealed that the metabolic enzyme 6-phosphogluconate dehydrogenase (PGD), which is a critical regulator of the pentose phosphate pathway (PPP), is significantly upregulated in the malignant epithelial cell subpopulation during malignant progression. However, the precise functional significance of PGD in LUAD and its underlying mechanisms remain elusive. Through the integration of TCGA database analysis and LUAD tissue microarray data, it was found that PGD expression was significantly upregulated in LUAD and closely correlated with a poor prognosis in LUAD patients. Moreover, in vitro and in vivo analyses demonstrated that PGD knockout and inhibition of its activity mitigated the proliferation, migration, and invasion of LUAD cells. Mechanistically, immunoprecipitation-mass spectrometry (IP-MS) revealed for the first time that IQGAP1 is a robust novel interacting protein of PGD. PGD decreased p-AMPK levels by competitively interacting with the IQ domain of the known AMPKα binding partner IQGAP1, which promoted glycolysis and fatty acid synthesis in LUAD cells. Furthermore, we demonstrated that the combination of Physcion (a PGD-specific inhibitor) and metformin (an AMPK agonist) could inhibit tumor growth more effectively both in vivo and in vitro. Collectively, these findings suggest that PGD is a potential prognostic biomarker and therapeutic target for LUAD.

The Hippo Coactivator TAZ Exacerbates Cisplatin-Induced Acute Renal Injury

Introduction: Transcriptional coactivator with PDZ-binding motif (TAZ), a Hippo signaling pathway effector, maintains the balance of cell proliferation, differentiation, and death. However, the role of TAZ in tubular cell survival and acute kidney injury (AKI) remains largely unknown. Methods: We used the RNA-seq database, Western blot, and immunohistochemistry to examine TAZ expression in kidneys from cisplatin-induced AKI. We generated tubular-specific TAZ knockout mice to assess the role of TAZ in cisplatin-induced renal toxicity. Immunoprecipitation-mass spectrometry followed standard procedures. Results: TAZ was activated in tubular cells in kidneys injected with cisplatin. Conditional deletion of TAZ in tubular cells confers ferroptosis resistance and protects kidneys from cisplatin-induced AKI, whereas overexpression of TAZ(S89A) exacerbates cisplatin-induced ferroptosis. Inhibition of ferroptosis with ferrostatin-1 potently preserves renal function and alleviates morphological injury and tubular cell ferroptosis induced by cisplatin. Mechanistically, in a PPARδ-dependent manner, but not TEAD, TAZ reduces the expression of glutathione peroxidase 4 (GPX4), thus exacerbating cisplatin-induced ferroptosis. Conclusions: Our findings show that cisplatin-induced AKI and tubular cell ferroptosis are mediated by TAZ-PPARδ interaction through regulation of GPX4, highlighting TAZ as a potential therapeutic candidate for AKI.

AMDHD1 acts as a tumor suppressor and contributes to activation of TGF-β signaling pathway in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a malignant tumor of the digestive system, characterized by its aggressive behavior and the absence of effective therapeutic biomarkers. Although recent studies have implicated AMDHD1 in tumor formation, its role in CCA development has been insufficiently explored. We utilized multiple bioinformatic datasets alongside 108 clinical samples to examine AMDHD1 expression in CCA. Then, in vitro and in vivo experiments were conducted to assess its impact on tumor growth and metastasis. Furthermore, proteomic analysis and immunoprecipitation mass spectrometry were employed to identify the downstream effectors of AMDHD1. We discovered that AMDHD1 was down-regulated in CCA and this down-regulation was associated with adverse clinicopathological features and prognosis. We also demonstrated that overexpression of AMDHD1 hindered G1/S progression in the cell cycle and promoted apoptosis, thereby inhibiting tumor growth and metastasis. Mechanistically, we found that AMDHD1 operated in a TGF-β-dependent manner and the inhibition of TGF-β signaling abrogated the effect of AMDHD1 overexpression on CCA cells. Specifically, AMDHD1 inhibited the ubiquitination and degradation of the SMAD4 protein through binding to the MH2 domain and synergistically enhanced SMAD2/3 phosphorylation, which activated of TGF-β signaling pathway and resulted in the suppression of CCA cell proliferation and migration. Our study identifies AMDHD1 as a significant prognostic biomarker and a tumor suppressor in CCA. It underscores the pivotal role of the AMDHD1/TGF-β signaling pathway in the development and progression of CCA.

Automated Immunoprecipitation, Sample Preparation, and Individual Ion Mass Spectrometry Platform for Proteoforms.

