HAP40 protein levels are huntingtin-dependent and decrease in Huntington disease.

The huntingtin-associated protein 40 (HAP40) is an abundant interactor of huntingtin (HTT). In complexes of these proteins, HAP40 tightly binds to HTT in a cleft formed by two larger domains rich in HEAT repeats, and a smaller bridge domain connecting the two. We show that HAP40 steady-state protein levels are directly dependent on HTT (both normal and mutant HTT) and that HAP40 is strongly stabilized by the interaction with HTT resulting in an at least 5-fold increase in HAP40's half-life when bound to HTT. Cellular HAP40 protein levels were reduced in primary fibroblasts and lymphoblasts of Huntington Disease (HD) patients and in brain tissue of a full-length HTT mouse model of HD, concomitant with decreased soluble HTT levels in these cell types. This data and our previous demonstration of coevolution between HTT and HAP40 and evolutionary conservation of their interaction suggest that HAP40 is an obligate interaction partner of HTT. Our observation of reduced HAP40 levels in HD invites further studies, whether HAP40 loss-of-function contributes to the pathophysiology of HD.

Mammalian ataxin-2 modulates translation control at the pre-initiation complex via PI3K/mTOR and is induced by starvation.

Ataxin-2 is a cytoplasmic protein, product of the ATXN2 gene, whose deficiency leads to obesity, while its gain-of-function leads to neural atrophy. Ataxin-2 affects RNA homeostasis, but its effects are unclear. Here, immunofluorescence analysis suggested that ataxin-2 associates with 48S pre-initiation components at stress granules in neurons and mouse embryonic fibroblasts, but is not essential for stress granule formation. Coimmunoprecipitation analysis showed associations of ataxin-2 with initiation factors, which were concentrated at monosome fractions of polysome gradients like ataxin-2, unlike its known interactor PABP. Mouse embryonic fibroblasts lacking ataxin-2 showed increased phosphorylation of translation modulators 4E-BP1 and ribosomal protein S6 through the PI3K-mTOR pathways. Indeed, human neuroblastoma cells after trophic deprivation showed a strong induction of ATXN2 transcript via mTOR inhibition. Our results support the notion that ataxin-2 is a nutritional stress-inducible modulator of mRNA translation at the pre-initiation complex.

Linear and extended: a common polyglutamine conformation recognized by the three antibodies MW1, 1C2 and 3B5H10.

A long-standing pathomechanistic model proposes that the polyglutamine (polyQ)-length-dependent toxicity threshold observed in all polyQ diseases is triggered by a conformational change within the monomer that occurs only above a certain polyQ length. If true, this yet undefined and elusive mutant-specific toxic conformation would constitute a direct therapeutic target. Three anti-polyQ antibodies-MW1, 1C2 and 3B5H10-have been extensively used to probe the conformation of polyQ. The crystal structure of the MW1 epitope reveals a linear, non-pathogenic polyQ. In contrast, although the detailed structure of its epitope is unknown, the 3B5H10 antibody is widely advertised and used as a conformational antibody that recognizes the toxic conformation of expanded polyQ. We solved the crystal structure of the 1C2 antigen-binding domain (1C2-Fab) and performed a direct comparison between the 1C2, MW1 and 3B5H10 structures. The MW1 and 1C2 antibodies have similar sequences and structures, consistent with their binding to short polyQ and their polyQ length-discrimination properties. Unexpectedly, the 3B5H10 antibody also shares striking features with MW1 and 1C2, which prompted us to revisit its binding properties. We show that the 3B5H10 epitope is actually a short, non-pathogenic polyQ. All three antibodies MW1, 1C2 and 3B5H10 interact similarly with polyQ of various lengths, and bind small polyQ epitopes in similar linear and extended conformations. Together with studies published during the recent years, our work argues against the hypothesis that a mutant-specific conformation in monomeric polyQ molecules is the toxic entity responsible for polyQ diseases.

