Biology subjects

Hevler, J. F.

Publications and source records attributed to Hevler, J. F..

6 recordsLinked to original sources

bioRxiv2025

Discovering Glycosylation-Dependent Protein Function by Thermal Proteome Profiling

Protein glycosylation regulates essential cellular processes including protein folding, stability, and cell-cell interactions; however, how aberrant glycosylation impacts protein function and interaction networks remains poorly understood. Here we combine mass spectrometry-based proteomics, chemical glycobiology, and molecular dynamics simulations to systematically investigate glycosylation dependent protein stability and function. By perturbing the secretory pathway at defined steps, we generated proteins with distinct glycan structures and analyzed their functional consequences using thermal proteome profiling. This approach revealed that cells mount convergent stress responses to diverse glycosylation perturbations, characterized by coordinated trafficking reorganization. This reorganization effectively redirects protein flux from secretion toward degradation. We further identified that perturbing terminal glycan modifications, particularly sialylation and fucosylation, exerts the most profound effects on cell surface protein function. We studied in detail how loss of fucosylation restricts the conformational dynamics of key domains in integrin alpha 4, reducing VCAM-1 binding affinity and suggesting a potential therapeutic strategy for multiple sclerosis. Overall, our systematic approach uncovered extensive complexity in how glycosylation regulates protein function beyond simple glycoform identification and provides a resource for dissecting essential glycan-protein relationships.

systems biology↗

bioRxiv2023

Preserved respiratory chain capacity and physiology in mice with profoundly reduced levels of mitochondrial respirasomes

The mammalian respiratory chain complexes I, III2 and IV (CI, CIII2 and CIV) are critical for cellular bioenergetics and form a stable assembly, the respirasome (CI- CIII2-CIV), that is biochemically and structurally well documented. The role of the respirasome in bioenergetics and regulation of metabolism is subject to intense debate and is difficult to study because the individual respiratory chain complexes coexist together with high levels of respirasomes. To critically investigate the in vivo role of the respirasome, we generated homozygous knock-in mice that have normal levels of respiratory chain complexes but profoundly decreased levels of respirasomes. Surprisingly, the mutant mice are healthy, with preserved respiratory chain capacity and normal exercise performance. Our findings show that high levels of respirasomes are dispensable for maintaining bioenergetics and physiology in the mouse, but raises questions about their alternate functions, such as relating to regulation of protein stability and prevention of age-associated protein aggregation.

molecular biology↗

bioRxiv2022

Juxtaposition of Bub1 and Cdc20 on phosphorylated Mad1 during catalytic mitotic checkpoint complex assembly

In response to improper kinetochore-microtubule attachments in mitosis, the spindle assembly checkpoint (SAC) assembles the mitotic checkpoint complex (MCC) to inhibit the anaphase- promoting complex/cyclosome, thereby delaying entry into anaphase. The MCC is a complex of Mad2, Cdc20 and BubR1:Bub3. Its assembly is catalysed by unattached kinetochores on a catalytic Mad1:Mad2 platform. Mad1-bound closed-Mad2 (C-Mad2) recruits open-Mad2 (O- Mad2) through self-dimerization. This interaction, combined with Mps1 kinase-mediated phosphorylation of Bub1 and Mad1, accelerates MCC assembly, in a process that requires conversion of O-Mad2 to C-Mad2 and concomitant binding of Cdc20. How Mad1 phosphorylation catalyses MCC assembly is poorly understood. In this study we characterized Mps1 phosphorylation of Mad1 and obtained structural insights into a phosphorylation-specific Mad1:Cdc20 interaction. This interaction, together with the Mps1-phosphorylation dependent association of Bub1 and Mad1, generates a tripartite assembly of Bub1 and Cdc20 onto the C- terminal domain of Mad1 (Mad1CTD). We additionally identified a folded state of the Mad1:Mad2 complex that suggests how the Cdc20:Mad1CTD interaction brings the Mad2- interacting motif (MIM) of Cdc20 (Cdc20MIM) into close proximity with O-Mad2. We also show that Cdc20MIM is sufficient to catalyse Mad2 conversion and its entrapment by the Mad2 safety- belt. Thus, Mps1-dependent formation of the MCC-assembly scaffold functions to position and orient Cdc20MIM near O-Mad2, thereby catalysing formation of C-Mad2:Cdc20.

biochemistry↗

bioRxiv2021

HAP40 orchestrates huntingtin structure for differential interaction with polyglutamine expanded exon 1

Huntingtons disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 [A] cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass-spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The polyglutamine tract containing N-terminal exon 1 region of HTT is dynamic, but shows greater conformational variety in the mutant than wildtype exon 1. By providing novel insight into the structural consequences of HTT polyglutamine expansion, our data provide a foundation for future functional and drug discovery studies targeting Huntingtons disease.

biochemistry↗

bioRxiv2021

Molecular characterization of a complex of Apoptosis Inducing Factor 1 (AIFM1) with cytochrome c oxidase of the mitochondrial respiratory chain

Combining mass spectrometry based chemical cross-linking and complexome profiling, we analyzed the interactome of heart mitochondria. We focused on complexes of oxidative phosphorylation and found that dimeric apoptosis inducing factor 1 (AIFM1) forms a defined complex with ~10% of monomeric cytochrome c oxidase (COX), but hardly interacts with respiratory chain supercomplexes. Multiple AIFM1 inter-crosslinks engaging six different COX subunits provided structural restraints to build a detailed atomic model of the COX-AIFM12 complex. Application of two complementary proteomic approaches thus provided unexpected insight into the macromolecular organization of the mitochondrial complexome. Our structural model excludes direct electron transfer between AIFM1 and COX. Notably however, the binding site of cytochrome c remains accessible allowing formation of a ternary complex. The discovery of the previously overlooked COX-AIFM12 complex and clues provided by the structural model hint at a role of AIFM1 in OXPHOS biogenesis and in programmed cell death.

molecular biology↗

bioRxiv2021

In-cell structures of a conserved supramolecular array at the mitochondria-cytoskeleton interface in mammalian sperm

Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image mammalian sperm, where mitochondria wrap around the ciliary cytoskeleton. We find that mitochondria are tethered to their neighbors through inter-mitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types where mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

cell biology↗