Proteomic analysis of induced human motor neurons revealed a markedly enhanced association between ΔE and exportin 1 (XPO1), a major nuclear export receptor, and an abnormally enhanced interaction between mutant TorsinA and exportin 1 (XPO1), a protein that transports cargo from the nucleus.
Mutations in several genes are known to cause familial forms of Parkinson's disease (PD), including mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene linked to late-onset, autosomal dominant PD. VPS35 encodes a core subunit of the retromer complex which functions in endosomal sorting and recycling. It remains unclear how the pathogenic D620N mutation in VPS35 disrupts retromer function to induce neurodegeneration in PD. Using cell- and rodent-based models expressing D620N VPS35, we performed interactome proteomics to identify alterations underlying the pathogenic effects of D620N VPS35 in PD. Using overexpression of VPS35 variants in HEK-293 T cells, we conducted tandem affinity purification (TAP) or co-immunoprecipitation (co-IP) with protein chemical crosslinking to determine the native and non-native protein interactomes of wild-type (WT) and D620N VPS35, respectively. Notably, we can confirm the reduced interaction of D620N VPS35 with components of the WASH complex. Additionally, using a viral-mediated gene transfer model of human D620N VPS35 overexpression in adult rat brain, we identify the first brain-specific protein interactome of VPS35. These overexpression models reveal remarkably similar interaction profiles of WT and D620N VPS35, suggesting that the D620N mutation has a subtle effect on the overall VPS35 protein interactome. We also conducted proteomic analysis of brain tissue from a D620N VPS35 knockin (KI) mouse model that expresses VPS35 at endogenous levels. Using co-IP from hemibrain or striatal extracts of WT and D620N VPS35 KI mice, we reveal a high degree of similarity between the brain interactomes of WT and D620N VPS35, further suggesting a subtle effect of the D620N mutation on VPS35 protein interactions. Notably, in both hemibrain and striatum, we find a selective decrease in the interaction of two known interactors, TBC1D5 and VPS29, with D620N VPS35. We also performed global proteomic analysis of striatal tissue from D620N VPS35 KI mice and reveal a high degree of similarity between WT and D620N, further suggesting a subtle effect of this mutation. Together, our study provides a comprehensive evaluation of the VPS35 protein interactome and reveals a selective effect of the PD-linked D620N mutation in mammalian cells and brain. Our study provides key insight into the mechanisms of retromer dysfunction in VPS35-linked PD.
Erin T. Williams, Maxwell Frye, Xi Chen et al.· Neurobiology of Disease· 0 citations
DYT1 dystonia is an incurable movement disorder caused by a loss-of-function mutation in Torsin1A, an endoplasmic reticulum (ER)-resident AAA+ ATPase. Here, we use Drosophila and human cells to shed light on Torsins' mode of action. Fly germ cells lacking dTorsin arrest in development with defects in nuclear pore complex (NPC) biogenesis due to impaired nuclear envelope membrane fusion. We identify the conserved membrane protein Chloride Channel CLIC-like protein 1 (CLCC1) as a Torsin1A interaction partner whose absence phenocopies membrane fusion defects caused by Torsin deletion. CLCC1 is enriched at membrane fusion sites, and molecular dynamics (MD) simulations suggest that CLCC1 rings induce bilayer remodeling and lipid flux to initiate fusion of the outer and inner nuclear membranes. Remarkably, CLCC1 overexpression rescues defects associated with loss of Torsins, indicating that a main role of dTorsin/Torsin1A is to sustain CLCC1 functionality. Our findings inform a model of nuclear envelope membrane fusion and imply that modulating CLCC1 expression is a promising therapeutic prospect for DYT1 dystonia.
D. Maslennikova, Harry J. M. Baird, Xinyue Ding et al.· Molecules and Cells· 2 citations· ⚡1
The first functional characterization of the cardiomyopathy-associated SMYD1 N101S variant identified in a child with severe infantile cardiomyopathy is provided, establishing a mechanistic link between SMYD1 dysfunction and infantile cardiomyopathy and highlighting the importance of integrating genomic and functional approaches in rare cardiovascular disease.
Marta W. Szulik, Clint Gwynn, Magnus Creed et al.· bioRxiv· 0 citations
Aggregation and cytoplasmic mislocalization of TDP-43 are defining features of several neurodegenerative disorders including Amyotrophic Lateral Sclerosis (ALS) and frontotemporal dementia (FTD). Yet the molecular interactions that regulate its transition from reversible assemblies to aggregation-prone states remain poorly understood. CHCHD10 is a mitochondrial protein genetically and pathologically linked to TDP-43 dysfunction, but the molecular basis connecting both proteins has remained unclear. Here, we combine solution Nuclear Magnetic Resonance (NMR) spectroscopy, biophysical assays and cellular imaging to characterize the interaction between human CHCHD10 and the C-terminal region of TDP-43 (TDP-43CTD). CHCHD10 comprises a dynamic N-terminal region and a folded CHCH domain that samples a reversible monomer–dimer equilibrium. Reciprocal NMR titrations show that the CHCH domain binds the conserved hydrophobic helix of TDP-43CTD through a dynamic submicromolar interaction that overlaps with self-association surfaces in both proteins. CHCHD10 alters the formation of ThT-reactive TDP-43CTD assemblies and reduces TDP-43CTD sedimentation under selected stoichiometric conditions, while itself becoming enriched in the sedimentable fraction. Equilibrium calculations, independently reproduced using a complete numerical mass-balance solution, identify the initial non-homodimeric CHCHD10 population as the strongest predictor of its subsequent sedimentation. In cells, full-length CHCHD10 shows stronger CHCH-dependent spatial association with TDP-43 than a construct lacking the CHCH domain. These findings define a CHCH–helix interface that couples homo- and heterotypic assembly equilibria and support an asymmetric interface-buffering model in which CHCHD10 can divert TDP-43CTD from self-association while increasing its own availability for recruitment into sedimentable assemblies.
Karen S. Alarcón-Morales, Sara Pozo, Mauro Aguilera-Toste et al.· bioRxiv· 0 citations
The Huntingtin gene (HTT) contains a conserved, yet expandable CAG repeat within exon 1. While the pathogenic expansion in Huntington’s Disease (HD) is well studied, the role of surrounding domains remains unclear. Using genome-edited mini-organoids and neurons, we dissected HTT exon 1 and found species-specific toxicity: the human variant caused more severe deficits than the mouse. Swapping the proline-rich domain (PRD) - the most divergent region - revealed its key role: the mouse PRD mitigated, while the human PRD worsened neuronal phenotypes. Omics profiling showed that pathogenic human exon 1 induced broad protein dysregulation, largely reversed by mouse PRD replacement. Bioinformatics implicated the actin cytoskeleton and transcriptional coactivator MKL2/MRTFB. We validated MKL2/MRTFB dysregulation in HD models and showed that restoring its expression rescued neuronal abnormalities. These findings highlight the PRD’s contribution to HD toxicity and point to MKL2/MRTFB and the cytoskeleton as candidate mediators.
R. Iennaco, Camilla Maffezzini, Simone Maestri et al.· bioRxiv· 1 citation
Tetratricopeptide Repeat Domain 14 plays a crucial role in RNA metabolism during neurodevelopment, and that the p.His30Arg variant impairs its function, possibly leading to a neurodevelopmental disorder within the lissencephaly spectrum.
S. R. Ahmad, M. Zeyaullah, Mohammad Suhail Khan et al.· Human Genetics· 0 citations
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