The tumour suppressor gene WWOX is mutated in autosomal recessive cerebellar ataxia with epilepsy and mental retardation.

We previously localized a new form of recessive ataxia with generalized tonic-clonic epilepsy and mental retardation to a 19 Mb interval in 16q21-q23 by homozygosity mapping of a large consanguineous Saudi Arabian family. We now report the identification by whole exome sequencing of the missense mutation changing proline 47 into threonine in the first WW domain of the WW domain containing oxidoreductase gene, WWOX, located in the linkage interval. Proline 47 is a highly conserved residue that is part of the WW motif consensus sequence and is part of the hydrophobic core that stabilizes the WW fold. We demonstrate that proline 47 is a key amino acid essential for maintaining the WWOX protein fully functional, with its mutation into a threonine resulting in a loss of peptide interaction for the first WW domain. We also identified another highly conserved homozygous WWOX mutation changing glycine 372 to arginine in a second consanguineous family. The phenotype closely resembled the index family, presenting with generalized tonic-clonic epilepsy, mental retardation and ataxia, but also included prominent upper motor neuron disease. Moreover, we observed that the short-lived Wwox knock-out mouse display spontaneous and audiogenic seizures, a phenotype previously observed in the spontaneous Wwox mutant rat presenting with ataxia and epilepsy, indicating that homozygous WWOX mutations in different species causes cerebellar ataxia associated with epilepsy.

ProteinCoLoc streamlines Bayesian analysis of colocalization in microscopic images.

Colocalization, the spatial overlap of molecular entities, is often key to support their involvement in common functions. Existing colocalization tools, however, face limitations, particularly because of their basic statistical analysis and their low-throughput manual entry processes making them unsuitable for automation and potentially introducing bias. These shortcomings underscore the need for user-friendly tools streamlining colocalization assessments and enabling their robust and automated quantitative analyses. We have developed ProteinCoLoc, an innovative software designed for automated high-throughput colocalization analyses and incorporating advanced statistical features such as Bayesian modelling, automatic background detection and localised correlation analysis. ProteinCoLoc rationalises colocalization assessments without manual input, comes with a user-friendly graphical user interface and provides various analytics allowing to study and locally quantify colocalization. This easy-to-use application presents numerous advantages, including a direct comparison with controls employing a Bayesian model and the analysis of local correlation patterns, while reducing hands-on time through automatic background detection. The software was validated while studying the colocalization pattern of two proteins forming a stable complex: the huntingtin protein (HTT) and its partner huntingtin-associated protein 40 (HAP40). Our results showcase the software's capacity to quantitatively assess colocalizations. ProteinCoLoc is available both as a Julia package and as a compiled software ( https://github.com/ma-seefelder/ProteinCoLoc ).

Huntingtin affinity for partners is not changed by polyglutamine length: aggregation itself triggers aberrant interactions.

Huntington's disease (HD) is caused by the expansion mutation above a length threshold of a polyglutamine (polyQ) stretch in the huntingtin (Htt) protein. Mutant Htt (mHtt) pathogenicity is proposed to rely on its malfunction and propensity to misfold and aggregate. Htt has scaffolding properties and has been reported to interact with hundreds of partners. Many interactors show apparent increased or decreased affinity (dysinteraction) for mHtt, which may account for selective malfunctions and striatal degeneration in HD. These dysinteractions are proposed to result from mutant polyQ conformational changes that remain elusive. To date, dysinteractions have only been studied using semi-quantitative techniques with their outcome potentially influenced by the presence of mHtt aggregates. Therefore, the molecular mechanism underlying these dysinteractions remains to be determined. Here, we have used purified proteins devoid of aggregates to quantify the interaction of normal and mHtt with two partners: SH3GL3, reported to have increased binding to mHtt, and the 2B4 antibody, a model partner. Using surface plasmon resonance and pull-down techniques, we show that in the absence of aggregation polyQ length has no effect on Htt interactions. We demonstrate that the presence of aggregates affects the spatial distribution and solubility of Htt partners and strongly influences the outcome of pull-down experiments. Our results show that expanded polyQ per se does not alter Htt interactions and suggest that aggregated mHtt form molecular platforms that influence the Htt interacting network. Modulating mHtt aggregation could thus have beneficial effects on specific cellular pathways deregulated in HD.