Charge detection mass spectrometry (CDMS) is a well-established technique that provides direct mass spectral outputs regardless of analyte heterogeneity or molecular weight. Over the past few years, it has been demonstrated that CDMS can be multiplexed on Orbitrap analyzers utilizing an integrated approach termed individual ion mass spectrometry (I2MS). To further increase adaptability, robustness, and throughput of this technique, here, we present a method that utilizes numerous integrated equipment components including a Kingfisher system, SampleStream platform, and Q Exactive mass spectrometer to provide a fully automated workflow for immunoprecipitation, sample preparation, injection, and subsequent I2MS acquisition. This automated workflow has been applied to a cohort of 58 test subjects to determine individualized patient antibody responses to SARS-CoV-2 antigens. Results from a range of serum donors include 37 subject I2MS spectra that contained a positive COVID-19 antibody response and 21 I2MS spectra that contained a negative COVID-19 antibody response. This high-throughput automated I2MS workflow can currently process over 100 samples per week and is general for making immunoprecipitation-MS workflows achieve proteoform resolution.

Transcription factor 4 is a key mediator of oncogenesis in neuroblastoma by promoting MYC activity.

Super-enhancer-associated transcription factor networks define cell identity in neuroblastoma (NB). Dysregulation of these transcription factors contributes to the initiation and maintenance of NB by enforcing early developmental identity states. We report that the class I basic helix-loop-helix (bHLH) transcription factor 4 (TCF4; also known as E2-2) is a critical NB dependency gene that significantly contributes to these identity states through heterodimerization with cell-identity-specific bHLH transcription factors. Knockdown of TCF4 significantly induces apoptosis in vitro and inhibits tumorigenicity in vivo. We used genome-wide expression profiling, TCF4 chromatin immunoprecipitation sequencing (ChIP-seq) and TCF4 immunoprecipitation-mass spectrometry to determine the role of TCF4 in NB cells. Our results, along with recent findings in NB for the transcription factors T-box transcription factor TBX2, heart- and neural crest derivatives-expressed protein 2 (HAND2) and twist-related protein 1 (TWIST1), propose a role for TCF4 in regulating forkhead box protein M1 (FOXM1)/transcription factor E2F-driven gene regulatory networks that control cell cycle progression in cooperation with N-myc proto-oncogene protein (MYCN), TBX2, and the TCF4 dimerization partners HAND2 and TWIST1. Collectively, we showed that TCF4 promotes cell proliferation through direct transcriptional regulation of the c-MYC/MYCN oncogenic program that drives high-risk NB. Mechanistically, our data suggest the novel finding that TCF4 acts to support MYC activity by recruiting multiple factors known to regulate MYC function to sites of colocalization between critical NB transcription factors, TCF4 and MYC oncoproteins. Many of the TCF4-recruited factors are druggable, giving insight into potential therapies for high-risk NB. This study identifies a new function for class I bHLH transcription factors (e.g., TCF3, TCF4, and TCF12) that are important in cancer and development.

Abstract We139: 3D Chromatin Architectures and Transcription Regulation in Diabetic Endothelial Dysfunction

Cardiovascular disease is the major cause of mortality in people with type 2 diabetes, primarily due to endothelial dysfunction. Recent studies have suggested that transcriptional reprogramming plays a central role in endothelial dysfunction, but its regulation in the context of diabetes remains poorly understood. In this study, we utilized 3D chromatin mapping analysis (H3K27ac HiChIP) to investigate the transcriptional regulation in aortic endothelial cells (db/db mice) and HUVEC cells treated with high glucose (HUVEC high-glu ). We found that these cells were characterized by a massive loss of enhancer-promoter loops, which was associated with transcriptional downregulation. Through transcription factor motif scan, ChIP-seq and ATAC-seq analysis, we uncovered that the lost loop anchors are demarcated by reduced JUNB (transcription factor) binding while their chromatin accessibility remains unchanged. Moreover, we demonstrated that overexpression of JUNB significantly restored enhancer-promoter loops lost in HUVEC high-glu and ameliorated endothelial dysfunctions both in vitro (cell scratch, Transwell), ex vivo (aorta relaxation/contraction) and in vivo (hindlimb ischemia) assays. We further showed that JUNB protein (but not RNA) expression is reduced in aortic endothelial cells (db/db), HUVEC high-glu and primary endothelial cells from diabetic patients. Through RIME (Rapid immunoprecipitation mass spectrometry of endogenous protein) assay, we identified that RBBP6 (RB Binding Protein 6, Ubiquitin Ligase) is a JUNB interacting protein in HUVEC high-glu . We further showed that overexpression of RBBP6 downregulated JUNB expression while proteasome inhibitor MG132 treatment largely abolished the inhibitory effect of RBBP6 on JUNB protein levels in normal HUVEC cells, suggesting that RPPB6 promoted JUNB degradation through ubiquitination. Notably, knockdown of RBBP6 in HUVEC high-glu significantly upregulated JUNB expression, enhancer-promoter loopings and improved endothelial dysfunction. Overall, our findings provide novel 3D chromatin insights into diabetic endothelial dysfunction and illustrate a model whereby RBBP6-mediated JUNB degradation leads to global loss of enhancer-promoter looping and transcriptional inhibition in diabetic endothelial dysfunction.

