Biology subjects

Sweredoski, M. J.

Publications and source records attributed to Sweredoski, M. J..

7 recordsLinked to original sources

bioRxiv2023

Functional glycoproteomics by integrated network assembly and partitioning

The post-translational modification (PTM) of proteins by O-linked {beta}-N-acetyl-D-glucosamine (O-GlcNAcylation) is widespread across the proteome during the lifespan of all multicellular organisms. However, nearly all functional studies have focused on individual protein modifications, overlooking the multitude of simultaneous O-GlcNAcylation events that work together to coordinate cellular activities. Here, we describe Networking of Interactors and SubstratEs (NISE), a novel, systems-level approach to rapidly and comprehensively monitor O-GlcNAcylation across the proteome. Our method integrates affinity purification-mass spectrometry (AP-MS) and site-specific chemoproteomic technologies with network generation and unsupervised partitioning to connect potential upstream regulators with downstream targets of O-GlcNAcylation. The resulting network provides a data-rich framework that reveals both conserved activities of O-GlcNAcylation such as epigenetic regulation as well as tissue-specific functions like synaptic morphology. Beyond O-GlcNAc, this holistic and unbiased systems-level approach provides a broadly applicable framework to study PTMs and discover their diverse roles in specific cell types and biological states.

cell biology↗

bioRxiv2021

A Systematic Interrogation of MHC Class I Antigen Presentation Identifies Constitutive and Compensatory Protein Degradation Pathways

The adaptive immune system distinguishes self from non-self by surveying peptides generated from degradation of intracellular proteins that are loaded onto MHC Class I molecules for display on the cell surface. While early studies reported that the bulk of cell surface MHC Class I complexes require the ubiquitin-proteasome system (UPS) for their generation, this conclusion has been challenged. To better understand MHC Class I peptide origins, we sought to carry out a comprehensive, quantitative census of the MHC Class I peptide repertoire in the presence and absence of UPS activity. We introduce optimized methodology to enrich for authentic Class I-bound peptides in silico and then quantify by mass spectrometry their relative amounts upon perturbation of the ubiquitin-proteasome system. Whereas most peptides are dependent on the proteasome and ubiquitination for their generation, a surprising 30% of the MHC Class I repertoire, enriched in peptides of mitochondrial origin, appears independent of these pathways. A further [~]10% of Class I-bound peptides were found to be dependent on the proteasome but independent of ubiquitination for their generation. Notably, clinically achievable partial inhibition of the proteasome resulted in display of novel peptides antigens, at least one of which promotes immune system activation. Our results suggest that generation of MHC Class I*peptide complexes is more complex than previously recognized and also provide evidence for compensatory peptide-generating pathways when canonical pathways are impaired.

molecular biology↗

bioRxiv2021

Targeted Neuronal Activation of the Gastrointestinal Tract Shapes the Environment of the Gut in Mice

The gastrointestinal (GI) tract is extensively innervated by intrinsic neurons of the enteric nervous system (ENS) and extrinsic neurons of the central nervous system and peripheral ganglia, which together regulate gut physiology. The GI tract also harbors a diverse microbiome, but interactions between the ENS and the microbiome remain poorly understood. Herein, we activate choline acetyltransferase (ChAT)-expressing or tyrosine hydroxylase (TH)-expressing gut-associated neurons in mice to determine effects on intestinal microbial communities and their metabolites, as well as on host physiology. The resulting multi-omics datasets support broad roles for discrete peripheral neuronal subtypes in shaping microbiome structure, including modulating bile acid profiles and fungal colonization. Physiologically, activation of either ChAT+ or TH+ neurons increases fecal output, while only ChAT+ activation results in increased colonic migrating motor complexes and diarrhea-like fluid secretion. These findings suggest that specific subsets of peripherally-activated ENS neurons differentially regulate the gut microbiome and GI physiology in mice, without involvement of signals from the brain.

