Protocol for plasma membrane enrichment by ultracentrifugation for mass spectrometry of cell lines, xenografts, and patient tumors.

The success of immunotherapies hinges on identifying targetable cell surface proteins expressed in the cancer of interest. Here, we present a protocol for enriching plasma membrane proteins for mass spectrometry analysis using a density gradient ultracentrifugation approach. We describe steps for cell lysis, membrane isolation, and preparation for downstream analysis.

Mapping a interactome by crosslinking mass spectrometry and machine learning.

, a widespread human parasite, persists in hosts through complex molecular interactions. Protein-protein interactions (PPIs) underpin essential biological processes, including parasite-host interactions and cellular invasion. Herein, we utilized advanced crosslinking mass spectrometry (XL-MS) techniques to map a tachyzoite cytosolic extract interactome.

ACR-16/α7-nicotinic receptor signaling mediates nicotine-induced mitochondrial dysfunction and suppression of host defense against gram-negative bacteria.

Smoking, alongside e-cigarette and heated tobacco use, impairs immune function and increases susceptibility to infections, primarily due to nicotine's disruption of cellular processes. While mitochondria are traditionally associated with bioenergetics, they also serve as critical regulators of immunity, with mitochondrial dysfunction linked to compromised host defenses. To investigate nicotine's effects on mitochondrial function and immunity, we utilized Caenorhabditis elegans infected with Pseudomonas aeruginosa or Staphylococcus aureus.

Fabry disease cardiomyopathy: A state-of-the-art review.

Fabry disease or Anderson-Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of α-galactosidase A (GLA), leading to systemic accumulation of globotriaosyl-ceramide (Gb3). Initially described in 1898 as a dermatological condition, Fabry disease is now recognized as a progressive multisystem disorder with significant cardiac involvement. Cardiomyopathy in Fabry disease arises from Gb3 accumulation in cardiac tissue, resulting in structural changes such as fibrosis and left ventricular hypertrophy (LVH), and functional impairments including diastolic dysfunction and heart failure.

Exploiting an Epigenetic Resistance Mechanism to PI3 Kinase Inhibition in Leukemic Stem Cells.

Acquired non-genetic resistance mechanisms to existing therapies contribute to poor outcomes for acute myeloid leukemia (AML) patients, and inability to target leukemic stem cells (LSCs) can lead to relapse. To overcome these challenges, we tested whether LSCs have dependencies on PI3 kinase (PI3K). We found that LSCs are susceptible to isoform-selective targeting of PI3K and are particularly dependent on the P110 alpha isoform of PI3K.

Altered Metabolism in Heterozygous Mice With a Mutation in the Motor Domain of Cytoplasmic Dynein, DYNC1H1 (AAA4; c9052C>T(P3018S)).

DYNC1H1 encodes the cytoplasmic dynein heavy chain, a key motor protein involved in intracellular transport and neural development. While mutations in DYNC1H1 are known to cause a range of neurodevelopmental and motor disorders, the molecular mechanisms linking genotype to phenotype remain poorly defined. Here, we investigated how a patient-derived missense mutation in the motor domain of DYNC1H1 (c.

The SWI/SNF-related protein SMARCA3 is a histone H3K23 ubiquitin ligase that regulates H3K9me3 in cancer.

Histone ubiquitination is a crucial post-translational modification (PTM) regulating chromatin function, yet many histone ubiquitination sites and the enzymes that control them remain poorly understood. Here, we identify SMARCA3, a SWI/SNF-related protein frequently downregulated in colorectal cancer (CRC), as an E3 ubiquitin ligase that targets histone H3 at lysine 23 (H3K23). We demonstrate that SMARCA3 histone ubiquitination activity is stimulated by the repressive H3K9me3 mark.

PRMT5 activity sustains histone production to maintain genome integrity.

Histone proteins package DNA into nucleosomes, forming chromatin and thereby safeguarding genome integrity. Proper histone expression is essential for cell proliferation and chromatin organization, yet the upstream regulators of histone supply remain incompletely understood. PRMT5-a cell essential type II protein arginine methyltransferase frequently overexpressed in cancer-catalyzes symmetric dimethylation of arginine residues.

Collagen hydroxylation couples NAD+/NADH dynamics to tumor dormancy and reactivation.

