Biology subjects

Hellerschmied, D.

Publications and source records attributed to Hellerschmied, D..

6 recordsLinked to original sources

bioRxiv2026

Selective proteasomal degradation from the Golgi apparatus membrane

To maintain cellular homeostasis, protein quality control (PQC) machineries target damaged proteins for degradation. However, the PQC machinery, operating at the major protein sorting and processing site of mammalian cells, the Golgi, has remained largely elusive. Here we used a chemical biology tool to induce misfolding and ubiquitination of Golgi-localized model substrates. Our tool recruits a Cullin-RING ubiquitin ligase complex by exposure of a degron, recognized by the substrate receptor KLHDC2. Using this tool, we found that ubiquitinated Golgi membrane proteins are targeted for proteasomal degradation, a process that is critical to avoid Golgi fragmentation. This process is facilitated by the p97-unfoldase, which assembles with its adaptors UFD1-NPL4 and FAF2 at the Golgi. We further identified the rhomboid pseudo-protease RHBDD2 as an important contributor to Golgi homeostasis as it binds misfolded, ubiquitinated Golgi membrane proteins and regulates Golgi morphology. Our findings establish a molecular framework for Golgi-PQC in mammalian cells. O_LIThe accumulation of misfolded Golgi proteins causes Golgi fragmentation. C_LIO_LIUbiquitinated Golgi membrane proteins are degraded by the proteasome. C_LIO_LIThe p97 unfoldase operates at the Golgi with its adaptors UFD1-NPL4 and FAF2. C_LIO_LIThe rhomboid pseudo-protease RHBDD2 contributes to Golgi protein quality control. C_LI

cell biology↗

bioRxiv2024

A non-autonomous protein quality control mechanism targeting tau aggregate propagation

Tauopathies such as Alzheimers disease, frontotemporal dementia with Parkinsonism, and other neurodegenerative disorders are characterized by the spread of tau pathology from an initial brain region to neuroanatomically connected areas. At the molecular level, spreading involves aggregation of tau in a donor cell, externalization of transmissible fragments of amyloid fibrils, internalization by an acceptor cell, followed by seeded aggregation of endogenous tau. However, the protein quality control mechanisms that counteract tau aggregation, and in particular its spreading process, are not well understood. In this context, a co-migrating factor performing location-independent interference of fibril formation and transmission would be an appropriate conceptual solution. Here, we show that the cell-to-cell transfer of the widely conserved serine protease HTRA1 impedes tau pathology by targeting multiple steps within the spreading process. Our results suggest a defense mechanism against the intercellular spread of pathogenic protein conformations.

biochemistry↗

bioRxiv2024

Induced degradation of SNAP-fusion proteins

Self-labeling protein tags are an efficient means to visualize, manipulate, and isolate engineered fusion proteins with suitable chemical probes. The SNAP-tag, which covalently conjugates to benzyl-guanine and -chloropyrimidine derivatives is used extensively in fluorescence microscopy, given the availability of suitable SNAP-ligand-based probes. Here, we extend the applicability of the SNAP-tag to targeted protein degradation. We developed a set of SNAP PROteolysis TArgeting Chimeras (SNAP-PROTACs), which recruit the VHL or CRBN-ubiquitin E3 ligases to induce the degradation of SNAP-fusion proteins. Endogenous tagging enabled the visualization and the selective depletion of a SNAP-clathrin light chain fusion protein using SNAP-PROTACs. The addition of PROTACs to the SNAP-tag reagent toolbox facilitates the comprehensive analysis of protein function with a single gene tagging event.

cell biology↗

bioRxiv2024

Conformational plasticity of a BiP-GRP94 chaperone complex

Hsp70/Hsp90-chaperones and their regulatory co-chaperones are critical for maintaining protein homeostasis. GRP94, the sole Hsp90-chaperone in the secretory pathway of mammalian cells, is essential for the maturation of important secretory and transmembrane proteins. Without the requirement of co-chaperones, the Hsp70-protein BiP controls regulatory conformational changes of GRP94 - the structural basis of which has remained elusive. Here, we biochemically and structurally characterize the formation of a BiP-GRP94 chaperone complex and its transition to a conformation expected to support the loading of substrate proteins from BiP onto GRP94. BiP initially binds to the open GRP94 dimer via an interaction interface that is conserved among Hsp70/90 paralogs. Subsequently, binding of a second BiP protein stabilizes a semi-closed GRP94 dimer, thereby advancing the chaperone cycle. Our findings highlight a fundamental mechanism of direct Hsp70/90 cooperation, independent of co-chaperones.

biochemistry↗

bioRxiv2022

High resolution analysis of proteolytic substrate processing

Proteolysis is a key catalytic event in protein and thus cellular homeostasis. Despite the importance and wide implications of proteolytic processing and degradation, methods describing the degradation of folded proteins at high temporal and spatial resolution are not well established. However, this information is required to obtain a deep mechanistic understanding of proteolytic events and their consequences. Here, we describe an integrated method comprising time-resolved mass spectrometry, circular dichroism spectroscopy and bioinformatics to reveal the sequential degradation and unfolding of the model substrate annexin A1 by the human serine protease HTRA1. This workflow represents a general strategy for obtaining precise molecular insights into protease-substrate interactions that can be conveniently adapted to studying other posttranslational modifications such as phosphorylation in dynamic protein complexes.

biochemistry↗

bioRxiv2021

Functional metabolic phenotyping of human pancreatic ductal adenocarcinoma

Pancreatic Ductal Adenocarcinoma (PDAC) lacks targeted treatment options. Although subtypes with transcriptome-based distinct lineage and differentiation features have been identified, deduced clinically actionable targets remain elusive. We here investigate functional metabolic features of the classical and QM (quasi-mesenchymal)/basal-like PDAC subtypes potentially exploitable for non-invasive subtype differentiation and therapeutic intervention. A collection of human PDAC cell lines, primary patient derived cells (PDC), patient derived xenografts (PDX) and patient PDAC samples were transcriptionally stratified into the classical and QM subtype. Functional metabolic analyses including targeted and non-targeted metabolite profiling (matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI)), seahorse metabolic flux assays and metabolic drug targeting were performed. Hyperpolarized 13C-magnetic resonance spectroscopy (HP-MRS) of PDAC xenografts was used for in vivo detection of intra-tumoral [1-13C]pyruvate and [1-13C]lactate metabolism. We identified glycolysis and lipid metabolism/fatty acid oxidation as transcriptionally preserved metabolic pathways in QM and classical PDAC subtype respectively. However, these metabolic cues were not unambiguously functionally linked to one subtype. Striking functional metabolic heterogeneity was observed especially in primary patient derived cells with only individual samples representing high dependence on glycolysis or mitochondrial oxidation. Of note, QM cells actively use the glycolytic product lactate as oxidative mitochondrial fuel. Using HP-MRS, we were able to non-invasively differentiate glycolytic tumor xenografts with high intratumoral [1-13C]pyruvate to [1-13C]lactate conversion in vivo. Although PDAC transcriptomes indicate molecular subtype-associated distinct metabolic pathways, we found substantial functional metabolic heterogeneity independent of the molecular subtype. Non-invasive identification of highly glycolytic tumors by [1-13C]pyruvate/lactate HP-MRS support individualized metabolic targeting approaches.

cancer biology↗