Plasma pTau217 predicts continuous brain amyloid levels in preclinical and early Alzheimer's disease.

This study investigated the potential of phosphorylated plasma Tau217 ratio (pTau217R) and plasma amyloid beta (Aβ) 42/Aβ40 in predicting brain amyloid levels measured by positron emission tomography (PET) Centiloid (CL) for Alzheimer's disease (AD) staging and screening. Quantification of plasma pTau217R and Aβ42/Aβ40 employed immunoprecipitation-mass spectrometry. CL prediction models were developed on a cohort of 904 cognitively unimpaired, preclinicaland early AD subjects and validated on two independent cohorts. Models integrating pTau217R outperformed Aβ42/Aβ40 alone, predicting amyloid levels up to 89.1 CL. High area under the receiver operating characteristic curve (AUROC) values (89.3% to 94.7%) were observed across a broad CL range (15 to 90). Utilizing pTau217R-based models for low amyloid levels reduced PET scans by 70.5% to 78.6%. pTau217R effectively predicts brain amyloid levels, surpassing cerebrospinal fluid Aβ42/Aβ40's range. Combining it with plasma Aβ42/Aβ40 enhances sensitivity for low amyloid detection, reducing unnecessary PET scans and expanding clinical utility. NCT02956486 (MissionAD1), NCT03036280 (MissionAD2), NCT04468659 (AHEAD3-45), NCT03887455 (ClarityAD) HIGHLIGHTS: Phosphorylated plasma Tau217 ratio (pTau217R) effectively predicts amyloid-PET Centiloid (CL) across a broad spectrum. Integrating pTau217R with Aβ42/Aβ40 extends the CL prediction upper limit to 89.1 CL. Combined model predicts amyloid status with high accuracy, especially in cognitively unimpaired individuals. This modelidentifies subjects above or below various CL thresholds with high accuracy. pTau217R-based models significantly reduce PET scans by up to 78.6% for screening out individuals with no/low amyloid.

Plasma biomarkers of neurodegeneration in patients and high risk subjects with Lewy body disease

Comorbid Alzheimer’s disease (AD) neuropathology is common in Lewy body disease (LBD); however, AD comorbidity in the prodromal phase of LBD remains unclear. This study investigated AD comorbidity in the prodromal and symptomatic phases of LBD by analyzing plasma biomarkers in patients with Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) and individuals at risk of LBD (NaT-PROBE cohort). Patients with PD (PD group, n = 84) and DLB (DLB group, n = 16) and individuals with LBD with ≥ 2 (high-risk group, n = 82) and without (low-risk group, n = 37) prodromal symptoms were enrolled. Plasma amyloid-beta (Aβ) composite was measured using immunoprecipitation-mass spectrometry assays. Plasma phosphorylated tau 181 (p-tau181), neurofilament light chain (NfL), and alpha-synuclein (aSyn) were measured using a single-molecule array. Plasma p-tau181 levels were higher in the PD and DLB groups than in the low-risk group. Aβ composite level was higher in the DLB group than in the high-risk group. AD-related biomarker levels were not elevated in the high-risk group. NfL levels were higher in the high-risk, PD, and DLB groups than in the low-risk group. In the PD group, Aβ composite was associated with cognitive function, p-tau181 with motor function and non-motor symptoms, and NfL with cognitive and motor functions and non-motor symptoms. In the high-risk group, NfL was associated with metaiodobenzylguanidine scintigraphy abnormalities. The PD and DLB groups exhibited comorbid AD neuropathology, though not in the prodromal phase. Elevated plasma NfL levels, even without elevated AD-related plasma biomarker levels, may indicate aSyn-induced neurodegeneration in the LBD prodromal phase.

RNA Helicase DDX5 Maintains Cardiac Function by Regulating CamkIIδ Alternative Splicing.