Heterologous DNA-prime/protein-boost immunization with a monomeric SARS-CoV-2 spike antigen redundantizes the trimeric receptor-binding domain structure to induce neutralizing antibodies in old mice.

A multitude of alterations in the old immune system impair its functional integrity. Closely related, older individuals show, for example, a reduced responsiveness to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines. However, systematic strategies to specifically improve the efficacy of vaccines in the old are missing or limited to simple approaches like increasing the antigen concentration or injection frequencies. We here asked whether the intrinsic, trimeric structure of the SARS-CoV-2 spike (S) antigen and/or a DNA- or protein-based antigen delivery platform affects priming of functional antibody responses particularly in old mice. The used S-antigens were primarily defined by the presence/absence of the membrane-anchoring TM domain and the closely interlinked formation/non-formation of a trimeric structure of the receptor binding domain (S-RBD). Among others, we generated vectors expressing prefusion-stabilized, cell-associated (TM+) trimeric "S2-P" or secreted (TM-) monomeric "S6-PΔTM" antigens. These proteins were produced from vector-transfected HEK-293T cells under mild conditions by Strep-tag purification, revealing that cell-associated but not secreted S proteins tightly bound Hsp73 and Grp78 chaperones. We showed that both, TM-deficient S6-PΔTM and full-length S2-P antigens elicited very similar S-RBD-specific antibody titers and pseudovirus neutralization activities in young (2-3 months) mice through homologous DNA-prime/DNA-boost or protein-prime/protein-boost vaccination. The trimeric S2-P antigen induced high S-RBD-specific antibody responses in old (23-24 months) mice through DNA-prime/DNA-boost vaccination. Unexpectedly, the monomeric S6-PΔTM antigen induced very low S-RBD-specific antibody titers in old mice through homologous DNA-prime/DNA-boost or protein-prime/protein-boost vaccination. However, old mice efficiently elicited an S-RBD-specific antibody response after heterologous DNA-prime/protein-boost immunization with the S6-PΔTM antigen, and antibody titers even reached similar levels and neutralizing activities as in young mice and also cross-reacted with different S-variants of concern. The old immune system thus distinguished between trimeric and monomeric S protein conformations: it remained antigen responsive to the trimeric S2-P antigen, and a simple change in the vaccine delivery regimen was sufficient to unleash its reactivity to the monomeric S6-PΔTM antigen. This clearly shows that both the antigen structure and the delivery platform are crucial to efficiently prime humoral immune responses in old mice and might be relevant for designing "age-adapted" vaccine strategies.

Mutation spectrum in the large GTPase dynamin 2, and genotype-phenotype correlation in autosomal dominant centronuclear myopathy.

Centronuclear myopathy (CNM) is a genetically heterogeneous disorder associated with general skeletal muscle weakness, type I fiber predominance and atrophy, and abnormally centralized nuclei. Autosomal dominant CNM is due to mutations in the large GTPase dynamin 2 (DNM2), a mechanochemical enzyme regulating cytoskeleton and membrane trafficking in cells. To date, 40 families with CNM-related DNM2 mutations have been described, and here we report 60 additional families encompassing a broad genotypic and phenotypic spectrum. In total, 18 different mutations are reported in 100 families and our cohort harbors nine known and four new mutations, including the first splice-site mutation. Genotype-phenotype correlation hypotheses are drawn from the published and new data, and allow an efficient screening strategy for molecular diagnosis. In addition to CNM, dissimilar DNM2 mutations are associated with Charcot-Marie-Tooth (CMT) peripheral neuropathy (CMTD1B and CMT2M), suggesting a tissue-specific impact of the mutations. In this study, we discuss the possible clinical overlap of CNM and CMT, and the biological significance of the respective mutations based on the known functions of dynamin 2 and its protein structure. Defects in membrane trafficking due to DNM2 mutations potentially represent a common pathological mechanism in CNM and CMT.

