Quantitative Interaction Proteomics of Neurodegenerative Disease Proteins

Several proteins have been linked to neurodegenerative disorders (NDDs), but their molecular function is not completely understood. Here, we used quantitative interaction proteomics to identify binding partners of Amyloid beta precursor protein (APP) and Presenilin-1 (PSEN1) for Alzheimer's disease (AD), Huntingtin (HTT) for Huntington's disease, Parkin (PARK2) for Parkinson's disease, and Ataxin-1 (ATXN1) for spinocerebellar ataxia type 1. Our network reveals common signatures of protein degradation and misfolding and recapitulates known biology. Toxicity modifier screens and comparison to genome-wide association studies show that interaction partners are significantly linked to disease phenotypes in vivo. Direct comparison of wild-type proteins and disease-associated variants identified binders involved in pathogenesis, highlighting the value of differential interactome mapping. Finally, we show that the mitochondrial protein LRPPRC interacts preferentially with an early-onset AD variant of APP. This interaction appears to induce mitochondrial dysfunction, which is an early phenotype of AD.Peer reviewe

Mutant huntingtin impairs Ku70-mediated DNA repair

Mutant huntingtin prevents interaction of the DNA damage repair complex component Ku70 with damaged DNA, blocking repair of double-strand breaks

Detection of Alpha-Rod Protein Repeats Using a Neural Network and Application to Huntingtin

A growing number of solved protein structures display an elongated structural domain, denoted here as alpha-rod, composed of stacked pairs of anti-parallel alpha-helices. Alpha-rods are flexible and expose a large surface, which makes them suitable for protein interaction. Although most likely originating by tandem duplication of a two-helix unit, their detection using sequence similarity between repeats is poor. Here, we show that alpha-rod repeats can be detected using a neural network. The network detects more repeats than are identified by domain databases using multiple profiles, with a low level of false positives (<10%). We identify alpha-rod repeats in approximately 0.4% of proteins in eukaryotic genomes. We then investigate the results for all human proteins, identifying alpha-rod repeats for the first time in six protein families, including proteins STAG1-3, SERAC1, and PSMD1-2 & 5. We also characterize a short version of these repeats in eight protein families of Archaeal, Bacterial, and Fungal species. Finally, we demonstrate the utility of these predictions in directing experimental work to demarcate three alpha-rods in huntingtin, a protein mutated in Huntington's disease. Using yeast two hybrid analysis and an immunoprecipitation technique, we show that the huntingtin fragments containing alpha-rods associate with each other. This is the first definition of domains in huntingtin and the first validation of predicted interactions between fragments of huntingtin, which sets up directions toward functional characterization of this protein. An implementation of the repeat detection algorithm is available as a Web server with a simple graphical output: http://www.ogic.ca/projects/ard. This can be further visualized using BiasViz, a graphic tool for representation of multiple sequence alignments

The ARFRP1-dependent Golgi scaffolding protein GOPC is required for insulin secretion from pancreatic β-cells

OBJECTIVE: Hormone secretion from metabolically active tissues, such as pancreatic islets, is governed by specific and highly regulated signaling pathways. Defects in insulin secretion are among the major causes of diabetes. The molecular mechanisms underlying regulated insulin secretion are, however, not yet completely understood. In this work, we studied the role of the GTPase ARFRP1 on insulin secretion from pancreatic β-cells. METHODS: A β-cell-specific Arfrp1 knockout mouse was phenotypically characterized. Pulldown experiments and mass spectrometry analysis were employed to screen for new ARFRP1-interacting proteins. Co-immunoprecipitation assays as well as super-resolution microscopy were applied for validation. RESULTS: The GTPase ARFRP1 interacts with the Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). Both proteins are co-localized at the trans-Golgi network and regulate the first and second phase of insulin secretion by controlling the plasma membrane localization of the SNARE protein SNAP25. Downregulation of both GOPC and ARFRP1 in Min6 cells interferes with the plasma membrane localization of SNAP25 and enhances its degradation, thereby impairing glucose-stimulated insulin release from β-cells. In turn, overexpression of SNAP25 as well as GOPC restores insulin secretion in islets from β-cell-specific Arfrp1 knockout mice. CONCLUSION: Our results identify a hitherto unrecognized pathway required for insulin secretion at the level of trans-Golgi sorting

Involvement of insulin-like growth factor-I in inner ear organogenesis and regeneration

The verterbrate inner ear is an excellent model system to study signalling mechanisms in embryonic development. During the last years, insulin-like growth factor-I (IGF-I) has attracted attention in relation to the regulation of inner ear ontogenesis. IGF-I and its high-affinity tyrosine-kinase receptor are expressed during early stages of inner ear development. IGF-I is a powerful mitogen for the otic vesicle, where it stimulates cell-division and mitogenic signalling cascades. Later in development, IGF-I also promotes survival and neurogenesis of the otic neurones in the cochleovestibular ganglion (CVG). The actions of IGF-I are associated with the generation of lipidic messengers and the activation of Raf kinase, which results in the rapid induction of the expression of the proliferative celt nuclear antigen (PCNA) and the nuclear proto-oncogenes c- fos and c-jun. Regulation of organogenesis involves a dynamic balance of the mechanisms regulating cell division, differentiation and death. A model is proposed where this balance is the consequence of the action of IGF-I and NGF, which converge in Raf activation or suppression. The combinatorial expression of Jun and Fos family members in particular domains of the otic vesicle would be the final result of such cascade. Some of these mechanisms may be also implicated in otic regeneration.Peer Reviewe