Biology subjects

Schriemer, D. C.

Publications and source records attributed to Schriemer, D. C..

9 recordsLinked to original sources

bioRxiv2026

Mapping the reactive cysteine landscape of the NOD-like receptor pyrin domain-containing 3 protein

NOD-like receptor pyring-containing protein 3 (NLRP3) is a cytosolic pattern recognition receptor that drives assembly of an oligomeric inflammasome complex to stimulate pro-inflammatory cytokine production and pyroptotic cell death. Extensive research has shown that NLRP3 activation is dependent on intracellular reactive oxygen species (ROS). Despite this, the precise targets of ROS in this process remain undefined. In this study, a combination of chemoproteomics, in vitro biochemical assays and molecular dynamics simulations were used to characterize reactive Cys and how they may impact NLRP3 conformation and activity status. Affinity-captured NLRP3 was subjected to reverse-thiol trapping to reveal a pool of reactive Cys residues that were identified in subsequent chemoproteomic analyses. C150, C196, and C884 were consistently detected with both in vitro and in cellulo experiments. Several NLRP3 mutants were generated; C150A and C196A (NACHT domain) as well as C933A and C941A (LRR domain) reduced IL-1b output in a reconstituted inflammasome assay. Molecular simulation of Cys sulfenylation in the LRR suggests structural alterations that reposition the trLRR loop to destabilize the inactive NLRP3 structure. Structural modelling provided insights for how Cys oxidation of the LRR may impact on NLRP3 structure. Collectively, the results establish a mechanistic understanding for how ROS may promote NLRP3 inflammasome activation.

biochemistry↗

bioRxiv2026

Microbiome-derived hydroxyphenyl propanoates enhance antitumour immunity by potentiating gasdermin D activity in tumour-associated myeloid cells

The microbiome plays a critical role in immune health and response to immune-stimulating treatments, including cancer immunotherapy. However, the molecular mechanisms by which commensal microbes influence cancer immunology remain poorly understood. Here, we report a class of microbiome-derived metabolites called hydroxyphenyl propanoates (HPP) that enhance tumour immune surveillance and response to immune checkpoint blockade (ICB) therapy in mice. HPPs function as potentiators of innate immune signalling in tumour-associated myeloid cells by promoting cleavage of the pore-forming protein gasdermin D (GSDMD), a critical effector of inflammasome function. This enhances the secretion of proinflammatory cytokines, such as IL-1 family members, while simultaneously protecting against pyroptosis. Secreted IL-1 family cytokines in turn elicit autocrine and paracrine inflammatory signaling in tumour-infiltrating leukocytes. HPP supplementation increases anticancer T cell function, improves disease control and extends overall survival (OS) in tumour-bearing mice harboring complex microbiomes or those treated with broad-spectrum antibiotics. In humans, GSDMD activation is associated with a favourable response to PD-1 blockade therapy in patients with advanced stage melanoma. Our study uncovers a molecular mechanism of regulation over host antitumour immunity that is modifiable and can be harnessed for improved cancer immunotherapy in a broad spectrum of patients.

cancer biology↗

bioRxiv2025

Signal Attrition in Whole Cell Crosslinking Mass Spectrometry

Crosslinking mass spectrometry (XL-MS) could replace traditional techniques for sampling the cellular interactome, such as affinity pulldown MS. It generates superior interaction data that can be used to better model cellular structure and function. However, the sampling depth of current XL-MS techniques is disappointing. Poor sampling is often blamed on a low-yielding crosslinking reaction, estimated to be [~]0.1% based on relative ion abundance in the mass spectrometer. Here, using a new two-step crosslinker installation process, we demonstrate that the yield of the chemical reaction is not the limiting factor in sampling the interactome. Low crosslink detection levels persist even when crosslink yields approach 30% of total peptide. We show that crosslinked peptides are not preferentially lost during sample workup; they enter the mass spectrometer at least as efficiently as linear peptides. Low detection rates arise mostly from severe signal splitting that reduces the S/N of crosslinked peptides, which is likely exacerbated by low-sensitivity database search tools and compounded by ion suppression.

biochemistry↗

bioRxiv2025

Recommendations for Automating Hydrogen/Deuterium Exchange Mass Spectrometry Measurements using Data-Independent Acquisition Methods

Hydrogen/deuterium exchange mass spectrometry (HX-MS) is a method used to study solution-phase protein structure and dynamics. Despite its many applications, HX-MS is limited in throughput because manual data analysis is still the norm. We previously developed HX-MS2 technology to add a second dimension of deuteration data and promote automated data processing. Data-independent acquisition (DIA) techniques enable this approach, but we require optimized methods for best performance. Using an Orbitrap Eclipse for illustration, we show that ion optics and collision energy settings typical of a proteomics DIA experiment generate maximal peptide retrieval from the DIA library. As few as three MS2 sequence ions are sufficient to generate a deuteration measurement with a precision that exceeds what is possible in traditional HX-MS. DIA window sizes are based on the chromatographic resolution of the method. An inter-scan window offset method is the recommended default configuration for most HX-DIA applications, but an intra-scan overlap method can be tuned for highest performance and is recommended when maximum peptide retrieval is desired. A fully automated HX-MS2 solution consists of Trajan HDX automation technology, an Orbitrap Eclipse mass spectrometer and AutoHX software. We demonstrate its robustness on an extensive time-course analysis of phosphorylase B and an epitope analysis of single domain antibodies (VHHs, nanobodies) specific to the receptor binding domain of SARS-CoV2 spike protein.