Heart failure (HF) is a leading cause of morbidity and mortality worldwide. RNA-binding proteins are identified as regulators of cardiac disease; DDX5 (dead-box helicase 5) is a master regulator of many RNA processes, although its function in heart physiology remains unclear. We assessed DDX5 expression in human failing hearts and a mouse HF model. To study the function of DDX5 in heart, we engineered cardiomyocyte-specific Ddx5 knockout mice. We overexpressed DDX5 in cardiomyocytes using adeno-associated virus serotype 9 and performed transverse aortic constriction to establish the murine HF model. The mechanisms underlined were subsequently investigated using immunoprecipitation-mass spectrometry, RNA-sequencing, alternative splicing analysis, and RNA immunoprecipitation sequencing. We screened transcriptome databases of murine HF and human dilated cardiomyopathy samples and found that DDX5 was significantly downregulated in both. Cardiomyocyte-specific deletion of Ddx5 resulted in HF with reduced cardiac function, an enlarged heart chamber, and increased fibrosis in mice. DDX5 overexpression improved cardiac function and protected against adverse cardiac remodeling in mice with transverse aortic constriction-induced HF. Furthermore, proteomics revealed that DDX5 is involved in RNA splicing in cardiomyocytes. We found that DDX5 regulated the aberrant splicing of Ca2+/calmodulin-dependent protein kinase IIδ (CamkIIδ), thus preventing the production of CaMKIIδA, which phosphorylates L-type calcium channel by serine residues of Cacna1c, leading to impaired Ca2+ homeostasis. In line with this, we found increased intracellular Ca2+ transients and increased sarcoplasmic reticulum Ca2+ content in DDX5-depleted cardiomyocytes. Using adeno-associated virus serotype 9 knockdown of CaMKIIδA partially rescued the cardiac dysfunction and HF in Ddx5 knockout mice. These findings reveal a role for DDX5 in maintaining calcium homeostasis and cardiac function by regulating alternative splicing in cardiomyocytes, identifying the DDX5 as a potential target for therapeutic intervention in HF.

M6A-mediated lnc-OXAR promotes oxaliplatin resistance by enhancing Ku70 stability in non-alcoholic steatohepatitis-related hepatocellular carcinoma

BackgroundThe escalating prevalence of metabolic diseases has led to a rapid increase in non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (NASH-HCC). While oxaliplatin (OXA)-based hepatic arterial infusion chemotherapy (HAIC) has shown promise in advanced-stage HCC patients, its efficacy in NASH-HCC remains uncertain. This study aims to assess the effectiveness of OXA-based HAIC and elucidate the mechanisms underlying OXA resistance in NASH-HCC.MethodsThe key lncRNAs were screened through RNA-seq analysis of NASH/non-NASH and OXA-sensitive/OXA-resistant (OXA-S/R) HCC tissues. The biological functions of the lnc-OXAR (OXA resistance–related lncRNA in NASH-HCC) in NASH-HCC were verified through a series of in vitro and in vivo experiments. The molecular mechanism of lnc-OXAR was elucidated by fluorescence in situ hybridization, immunoprecipitation-mass spectrometry (FISH), Immunoprecipitation-Mass Spectrometry (IP-MS), RNA pulldown, RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and a dual-luciferase reporter assay.ResultsNASH-HCC exhibited reduced responsiveness to OXA-based HAIC compared to non-NASH HCC. We identified and validated a novel transcript namedlnc-OXAR, which played a crucial role in conferring OXA resistance to NASH-HCC. Inhibition of lnc-OXAR suppressed HCC cell growth and restored OXA sensitivity both in NASH-HCC mouse models and in vitro. Mechanistically, lnc-OXAR recruited Ku70 and cystatin A (CSTA), preventing Ku70 degradation and facilitating DNA double-strand break (DSB) repair, thereby promoting OXA resistance in NASH-HCC. Additionally, WTAP-mediated m6A modification enhanced the stability of lnc-OXAR in an IGF2BP2-dependent manner. Notably, silencing lnc-OXAR significantly enhanced the response to OXA in patient-derived xenograft (PDX) models derived from NASH-HCC.ConclusionsThe reduced responsiveness of NASH-HCC to OXA treatment can be attributed to the upregulation of lnc-OXAR. Our findings provide a rationale for stratifying HCC patients undergoing OXA-based HAIC based on etiology. Lnc-OXAR holds promise as a novel target for overcoming OXA resistance in NASH-HCC and improving prognosis.

