Oliver Popp

Huntingtin-associated protein 40 (HAP40) forms a stable protein complex with huntingtin (HTT). Its cellular function and how HAP40 loss influences mutant HTT (mHTT) abundance and pathobiology are currently unclear. Here, using diverse cellular models and omics methods, we demonstrate that HAP40 is an obligate interaction partner of HTT and through its binding controls HTT interactions and the abundance of HTT-associated proteins, indicating that it functions as a HTT interaction regulatory unit. Also, loss of HAP40 in mHTT-expressing striatal cells impairs autophagic protein degradation and triggers transcriptional activation of the CLEAR network, demonstrating that it functions as a proteostasis regulator that acts on quality control pathways. Finally, mHTT-expressing cells lacking HAP40 showed increased secretion of a ~205 kDa mHTT fragment through an endoplasmic reticulum (ER)/Golgi-associated route, indicating that striatal cells reduce intracellular mHTT-induced proteotoxicity through activation of secretory pathways. Together, these results establish HAP40 as a critical proteostasis regulator that through controlling HTT interactions maintains cellular homeostasis.

Lysine hydroxylation is increasingly recognised as a widespread post-translational modification in human cells, with more than 100 modified sites identified to date. Jumonji domain-containing protein 6 (JMJD6) catalyses hydroxylation at multiple lysines within lysine-rich regions and is a major contributor to this modification across the proteome. As JMJD6 requires oxygen as a co-substrate, lysine hydroxylation has been proposed to couple oxygen availability to cellular function. However, the biological significance of this modification remains incompletely understood, in part due to technical challenges associated with the detection of hydroxylation within lysine-rich regions by mass spectrometry. To address limitations of conventional approaches, we systematically evaluated key steps in the analysis of proteomic data from lysine-derivatised samples and developed a workflow for comprehensive, accurate, and quantitative analysis of lysine hydroxylation. Methodological improvements included optimisation of database search strategies to increase peptide coverage in lysine-rich regions and incorporation of immonium ion signatures to substantially improve confidence in hydroxylysine identification. We further demonstrated that stoichiometry derived from peptide precursor ion intensity faithfully captures hypoxia-responsive changes in lysine hydroxylation at amino acid resolution. Application of this workflow to bromodomain (BRD) proteins — epigenetic readers containing lysine-rich regions extensively hydroxylated by JMJD6 — revealed marked heterogeneity in the apparent kinetics of hydroxylation among target lysines, with evidence of interdependence between neighbouring sites. Hypoxia suppressed hydroxylation in a site-dependent manner, with greater suppression observed at sites displaying slower rates of hydroxylation. Together, the development and application of this workflow establish a methodological and biological framework for understanding how oxygen availability regulates protein function through lysine hydroxylation.

Skeletal muscle atrophy is a pathological condition characterized by the loss of muscle mass and strength, driven by diverse stimuli including disuse, malnutrition, acute and chronic inflammation, ...

Huntingtin-associated protein 40 (HAP40) forms a stable protein complex with huntingtin (HTT). Its cellular function and how HAP40 loss influences mutant HTT (mHTT) abundance and pathobiology are currently unclear. Here, using diverse cellular models and OMICs methods, we demonstrate that HAP40 is an obligate interaction partner of full-length HTT and through its binding controls the abundance of HTT-associated proteins, indicating that it functions as HTT interaction regulatory unit. Also, loss of HAP40 in mHTT-expressing striatal cells impairs autophagosome-lysosome flux, triggers massive transcriptional dysregulation, including the activation of the CLEAR network, demonstrating that it functions as proteostasis regulator that acts on quality control pathways. Finally, mHTT-expressing cells lacking HAP40 showed increased secretion of mHTT through the ER-to-Golgi route, indicating that striatal cells reduce intracellular mHTT-induced proteotoxicity through activation of secretory pathways. Together, these results establish HAP40 as a critical proteostasis regulator that through controlling HTT interactions maintains cellular homeostasis.

Skeletal muscle atrophy studies rely on in vivo mouse data as well as in vitro data. Our deep coverage transcriptome and proteome data reveal that the commonly used C2C12 cells faithfully recapitulate differentiation and atrophy markers, with significant alternative splicing occurring under dexamethasone atrophy. Using published mouse tissue data of comparable methods, we provide an up-to-date resource for skeletal muscle atrophy to complement animal studies.

Skeletal muscle atrophy and weakness are major contributors to morbidity, prolonged recovery, and long-term disability across a wide range of diseases. Atrophy is caused by breakdown of sarcomeric proteins resulting in loss of muscle mass and strength. Molecular mechanism underlying the onset of muscle atrophy and its progression have been analysed in patients, mice, and cell culture but the complementarity of these model systems remains to be explored. Here, we applied deep-coverage transcriptomic and proteomic profiling to characterize dynamic changes during dexamethasone-induced atrophy in the widely used murine skeletal muscle cell line C2C12. Comparison with published datasets confirmed that muscle differentiation is well recapitulated in C2C12 myotubes. Under dexamethasone treatment, this model was particularly suited to capture early atrophy events. We identified alterations in mitochondrial gene expression and differential alternative splicing events during early-stage myotube atrophy. This dataset complements existing in vivo data and provides novel insights into the regulatory processes during skeletal muscle wasting.