Metastasis remains the leading cause of cancer-related mortality. Disseminated tumor cells (DTCs) colonize distant organs where they enter a prolonged state of quiescence, named cellular dormancy, within collagen-rich extracellular matrix (ECM) niches. How dormant cells regulate the formation of collagen-rich niches and the mechanisms maintaining collagen proteostasis during dormancy and reactivation are not understood.

Identifying Space-Resolved Proteins of the Murine Thymus, by Combining MALDI Mass Spectrometry Imaging and Proteomics.

The identification of spatially resolved proteomes has recently seen significant breakthroughs, yet challenges persist, particularly in integrating protein identification with precise spatial localization within a single experimental workflow. Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) enables spatial protein mapping on tissue sections without the need for antibodies. While MALDI-MSI addresses several obstacles in proteomic mapping with spatial resolution, it remains limited in its capacity for definitive protein identification.

The H3 oncohistone inhibits NSD2 to activate a SETD2-dependent antiviral-like immune response in KRAS-driven lung cancer.

Mutations in histone 3 at or near lysine 36 (H3K36) have dominantly acting oncogenic effects in multiple tumor types by limiting H3K36-directed methyltransferases. Paradoxically, we find that expression of the oncohistone unexpectedly inhibits tumor formation in KRAS-driven lung adenocarcinoma by inducing a potent immune-mediated tumor clearance. Mechanistically, oncohistone expression derepresses endogenous retroviral element transcription, results in the accumulation of double-stranded RNA (dsRNA), and activates an innate antiviral-like immune response that eradicates tumor growth.

Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer.

Chaperone-mediated autophagy (CMA), a type of selective degradation of cytosolic proteins in lysosomes, is commonly upregulated in cancer cells, contributing to their survival and growth. The lack of a specific target for CMA inhibition has limited CMA blockage to genetic manipulations or global lysosomal function inhibition. Here, using genetic modulation, transcriptional analysis, and functional studies, we demonstrate a regulatory role for the interaction of the retinoic acid receptor alpha (RARα) and its corepressor, the nuclear receptor corepressor 1 (NCoR1), on CMA in non-small cell lung cancer (NSCLC).

Multi-Omics Identification of Extracellular Components of the Fetal Monkey and Human Neocortex.

During development, precursor cells are continuously and intimately interacting with their extracellular environment, which guides their ability to generate functional tissues and organs. Much is known about the development of the neocortex in mammals. This information has largely been derived from histological analyses, heterochronic cell transplants, and genetic manipulations in mice, and to a lesser extent from transcriptomic and histological analyses in humans.

3D Biomimetic Liver Cancer Model: Diethylnitrosamine-Induced Proteomic Dysregulations in Stromal-Epithelial Milieu.

Hydrogel-based three-dimensional (3D) co-culture systems are emerging as biomimetic platforms that more accurately recapitulate tissue architecture and microenvironmental interactions compared to conventional two-dimensional (2D) cultures. This study introduces an engineered 3D liver-like model to investigate compartment-specific responses to the potent hepatocarcinogen Diethylnitrosamine (DEN), with a focus on early events in carcinogenesis and tumor-stroma interactions. AML12 and 3T3 cell lines were treated with DEN or vehicle either in 2D culture or in 3D hydrogels in four experimental groups: (1) DEN-treated AML12 with vehicle-treated 3T3, (2) DEN-treated 3T3 with vehicle-treated AML12, (3) both cell types DEN-treated, and (4) both vehicle-treated.

Widespread discordance between mRNA expression, protein abundance and lipogenesis activity in hepatocytes during the fed-starvation transition.

The mammalian liver plays a critical role in maintaining metabolic homeostasis during fasting and feeding. Liver function is further shaped by sex dimorphism and zonation of hepatocytes. To explore how these factors interact, we performed deep RNA-sequencing and label-free proteomics on periportal and pericentral hepatocytes isolated from male and female mice under fed and starved conditions.

Loss of CARM1 alters the developmental programming of Glioma stem-like cells and creates a druggable NGFR/NTRK dependency.

A key driver of Glioblastoma (GBM) heterogeneity and therapy resistance is the capacity of glioma stem-like cells (GSCs) to hijack developmental signaling programs. However, it remains unclear how GSCs regulate these adapted developmental signaling pathways and how these pathways might be therapeutically exploited. The arginine methyltransferase, CARM1, has been shown to play critical roles in maintaining stem cell pluripotency, preventing differentiation, and recently was discovered to be upregulated in Glioblastoma.