Tau protein profiling in tauopathies: a human brain study

Abnormal accumulation of misfolded and hyperphosphorylated tau protein in brain is the defining feature of several neurodegenerative diseases called tauopathies, including Alzheimer’s disease (AD). In AD, this pathological change is reflected by highly specific cerebrospinal fluid (CSF) tau biomarkers, including both phosphorylated and non-phosphorylated variants. Interestingly, despite tau pathology being at the core of all tauopathies, CSF tau biomarkers remain unchanged in certain tauopathies, e.g., progressive supranuclear palsy (PSP), Pick’s disease (PiD), and corticobasal neurodegeneration (CBD). To better understand commonalities and differences between tauopathies, we report a multiplex assay combining immunoprecipitation and high-resolution mass spectrometry capable of detecting and quantifying peptides from different tau protein isoforms as well as non-phosphorylated and phosphorylated peptides, including those carrying multiple phosphorylations. We investigated the tau proteoforms in soluble and insoluble fractions of brain tissue from subjects with autopsy-confirmed tauopathies, including sporadic AD (n = 10), PSP (n = 11), PiD (n = 10), and CBD (n = 10), and controls (n = 10). Our results demonstrate that non-phosphorylated tau profiles differ across tauopathies, generally showing high abundance of microtubule-binding region (MTBR)-containing peptides in insoluble protein fractions compared with controls; the AD group showed 12–72 times higher levels of MTBR-containing aggregates. Quantification of tau isoforms showed the 3R being more abundant in PiD and the 4R isoform being more abundant in CBD and PSP in the insoluble fraction. Twenty-three different phosphorylated peptides were quantified. Most phosphorylated peptides were measurable in all investigated tauopathies. All phosphorylated peptides were significantly increased in AD insoluble fraction. However, doubly and triply phosphorylated peptides were significantly increased in AD even in the soluble fraction. Results were replicated using a validation cohort comprising AD (n = 10), CBD (n = 10), and controls (n = 10). Our study demonstrates that abnormal levels of phosphorylation and aggregation do indeed occur in non-AD tauopathies, however, both appear pronouncedly increased in AD, becoming a distinctive characteristic of AD pathology.

ACADM inhibits AMPK activation to modulate PEDV-induced lipophagy and β-oxidation for impairing viral replication

Porcine epidemic diarrhea virus (PEDV) belongs to the Alphacoronavirus genus within the Coronavirus family, causing severe watery diarrhea in piglets and resulting in significant economic losses. Medium-chain acyl-CoA dehydrogenase (ACADM) is an enzyme participating in lipid metabolism associated with metabolic diseases and pathogen infections. Nonetheless, the precise role of ACADM in regulating PEDV replication remains uncertain. In this study, we identified ACADM as the host binding partner of NSP4 via immunoprecipitation-mass spectrometry analysis. The interaction between ACADM and NSP4 was subsequently corroborated through coimmunoprecipitation and laser confocal microscopy. Following this, a notable upsurge in ACADM expression was observed during PEDV infection. ACADM overexpression effectively inhibited virus replication, whereas ACADM knockdown facilitated virus replication, suggesting ACADM has negative regulation effect on PEDV infection. Furthermore, we demonstrated fatty acid β-oxidation affected PEDV replication for the first time, inhibition of fatty acid β-oxidation reduced PEDV replication. ACADM decreased PEDV-induced β-oxidation to suppress PEDV replication. Mechanistically, ACADM reduced cellular free fatty acid levels and subsequent β-oxidation by hindering AMPK-mediated lipophagy. In summary, our results reveal that ACADM plays a negative regulatory role in PEDV replication by regulating lipid metabolism. The present study introduces a novel approach for the prevention and control of PEDV infection.

Single-point and kinetics of peripheral residual disease by mass spectrometry to predict outcome in patients with high risk smoldering multiple myeloma included in the GEM-CESAR trial.

The value of quantitative immunoprecipitation mass spectrometry (QIP-MS) to identify the M-protein is being investigated in patients with monoclonal gammopathies but no data are yet available in high-risk smoldering myeloma (HRsMM). We have therefore investigated QIP-MS to monitor peripheral residual disease (PRD) in 62 HRsMM patients enrolled in the GEM-CESAR trial. After 24 cycles of maintenance, detecting the M-protein by MS or clonal plasma cells by NGF identified cases with a significantly shorter median PFS (mPFS; MS: not reached vs 1,4 years, p=0.001; NGF: not reached vs 2 years, p=0.0002) but reaching CR+sCR did not discriminate patients with different outcome. With NGF as a reference, the combined results of NGF and MS showed a high negative predictive value (NPV) of MS: 81% overall and 73% at treatment completion. When sequential results were considered, sustained negativity by MS or NGF was associated with a very favorable outcome with a mPFS not yet reached vs 1.66 years and 2.18 years in cases never attaining PRD or minimal residual disease (MRD) negativity, respectively. We can thus conclude that 1) the standard response categories of the IMWG do not seem to be useful for treatment monitoring in HRsMM patients, 2) MS could be used as a non-invasive, clinical valuable tool with the capacity of guiding timely bone marrow evaluations (based on its high NPV with NGF as a reference) and 3) similarly to NGF, sequential results of MS are able identify a subgroup of HRsMM patients with long-term disease control. This study was registered at www.clinicaltrials.gov (ClinicalTrials.gov identifier: NCT02415413).