biochemistry↗

bioRxiv2024

Click-linking: a cell-compatible protein crosslinking method based on click chemistry

Crosslinking mass spectrometry (XL-MS) has the potential to map the human interactome at high resolution and with high fidelity, replacing indirect, error prone sampling methods such as affinity pulldown MS. However, the sampling depth of XL-MS remains stubbornly low. We present a novel crosslinking strategy that splits the crosslinking reaction into two sequential and orthogonal coupling events. The method involves pre-stabilizing the spatial proteome with a fixation protocol inspired by immunofluorescence imaging, followed by a stepwise process that begins with extensively labeling surface-accessible lysines in the cell with N-hydroxysuccinimide (NHS)-modified click reagents. We show that a subsequent copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction of the installed precursors generates crosslinks at levels approaching 30% of the total signal, as demonstrated by a subtractive approach. The method generates no detectable side reactions or obvious distortions of the spatial proteome. Protein-protein interactions (PPIs) are detected at levels approximately 20 times higher than a conventional DSS-based method, outperforming even enrichable crosslinkers.

biochemistry↗

bioRxiv2024

Cell fixation improves performance of in situ crosslinking mass spectrometry while preserving cellular ultrastructure

Crosslinking mass spectrometry (XL-MS) has the potential to map the interactome of the cell with high resolution and depth of coverage. However, current in vivo XL-MS methods are hampered by crosslinkers that demonstrate low cell permeability and require long reaction times. Consequently, interactome sampling is not high and long incubation times can distort the cell, bringing into question the validity any protein interactions identified by the method. We address these issues with a fast formaldehyde-based fixation method applied prior to the introduction of secondary crosslinkers. Using human A549 cells and a range of reagents, we show that 4% formaldehyde fixation with membrane permeabilization preserves cellular ultrastructure and simultaneously improves reaction conditions for in situ XL-MS. Protein labeling yields can be increased even for nominally membrane-permeable reagents, and surprisingly, formaldehyde does not compete with conventional amine-reactive crosslinking reagents. Prefixation with permeabilization uncouples cellular dynamics from crosslinker dynamics, enhancing control over crosslinking yield and permitting the use of any chemical crosslinker.

biochemistry↗

bioRxiv2023

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology

We developed a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS2) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-validation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS1-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-validation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.

biochemistry↗

bioRxiv2023

mosaicMPI: a framework for modular data integration across cohorts and -omics modalities

Advances in molecular profiling have facilitated generation of large multi-modal datasets that can potentially reveal critical axes of biological variation underlying complex diseases. Distilling biological meaning, however, requires computational strategies that can perform mosaic integration across diverse cohorts and datatypes. Here, we present mosaicMPI, a framework for discovery of low to high-resolution molecular programs representing both cell types and states, and integration within and across datasets into a network representing biological themes. Using existing datasets in glioblastoma, we demonstrate that this approach robustly integrates single cell and bulk programs across multiple platforms. Clinical and molecular annotations from cohorts are statistically propagated onto this network of programs, yielding a richly characterized landscape of biological themes. This enables deep understanding of individual tumor samples, systematic exploration of relationships between modalities, and generation of a reference map onto which new datasets can rapidly be mapped. mosaicMPI is available at https://github.com/MorrissyLab/mosaicMPI.

bioinformatics↗

bioRxiv2021

Doublecortin engages the microtubule lattice through a cooperative binding mode involving its C- terminal domain

Doublecortin (DCX) is a microtubule (MT) associated protein that regulates MT structure and function during neuronal development and mutations in DCX lead to a spectrum of neurological disorders. The structural properties of MT-bound DCX remain poorly resolved. Here, we describe the molecular architecture of the DCX-MT complex through an integrative modeling approach that combines data from X-ray crystallography, cryo-EM and a high-fidelity chemical crosslinking method. We demonstrate that DCX interacts with MTs through its N-terminal domain and induces a lattice-dependent self-association involving both the C-terminal structured domain and the C-tails, in a conformation that favors an open, domain-swapped state. The networked state can accommodate multiple different attachment points on the MT lattice, all of which orient the C-tails away from the lattice. As numerous disease mutations cluster in the C-terminus, and regulatory phosphorylations cluster in the C-tail, our study shows that lattice-driven self-assembly is an important property of DCX.

biochemistry↗