Skeletal muscle atrophy is a pathological condition characterized by the progressive loss of muscle mass and function, driven by factors such as disuse, inflammation, and aging. While the ubiquitin-proteasome system is established as the central mediator of myofibrillar protein degradation, the role of autophagy in selective protein turnover remains largely unexplored. To address this, we employed a quantitative, time-resolved analysis of protein synthesis and degradation in C2C12 myotubes undergoing TNF-α-induced atrophy, using dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (dynamic SILAC) coupled with LC-MS/MS. Our data challenges the classical view of atrophy as a uniform, degradation-centric process. Instead, we reveal temporally distinct patterns of selective protein turnover, including differential degradation of myofibrillar, ribosomal, and endoplasmic reticulum (ER)-resident proteins. Early atrophy is characterized by suppressed short-term protein synthesis, increased ubiquitin-ligase expression, proteasomal activation, and ribosome turnover. In contrast, late atrophy features proteasome-dependent myofibrillar protein degradation, selective synthesis of mitochondrial ribosomes and cytoplasmic ribosome degradation, indicative of metabolic adaptation. Moreover, we identify a temporal shift in autophagic selectivity: from ER homeostasis maintenance to a stress-induced ER-degradation program. Notably, inhibition of autophagy during atrophy leads to accumulation of ER-phagy receptors Tex264 and Calcoco1, implicating ER-phagy as a key contributor to atrophic remodeling, underscoring an underexplored regulatory mechanism in muscle proteostasis. By elucidating the role of autophagy in degradation of the ER, this study opens new avenues for therapeutic interventions targeting proteostasis regulation in inflammation-induced muscle-wasting disorders, ultimately contributing to a more refined understanding of muscle atrophy beyond proteasomal degradation.

Long non-coding RNAs (lncRNAs) are well-established as key regulators of gene expression. However, emerging evidence reveals that some lncRNAs can also encode functional microproteins. In this study, we report the identification of an evolutionarily young microprotein encoded by LINC01503 , expressed across several human tissues. This microprotein, designated as LINC01503-MP, localises to the mitochondria and exerts a proliferative effect on HCT116 colorectal cancer (CRC) cells. Functional studies reveal that LINC01503-MP regulates mitochondrial oxygen consumption rate, linking its activity to enhanced metabolic functions and cell proliferation. Interactome analyses identified multiple mitochondrial metabolism-related proteins as potential interaction partners. Our findings show that LINC01503-MP plays a role in the proliferative phenotype associated with LINC01503 upregulation in CRC, suggesting the functional significance of evolutionarily young, lncRNA-derived microproteins in cancer progression.

Local mRNA translation is required for adaptive changes in the synaptic proteome. The mitogen-activated protein kinase (MAPK) interacting protein kinases 1 and 2 (MNK1 and MNK2) have emerged as key regulators of neuronal translation, primarily through phosphorylation of the eukaryotic initiation factor 4E (eIF4E). The therapeutic benefits of targeting the MNKs are being investigated for nervous system conditions and disorders that affect translation, including autism, pain, and cancer. However, it is still unclear if MNK1 and MNK2 regulate distinct aspects of neuronal translation and how the activity of each kinase is associated with the synaptic and behavioral features linked to MNK signaling. To examine the individual neurobiological functions of each kinase, we used knockout mice lacking either MNK1 or MNK2. We found that the knockout of MNK1 and MNK2 leads to different social and cognitive behavioral profiles and distinct alterations of the cortical synaptic proteome, transcriptome, and phosphoproteome. Loss of MNK1 was associated with an increase in ribosomal protein expression, whereas deletion of MNK2 decreased the expression and phosphorylation of synaptic proteins. Together, our findings provide evidence for a high degree of functional specialization of the MNKs in synaptic compartments and suggest that pharmacological inhibition of individual MNK may provide more specific targets for neurological disorders.

SUMMARY: Gentle touch is conveyed to the brain by fast-conducting sensory fibers. Mechanosensitive ion channels at the terminals of these neurons are thought to be gated by extracellular tethers that transmit force from the surrounding matrix to the channel complex, but the molecular identity of such tethers has remained unknown. Here, we identify Teneurin-4 (TENM4) as an essential extracellular linker protein for mechanotransduction in mechanoreceptors. Sensory neuron–specific deletion of Tenm4 in mice caused profound touch insensitivity, while acute and reversible proteolytic disassembly of TENM4 at sensory endings confirmed its direct role in force transduction. Ultrastructural analyses revealed TENM4 localization to filamentous structures at the neurite–laminin interface, defining it as a structural component of the mechanosensory tether. These findings identify TENM4 as a core element of fast somatic sensation and provide molecular insight into how extracellular forces are coupled to ion channel activation.