Biology subjects

Plumb, R.

Publications and source records attributed to Plumb, R..

4 recordsLinked to original sources

bioRxiv2026

Insight into the scaffolding function of USP18 from a high resolution cryo-EM structure of STAT2-USP18-ISG15 ternary complex

USP18 is a primary negative regulator of the type I interferon (IFN-I) signaling which regulates hundreds of IFN-stimulated genes for viral protection and anti-cancer immunity. USP18 plays dual roles in the IFN-I signaling: 1) deubiquitinase enzymatic function which cleaves ISG15 from its substrates and 2) scaffolding function through forming a complex with STAT2 to suppress IFN-I signaling. Targeting the scaffolding function of USP18, instead of its enzyme activity, is crucial for reducing cancer cell fitness and boosting anti-tumor immunity. However, the molecular basis of USP18s scaffolding function remains unclear due to the lack of structural information. Here, using a fusion tag strategy, we captured the transient USP18-STAT2 complex and determined a ternary complex structure of STAT2-USP18-ISG15 at 3.05 [A] resolution by cryogenic electron microscopy (cryo-EM) that delineated detailed USP18-STAT2 interactions. Remarkably, the ternary complex impairs USP18s enzymatic function by STAT2-mediated disruption of its catalytic triad. Structural analysis and mutagenesis identify specific USP18 point mutations, facilitating further investigation into the role of USP18 in IFN-I signaling. Taken together, our findings suggest that USP18s scaffolding function could present an untapped opportunity for cancer therapy.

molecular biology↗

bioRxiv2025

Development and validation of a streamlined workflow for proteomic analysis of proteins and post-translational modifications from dried blood

It is increasingly recognized that the omic analysis of whole blood has applications for precision medicine and disease phenotyping. Despite this realization, whole blood is generally viewed as a challenging analytical matrix in comparison to plasma or serum. Moreover, proteomic analyses of whole blood proteomics have almost exclusively focused on (non)targeted analyses of protein abundances and much less on post-translational modifications (PTMs). Here, we developed a streamlined workflow for processing twenty microliters of venous blood collected by volumetric absorptive microsampling that incorporates serial trypsinization, N-glycopeptide and phosphopeptide enrichment and avoids laborious sample dry-down or cleanup steps. Up to 10,000 analytes (reported as protein groups, glycopeptidoforms and phosphosites) were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in approximately 2 h of MS acquisition time. Using these methods, we explored the stability of "dried" and "wet" blood proteomes, as well as effects of ex vivo inflammatory stimulus or phosphatase inhibition. Multi-omics factor analysis enabled facile identification of analytes that contributed to inter-individual variability of the blood proteomes, including N-glycopeptides that distinguish immunoglobulin heavy constant alpha 2 allotypes. Collectively, our results help to establish feasibility and best practices for the integrated MS-based quantification of proteins and PTMs from dried blood.

biochemistry↗

bioRxiv2023

Three-dimensional reconstructions of mechanosensory end organs suggest a unifying mechanism underlying dynamic, light touch

Specialized mechanosensory end organs within mammalian skin--hair follicle-associated lanceolate complexes, Meissner corpuscles, and Pacinian corpuscles--enable our perception of light, dynamic touch1. In each of these end organs, fast-conducting mechanically sensitive neurons, called A{beta} low-threshold mechanoreceptors (A{beta} LTMRs), associate with resident glial cells, known as terminal Schwann cells (TSCs) or lamellar cells, to form complex axon ending structures. Lanceolate-forming and corpuscle-innervating A{beta} LTMRs share a low threshold for mechanical activation, a rapidly adapting (RA) response to force indentation, and high sensitivity to dynamic stimuli1-6. How mechanical stimuli lead to activation of the requisite mechanotransduction channel Piezo27-15 and A{beta} RA-LTMR excitation across the morphologically dissimilar mechanosensory end organ structures is not understood. Here, we report the precise subcellular distribution of Piezo2 and high-resolution, isotropic 3D reconstructions of all three end organs formed by A{beta} RA-LTMRs determined by large volume enhanced Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging. We found that within each end organ, Piezo2 is enriched along the sensory axon membrane and is minimally or not expressed in TSCs and lamellar cells. We also observed a large number of small cytoplasmic protrusions enriched along the A{beta} RA-LTMR axon terminals associated with hair follicles, Meissner corpuscles, and Pacinian corpuscles. These axon protrusions reside within close proximity to axonal Piezo2, occasionally contain the channel, and often form adherens junctions with nearby non-neuronal cells. Our findings support a unified model for A{beta} RA-LTMR activation in which axon protrusions anchor A{beta} RA-LTMR axon terminals to specialized end organ cells, enabling mechanical stimuli to stretch the axon in hundreds to thousands of sites across an individual end organ and leading to activation of proximal Piezo2 channels and excitation of the neuron.

neuroscience↗

bioRxiv2020

The Sec63/BiP complex suppresses higher-order oligomerization and RNase activity of IRE1α during ER stress

Misfolded proteins in the endoplasmic reticulum (ER) activate IRE1 endoribonuclease in mammalian cells, which mediates XBP1 mRNA splicing to produce an active transcription factor. This promotes the expression of specific genes to alleviate ER stress and thereby attenuating IRE1. Although sustained activation of IRE1 is linked to human diseases, it is not clear how IRE1 is attenuated during ER stress. Here, we identify that Sec63 is a subunit of the previously identified IRE1/Sec61 translocon complex. We find that Sec63 recruits and activates BiP ATPase through its luminal J-domain to bind onto IRE1. This leads to inhibition of higher-order oligomerization and attenuation of IRE1 RNase activity during prolonged ER stress. In Sec63 deficient cells, IRE1 remains activated for a long time despite the presence of excess BiP in the ER. Thus, our data suggest that the Sec61 translocon bridges IRE1 with Sec63/BiP to regulate the dynamics of IRE1 signaling in cells.

cell biology↗