Unveiling epithelial plasticity regulation in lung cancer: Exploring the cross-talk among Tks4 scaffold protein partners.

The epithelial-to-mesenchymal transition (EMT) represents a hallmark event in the evolution of lung cancer. This work aims to study a recently described EMT-regulating protein, Tks4, and to explore its potential as a prognostic biomarker in non-small cell lung cancer. In this study, we used CRISPR/Cas9 method to knockout (KO) Tks4 to study its functional roles in invadopodia formation, migration, and regulation of EMT marker expressions and we identified Tks4-interacting proteins. Tks4-KO A549 cells exhibited an EMT-like phenotype characterized by elongated morphology and increased expression of EMT markers. Furthermore, analyses of a large-scale lung cancer database and a patient-derived tissue array data revealed that the Tks4 mRNA level was decreased in more aggressive lung cancer stages. To understand the regulatory role of Tks4 in lung cancer, we performed a Tks4-interactome analysis via Tks4 immunoprecipitation-mass spectrometry on five different cell lines and identified CAPZA1 as a novel Tks4 partner protein. Thus, we propose that the absence of Tks4 leads to disruption of a connectome of multiple proteins and that the resulting undocking and likely mislocalization of signaling molecules impairs actin cytoskeleton rearrangement and activates EMT-like cell fate switches, both of which likely influence disease severity.

A multicenter, randomized, double-blind, placebo-controlled ascending dose study to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamic (PD) effects of Posiphen in subjects with early Alzheimer’s Disease

BackgroundAmyloid beta protein (Aβ) is a treatment target in Alzheimer’s Disease (AD). Lowering production of its parent protein, APP, has benefits in preclinical models. Posiphen, an orally administered small molecule, binds to an iron-responsive element in APP mRNA and decreases translation of APP and Aβ. To augment human data for Posiphen, we evaluated safety, tolerability and pharmacokinetic and pharmacodynamic (PD) effects on Aβ metabolism using Stable Isotope Labeling Kinetic (SILK) analysis.MethodsDouble-blind phase 1b randomized ascending dose clinical trial, at five sites, under an IRB-approved protocol. Participants with mild cognitive impairment or mild AD (Early AD) confirmed by low CSF Aβ42/40 were randomized (within each dose arm) to Posiphen or placebo. Pretreatment assessment included lumbar puncture for CSF. Participants took Posiphen or placebo for 21–23 days, then underwent CSF catheter placement, intravenous infusion of 13C6-leucine, and CSF sampling for 36 h. Safety and tolerability were assessed through participant reports, EKG and laboratory tests. CSF SILK analysis measured Aβ40, 38 and 42 with immunoprecipitation-mass spectrometry. Baseline and day 21 CSF APP, Aβ and other biomarkers were measured with immunoassays. The Mini-Mental State Exam and ADAS-cog12 were given at baseline and day 21.ResultsFrom June 2017 to December 2021, 19 participants were enrolled, randomized within dose cohorts (5 active: 3 placebo) of 60 mg once/day and 60 mg twice/day; 1 participant was enrolled and completed 60 mg three times/day. 10 active drug and 5 placebo participants completed all study procedures. Posiphen was safe and well-tolerated. 8 participants had headaches related to CSF catheterization; 5 needed blood patches. Prespecified SILK analyses of Fractional Synthesis Rate (FSR) for CSF Aβ40 showed no significant overall or dose-dependent effects of Posiphen vs. placebo. Comprehensive multiparameter modeling of APP kinetics supported dose-dependent lowering of APP production by Posiphen. Cognitive measures and CSF biomarkers did not change significantly from baseline to 21 days in Posiphen vs. placebo groups.ConclusionsPosiphen was safe and well-tolerated in Early AD. A multicenter SILK study was feasible. Findings are limited by small sample size but provide additional supportive safety and PK data. Comprehensive modeling of biomarker dynamics using SILK data may reveal subtle drug effects.Trial registrationNCT02925650 on clinicaltrials.gov (registered on 10-24-2016).