physiology↗

bioRxiv2020

Regulated assembly of the ER membrane protein complex

The assembly of nascent proteins into multi-subunit complexes is tightly regulated to maintain cellular homeostasis. The ER membrane protein complex (EMC) is an essential insertase that requires seven membrane-spanning and two soluble subunits for function. Here we show that the kinase With no lysine 1 (WNK1), known for its role in hypertension and neuropathy, is required for assembly of the human EMC. WNK1 uses a conserved amphipathic helix to stabilize the soluble subunit, EMC2, by binding to the EMC2-8 interface. Shielding this hydrophobic surface prevents promiscuous interactions of unassembled EMC2 and precludes binding of ubiquitin ligases, permitting assembly. Using biochemical reconstitution, we show that after EMC2 reaches the membrane, its interaction partners within the EMC displace WNK1, and similarly shield its exposed hydrophobic surfaces. This work describes an unexpected role for WNK1 in protein biogenesis, and defines the general requirements of an assembly factor that will apply across the proteome.

molecular biology↗

bioRxiv2020

Deciphering the regulatory genome of Escherichiacoli, one hundred promoters at a time

Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for {approx} 65% of the promoters we remain completely ignorant of their regulation. Until we have cracked this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method (Reg-Seq) linking a massively-parallel reporter assay and mass spectrometry to produce a base pair resolution dissection of more than 100 promoters in E. coli in 12 different growth conditions. First, we show that our method recapitulates regulatory information from known sequences. Then, we examine the regulatory architectures for more than 80 promoters in the E. coli genome which previously had no known regulation. In many cases, we also identify which transcription factors mediate their regulation. The method introduced here clears a path for fully characterizing the regulatory genome of model organisms, with the potential of moving on to an array of other microbes of ecological and medical relevance.

biophysics↗

bioRxiv2019

Bacterial flagellar motor PL-ring disassembly sub-complexes are widespread and ancient

The bacterial flagellar motor is an amazing nanomachine. Understanding how such complex structures arose is crucial to our understanding of cellular evolution. We and others recently reported that in several Gammaproteobacterial species, a relic sub-complex comprising the decorated P- and L-rings persists in the outer membrane after flagellum disassembly. Imaging nine additional species with cryo-electron tomography, here we show that this sub-complex persists after flagellum disassembly in other phyla as well. Bioinformatic analyses fail to show evidence of any recent horizontal transfers of the P- and L-ring genes, suggesting that this sub-complex and its persistence is an ancient and conserved feature of the flagellar motor. We hypothesize that one function of the P- and L-rings is to seal the outer membrane after motor disassembly.

evolutionary biology↗

bioRxiv2019

Single subunit degradation of WIZ, a lenalidomide- and pomalidomide dependent substrate of E3 ubiquitin ligase CRL4CRBN

Immunomodulators (IMiDs) are an effective class of drugs used to treat blood cancers. IMiDs are believed to work by recruiting protein targets containing a {beta}-hairpin motif for ubiquitination by E3 ubiquitin ligase complexes composed of cereblon (CRBN), Cullin-4a (CUL4a), DNA Damage Binding protein-1 (DDB1), and Ring Box-1 (RBX1). The ubiquitinated protein is subsequently degraded by the proteasome. By characterizing the repertoire of proteins that show an increased physical association with CRBN after IMiD treatment, we identified a novel IMiD substrate, Widely Interspaced Zinc Finger Motifs (WIZ). WIZ contains a C2H2 zinc finger domain, like several other substrates that were previously characterized. We demonstrate that IMiDs stabilize physical association of WIZ with CRBN, deplete WIZ steady state protein levels in a way that is dependent on E3 ligase activity, and enhance the rate of its degradation. Notably, proteins that assemble with WIZ are co-recruited to CRBN by IMiDs but are not degraded, illustrating the potential of targeted protein degradation to eliminate individual subunits of a protein complex. These findings suggest that systematic characterization of the full repertoire of proteins that are targeted for degradation by IMiD compounds will be required to better understand their biological effects.\n\nSynopsisProteolysis Targeting Chimeras (PROTACs) can be used to precisely target a subunit of a transcriptional complex for degradation in subpopulations of cells.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC=\"FIGDIR/small/595389_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (18K):\norg.highwire.dtl.DTLVardef@186fed8org.highwire.dtl.DTLVardef@1c522f9org.highwire.dtl.DTLVardef@17b19baorg.highwire.dtl.DTLVardef@d626ff_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