Biology subjects

Meinhart, A.

Publications and source records attributed to Meinhart, A..

9 recordsLinked to original sources

bioRxiv2025

A fluorescent folding reporter uncovers myosin misfolding as a driver of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a fatal genetic disorder causing the thickening of ventricular walls in the heart muscle. While certain mutations in cardiac myosin deregulate ATPase activity, the pathology mechanism of most HCM mutations is not known. Here, by designing a fluorescent reporter to monitor myosin folding in cells, we uncovered a distinct class of HCM mutations that cause graded defects in myosin maturation. Using C. elegans as a disease model, we found that folding deficient HCM variants cause myofilament disruption, impaired motility, and reduced lifespan. Dietary restrictions resulted in a near-complete recovery from these detrimental defects by activating autophagy pathways through insulin/TOR signaling. In conclusion, our study identifies myosin misfolding as an important driver of HCM, revealing therapeutic opportunities to counteract muscle protein disorders.

cell biology↗

bioRxiv2024

Architecture of the UBR4 complex, a giant E4 ligase central to eukaryotic protein quality control

Eukaryotic cells have evolved sophisticated quality control mechanisms to eliminate aggregation-prone proteins that compromise cellular health. Central to this defense is the ubiquitin-proteasome system, where UBR4 acts as essential E4 ubiquitin ligase, amplifying degradation marks on defective proteins. Our cryo-EM analysis of UBR4 in complex with its cofactors KCMF1 and CALM1 reveals a massive 1.3 MDa ring structure, featuring a central substrate-binding arena and flexibly attached catalytic units. Structural data illustrate how UBR4 binds substrate and extends K48-specific ubiquitin chains. Importantly, efficient substrate targeting depends on both pre-ubiquitination and specific N-degrons, with KCMF1 acting as key substrate filter. Furthermore, we show that the architecture of the E4 megacomplex is conserved across eukaryotes but with species specific adaptations, allowing UBR4 to perform its precisely tuned quality-control function in diverse cellular environments.

biochemistry↗

bioRxiv2024

Cross-species interactome analysis uncovers a conserved selective autophagy mechanism for protein quality control in plants

Selective autophagy is a fundamental protein quality control pathway that safeguards proteostasis by degrading damaged or surplus cellular components, particularly under stress. This process is orchestrated by selective autophagy receptors (SARs) that direct specific cargo for degradation. While significant strides have been made in understanding the molecular framework of selective autophagy, the diversity of SAR repertoires across species remain largely unexplored. Through a comparative interactome analysis across five model organisms, we identified a suite of conserved and lineage-specific SAR candidates. Among these, we validated CESAR as a conserved SAR critical for proteostasis under heat stress. CESAR specifically facilitates the degradation of hydrophobic, ubiquitinated protein aggregates and is indispensable for heat stress tolerance. Our study offers a rich resource for SAR discovery and positions CESAR as a pivotal regulator of proteostasis, with broad implications for improving stress resilience in plants.

plant biology↗

bioRxiv2022

Structural basis of how the BIRC6/SMAC complex regulates apoptosis and autophagy

Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. BIRC6 is an exceptionally large, multidomain IAP that inhibits its targets by means of its atypical ubiquitin ligase activity and in addition, functions as an inhibitor of autophagy by depleting LC3B. Little is known of the mechanisms by which BIRC6 interacts with its targets and fulfills these two roles. Here, we determined the cryo-EM structure of BIRC6 alone and in complex with two mitochondrial pro-apoptotic proteins, HTRA2 and SMAC. We show BIRC6 is an antiparallel homodimer that forms a crescent shape that arcs around a spacious cavity. The cavity is surrounded by binding sites for client proteins, where they interact with the flexible UBC domain that mediates ubiquitin ligation. Functional data reveal that multivalent binding of SMAC in the central cavity obstructs substrate binding, impeding ubiquitination of both autophagy and apoptotic target proteins. Together our data reveal the molecular mechanisms of how SMAC specifically binds and inhibits BIRC6 to promote apoptosis, and how this regulatory mechanism also extends to autophagy substrates. The interaction sites are hot spots of cancer and atrophy mutations, highlighting the importance of carefully balancing the interplay between BIRC6 and SMAC.