Advances in Blood Biomarkers for Alzheimer's Disease: Ultra-Sensitive Detection Technologies and Impact on Clinical Diagnosis.

Alzheimer's disease has escalated into a critical public health concern, marked by its neurodegenerative nature that progressively diminishes cognitive abilities. Recognized as a continuously advancing and presently incurable condition, AD underscores the necessity for early-stage diagnosis and interventions aimed at delaying the decline in mental function. Despite the proven efficacy of cerebrospinal fluid and positron emission tomography in diagnosing AD, their broader utility is constrained by significant costs and the invasive nature of these procedures. Consequently, the innovation of blood biomarkers such as Amyloid-beta, phosphorylated-tau, total-tau et al, distinguished by their high sensitivity, minimal invasiveness, accessibility, and cost-efficiency, emerges as a promising avenue for AD diagnosis. The advent of ultra-sensitive detection methodologies, including single-molecule enzyme-linked immunosorbent assay and immunoprecipitation-mass spectrometry, has revolutionized the detection of AD plasma biomarkers, supplanting previous low-sensitivity techniques. This rapid advancement in detection technology facilitates the more accurate quantification of pathological brain proteins and AD-associated biomarkers in the bloodstream. This manuscript meticulously reviews the landscape of current research on immunological markers for AD, anchored in the National Institute on Aging-Alzheimer's Association AT(N) research framework. It highlights a selection of forefront ultra-sensitive detection technologies now integral to assessing AD blood immunological markers. Additionally, this review examines the crucial pre-analytical processing steps for AD blood samples that significantly impact research outcomes and addresses the practical challenges faced during clinical testing. These discussions are crucial for enhancing our comprehension and refining the diagnostic precision of AD using blood-based biomarkers. The review aims to shed light on potential avenues for innovation and improvement in the techniques employed for detecting and investigating AD, thereby contributing to the broader field of neurodegenerative disease research.

Shikonin blocks CAF-induced TNBC metastasis by suppressing mitochondrial biogenesis through GSK-3β/NEDD4-1 mediated phosphorylation-dependent degradation of PGC-1α

BackgroundTriple-negative breast cancer (TNBC) is characterized by its high metastatic potential, which results in poor patient survival. Cancer-associated fibroblasts (CAFs) are crucial in facilitating TNBC metastasis via induction of mitochondrial biogenesis. However, how to inhibit CAF-conferred mitochondrial biogenesis is still needed to explore.MethodsWe investigated metastasis using wound healing and cell invasion assays, 3D-culture, anoikis detection, and NOD/SCID mice. Mitochondrial biogenesis was detected by MitoTracker green FM staining, quantification of mitochondrial DNA levels, and blue-native polyacrylamide gel electrophoresis. The expression, transcription, and phosphorylation of peroxisome-proliferator activated receptor coactivator 1α (PGC-1α) were detected by western blotting, chromatin immunoprecipitation, dual-luciferase reporter assay, quantitative polymerase chain reaction, immunoprecipitation, and liquid chromatography-tandem mass spectrometry. The prognostic role of PGC-1α in TNBC was evaluated using the Kaplan–Meier plotter database and clinical breast cancer tissue samples.ResultsWe demonstrated that PGC-1α indicated lymph node metastasis, tumor thrombus formation, and poor survival in TNBC patients, and it was induced by CAFs, which functioned as an inducer of mitochondrial biogenesis and metastasis in TNBC. Shikonin impeded the CAF-induced PGC-1α expression, nuclear localization, and interaction with estrogen-related receptor alpha (ERRα), thereby inhibiting PGC-1α/ERRα-targeted mitochondrial genes. Mechanistically, the downregulation of PGC-1α was mediated by synthase kinase 3β-induced phosphorylation of PGC-1α at Thr295, which associated with neural precursor cell expressed developmentally downregulated 4e1 recognition and subsequent degradation by ubiquitin proteolysis. Mutation of PGC-1α at Thr295 negated the suppressive effects of shikonin on CAF-stimulated TNBC mitochondrial biogenesis and metastasis in vitro and in vivo.ConclusionsOur findings indicate that PGC-1α is a viable target for blocking TNBC metastasis by disrupting mitochondrial biogenesis, and that shikonin merits potential for treatment of TNBC metastasis as an inhibitor of mitochondrial biogenesis through targeting PGC-1α.