Spatial hepatocyte plasticity of gluconeogenesis during the metabolic transitions between fed, fasted and starvation states.

Hepatocytes are organized along a spatial axis between the portal triad and the central vein to form functionally repetitive units known as lobules. The hepatocytes perform distinct metabolic functions depending on their location within the lobule. Single-cell analysis of hepatocytes across the liver lobule demonstrates that gluconeogenic gene expression is relatively low in the fed state and gradually increases in the periportal hepatocytes during the initial fasting period.

Identification of Chromatin Readers Using a Peptide Pull-Down and Mass Spectrometry Integrated Approach.

Histone proteins regulate essential cellular processes by being decorated with a myriad of posttranslational modifications (PTMs). These processes are mostly led by the ability of these PTMs to recruit protein readers involved in gene transcription, DNA replication, DNA damage, chromatin remodeling, and other functions. Identifying histone readers is critical for the understanding of mechanisms leading to these functions and potentially predict targets for treatment in anomalous phenotypes.

Automated Sample Preparation for the Unbiased Analysis of Histone Posttranslational Modifications Via Mass Spectrometry.

Histone proteins are the structural components of nucleosomes, which form chromatin. Histone proteins are typically modified with many posttranslational modifications (PTMs), which affect chromatin accessibility, and by extension, modulate gene transcription, and other DNA-related processes. Mass spectrometry has become the reference technology to quantify global levels of hundreds of histone PTMs in single experiments.

Myocardial Proteome in Human Heart Failure With Preserved Ejection Fraction.

Background: Heart failure with preserved ejection fraction (HFpEF) constitutes more than half of all HF but has few effective therapies. Recent human myocardial transcriptomics and metabolomics have identified major differences between HFpEF and controls. How this translates at the protein level is unknown.

Extracellular Histones as Exosome Membrane Proteins Regulated by Cell Stress.

Histones are conserved nuclear proteins that function as part of the nucleosome in the regulation of chromatin structure and gene expression. Interestingly, extracellular histones populate biofluids from healthy individuals, and when elevated, may contribute to various acute and chronic diseases. It is generally assumed that most extracellular histones exist as nucleosomes, as components of extracellular chromatin.

Hydrogen/Deuterium Exchange and Protein Oxidative Footprinting with Mass Spectrometry Collectively Discriminate the Binding of Small-Molecule Therapeutics to Bcl-2.

Characterizing protein-ligand interactions is crucial to understanding cellular metabolism and guiding drug discovery and development. Herein, we explore complementing hydrogen/deuterium exchange mass spectrometry (HDX-MS) with a recently developed Fenton chemistry-based approach to protein oxidative footprinting mass spectrometry (OX-MS) to discriminate the binding of small-molecule therapeutics. Using drug-dependent perturbation as the experimental report, this combination of techniques more clearly differentiates the in-solution binding profiles of Venetoclax (ABT-199, GDC-0199-AbbVie and Genentech) and a drug candidate S55746 (Servier) to the apoptotic regulatory protein Bcl-2 than either technique alone.

The Power of Three: Dynactin associates with three dyneins under load for greater force production.

Cytoplasmic dynein is an essential microtubule motor protein that powers organelle transport and mitotic spindle assembly. Its activity depends on dynein-dynactin-cargo adaptor complexes, such as dynein-dynactin-BicD2 (DDB), which typically function with two dynein motors. We show that mechanical tension recruits a third dynein motor via an auxiliary BicD adaptor binding the light intermediate chain of the third dynein, stabilizing multi-dynein assemblies and enhancing force generation.

Analogue-Sensitive Inhibition of Histone Demethylases Uncovers Member-Specific Function in Ribosomal Protein Synthesis.

Lysine demethylases (KDMs) catalyze the oxidative removal of the methyl group from histones using earth-abundant iron and the metabolite 2-oxoglutarate (2OG). KDMs have emerged as master regulators of eukaryotic gene expression and are novel drug targets; small-molecule inhibitors of KDMs are in the clinical pipeline for the treatment of human cancer. Yet, mechanistic insights into the functional heterogeneity of human KDMs are limited, necessitating the development of chemical probes for precision targeting.