biochemistry↗

bioRxiv2022

UNC-45 assisted myosin folding depends on a conserved FX3HY motif implicated in Freeman Sheldon Syndrome

Myosin motors are critical for diverse motility functions ranging from cytokinesis and endocytosis to muscle contraction. The UNC-45 chaperone controls myosin function mediating the folding, assembly, and degradation of the muscle protein. Here, we analyze the molecular mechanism of UNC-45 as a hub in myosin quality control. We show that UNC-45 forms discrete complexes with folded and unfolded myosin, forwarding them to downstream chaperones and E3 ligases. Structural analysis of a minimal chaperone:substrate complex reveals that UNC-45 binds to a conserved FX3HY motif in the myosin motor domain. Disrupting the observed interface by mutagenesis prevents myosin maturation leading to protein aggregation in vivo. We also show that a mutation in the FX3HY motif linked to the Freeman Sheldon Syndrome impairs UNC-45 assisted folding, reducing the level of functional myosin. These findings demonstrate that a faulty myosin quality control is a critical yet unexplored cause of human myopathies.

biochemistry↗

bioRxiv2021

A molecular network of conserved factors keeps ribosomes dormant in the egg

Ribosomes are produced in large quantities during oogenesis and stored in the egg. However, the egg and early embryo are translationally repressed. Using mass-spectrometry and cryo-EM analyses of ribosomes isolated from zebrafish and Xenopus eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules and occupy functionally important sites of the ribosome: a Habp4-eEF2 module that stabilizes ribosome levels and a Dap1b/Dapl1-eIF5a module that represses translation. Dap1b/Dapl1 is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Thus, a developmentally programmed, conserved ribosome state plays a key role in ribosome storage and translational repression in the egg.

biochemistry↗

bioRxiv2021

BacPROTACs mediate targeted protein degradation in bacteria

Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis targeting chimeras (PROTACs). Despite their superior properties over classical inhibitors, it has so far not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, highlighting the potential of the technology to provide next generation antibiotics.

biochemistry↗

bioRxiv2020

McsB forms a gated kinase chamber to mark aberrant bacterial proteins for degradation

In Gram-positive bacteria, the McsB protein arginine kinase is central to protein quality control, labelling aberrant molecules for degradation by the ClpCP protease. Despite its importance for stress response and pathogenicity, it is still elusive how the bacterial degradation labelling is regulated. Here, we delineate the mechanism how McsB targets aberrant proteins during stress conditions. Structural data reveal a self-compartmentalized kinase, in which the active sites are sequestered in a molecular cage. The "closed" octamer interconverts with other oligomers in a phosphorylation-dependent manner and, contrary to these "open" forms, preferentially labels unfolded proteins. In vivo data show that heat-shock triggers accumulation of higher-order oligomers, of which the octameric McsB is essential for surviving stress situations. The interconversion of open and closed oligomers represents a distinct regulatory mechanism of a degradation labeler, allowing the McsB kinase to adapt its potentially dangerous enzyme function to the needs of the bacterial cell.

molecular biology↗

bioRxiv2019

Native mass spectrometry can effectively predict PROTAC efficacy

Protein degraders, also known as proteolysis targeting chimeras (PROTACs), are bifunctional small molecules that bring an E3 ubiquitin ligase and a protein of interest (POI) into proximity, thus promoting ubiquitination and degradation of the targeted POI [1-3]. Despite their great promise as next-generation pharmaceutical drugs, the development of new PROTACs is challenged by the complexity of the system, which involves binary and ternary interactions between components. Here, we demonstrate the strength of native mass spectrometry (nMS), a label-free technique, to provide novel insight into PROTAC-mediated protein interactions. We show that nMS can monitor the formation of ternary E3-PROTAC-POI complexes and detect various intermediate species in a single experiment. A unique benefit of the method is its ability to reveal preferentially formed E3-PROTAC-POI combinations in competition experiments with multiple substrate proteins, thereby positioning it as an ideal high-throughput screening strategy during the development of new PROTACs.

biochemistry↗