UBQLN1 links proteostasis and mitochondria function to telomere maintenance in human embryonic stem cells

BackgroundTelomeres consist of repetitive DNA sequences at the chromosome ends to protect chromosomal stability, and primarily maintained by telomerase or occasionally by alternative telomere lengthening of telomeres (ALT) through recombination-based mechanisms. Additional mechanisms that may regulate telomere maintenance remain to be explored. Simultaneous measurement of telomere length and transcriptome in the same human embryonic stem cell (hESC) revealed that mRNA expression levels of UBQLN1 exhibit linear relationship with telomere length.MethodsIn this study, we first generated UBQLN1-deficient hESCs and compared with the wild-type (WT) hESCs the telomere length and molecular change at RNA and protein level by RNA-seq and proteomics. Then we identified the potential interacting proteins with UBQLN1 using immunoprecipitation-mass spectrometry (IP-MS). Furthermore, the potential mechanisms underlying the shortened telomeres in UBQLN1-deficient hESCs were analyzed.ResultsWe show that Ubiquilin1 (UBQLN1) is critical for telomere maintenance in human embryonic stem cells (hESCs) via promoting mitochondrial function. UBQLN1 deficiency leads to oxidative stress, loss of proteostasis, mitochondria dysfunction, DNA damage, and telomere attrition. Reducing oxidative damage and promoting mitochondria function by culture under hypoxia condition or supplementation with N-acetylcysteine partly attenuate the telomere attrition induced by UBQLN1 deficiency. Moreover, UBQLN1 deficiency/telomere shortening downregulates genes for neuro-ectoderm lineage differentiation.ConclusionsAltogether, UBQLN1 functions to scavenge ubiquitinated proteins, preventing their overloading mitochondria and elevated mitophagy. UBQLN1 maintains mitochondria and telomeres by regulating proteostasis and plays critical role in neuro-ectoderm differentiation.

Serum mass spectrometry for treatment monitoring in patients with multiple myeloma receiving ARI0002h CAR T-cells.

Chimeric antigen receptor (CAR) T-cell therapies have increased the patients with relapsed/refractory multiple myeloma (RRMM) in whom standard electrophoretic techniques fail to detect the M-protein. Quantitative immunoprecipitation mass spectrometry (QIP-MS) can accurately measure serum M-protein with high sensitivity, and identify interferences caused by therapeutic monoclonal antibodies. Here, we investigate the outcome of QIP-MS in 33 patients treated with the academic BCMA-directed CAR T-cell ARI0002h (Cesnicabtagene Autoleucel). QIP-MS offered more detailed insights than serum immunofixation (sIFE), identifying glycosylated M-proteins and minor additional peaks. Moreover, the potential interferences owing to daratumumab or tocilizumab treatments were successfully detected. When analysing different assay platforms during patient's monitoring after ARI0002h administration, we observed that QIP-MS showed a high global concordance (78.8%) with sIFE, whereas it was only moderate (55.6%) with bone marrow (BM)-based next-generation flow cytometry (NGF). Furthermore, QIP-MS consistently demonstrated the lowest negativity rate across the different timepoints (27.3% vs. 60.0% in months 1 and 12, respectively). Patients with QIP-MS(+)/BM-based NGF(-) showed a non-significant shorter median progression free survival than those with QIP-MS(-)/BM-based NGF(-). In summary, we show the first experience to our knowledge demonstrating that QIP-MS could be particularly useful as a non-invasive technique when evaluating response after CAR T-cell treatment in MM.

NCF4 attenuates colorectal cancer progression by modulating inflammasome activation and immune surveillance

The spatiotemporal regulation of inflammasome activation remains unclear. To examine the mechanism underlying the assembly and regulation of the inflammasome response, here we perform an immunoprecipitation-mass spectrometry analysis of apoptosis-associated speck-like protein containing a CARD (ASC) and identify NCF4/1/2 as ASC-binding proteins. Reduced NCF4 expression is associated with colorectal cancer development and decreased five-year survival rate in patients with colorectal cancer. NCF4 cooperates with NCF1 and NCF2 to promote NLRP3 and AIM2 inflammasome activation. Mechanistically, NCF4 phosphorylation and puncta distribution switches from the NADPH complex to the perinuclear region, mediating ASC oligomerization, speck formation and inflammasome activation. NCF4 functions as a sensor of ROS levels, to establish a balance between ROS production and inflammasome activation. NCF4 deficiency causes severe colorectal cancer in mice, increases transit-amplifying and precancerous cells, reduces the frequency and activation of CD8+ T and NK cells, and impairs the inflammasome-IL-18-IFN-γ axis during the early phase of colorectal tumorigenesis. Our study implicates NCF4 in determining the spatial positioning of inflammasome assembly and contributing to inflammasome-mediated anti-tumor responses.