Huntingtin and Its Partner Huntingtin-Associated Protein 40: Structural and Functional Considerations in Health and Disease.

Since the discovery of the mutation causing Huntington's disease (HD) in 1993, it has been debated whether an expanded polyglutamine (polyQ) stretch affects the properties of the huntingtin (HTT) protein and thus contributes to the pathological mechanisms responsible for HD. Here we review the current knowledge about the structure of HTT, alone (apo-HTT) or in a complex with Huntingtin-Associated Protein 40 (HAP40), the influence of polyQ-length variation on apo-HTT and the HTT-HAP40 complex, and the biology of HAP40. Phylogenetic analyses suggest that HAP40 performs essential functions. Highlighting the relevance of its interaction with HTT, HAP40 is one of the most abundant partners copurifying with HTT and is rapidly degraded, when HTT levels are reduced. As the levels of both proteins decrease during disease progression, HAP40 could also be a biomarker for HD. Whether declining HAP40 levels contribute to disease etiology is an open question. Structural studies have shown that the conformation of apo-HTT is less constrained but resembles that adopted in the HTT-HAP40 complex, which is exceptionally stable because of extensive interactions between HAP40 and the three domains of HTT. The complex- and to some extent apo-HTT- resists fragmentation after limited proteolysis. Unresolved regions of apo-HTT, constituting about 25% of the protein, are the main sites of post-translational modifications and likely have major regulatory functions. PolyQ elongation does not substantially alter the structure of HTT, alone or when associated with HAP40. Particularly, polyQ above the disease length threshold does not induce drastic conformational changes in full-length HTT. Therefore, models of HD pathogenesis stating that polyQ expansion drastically alters HTT properties should be reconsidered.

Pathogenic and non-pathogenic polyglutamine tracts have similar structural properties: towards a length-dependent toxicity gradient.

Abnormally expanded polyglutamine (polyQ) tracts provide a gain of toxic functions to nine otherwise unrelated human proteins and induce progressive neurodegenerative diseases. Over the past ten years, it was suggested that only polyQ tracts longer than a specific threshold adopt a particular structure, which would be the cause of the apparent polyQ length-dependent toxicity threshold observed in polyQ diseases. We have used a combination of biochemical and biophysical approaches to compare the structural properties of polyQ of pathogenic and non-pathogenic lengths under various conditions. We observe that pathogenic and non-pathogenic polyQ, as soluble species and upon interaction with a partner, during aggregation, or as mature aggregates, display very similar structural properties. PolyQ length only influences the aggregation kinetics and, to a lesser extent, the stability of the aggregates. We thus propose that polyQ toxicity does not depend on a structural transition occurring above a specific threshold, but rather that polyQ tracts are inherently toxic sequences, whose deleterious effect gradually increases with their length. We discuss how polyQ properties and other cellular factors may explain the existence of an apparent polyQ length-dependent toxicity threshold.

SynAggreg: A Multifunctional High-Throughput Technology for Precision Study of Amyloid Aggregation and Systematic Discovery of Synergistic Inhibitor Compounds.

Numerous proteins can coalesce into amyloid self-assemblies, which are responsible for a class of diseases called amyloidoses, but which can also fulfill important biological functions and are of great interest for biotechnology. Amyloid aggregation is a complex multi-step process, poorly prone to detailed structural studies. Therefore, small molecules interacting with amyloids are often used as tools to probe the amyloid aggregation pathway and in some cases to treat amyloidoses as they prevent pathogenic protein aggregation. Here, we report on SynAggreg, an in vitro high-throughput (HT) platform dedicated to the precision study of amyloid aggregation and the effect of modulator compounds. SynAggreg relies on an accurate bi-fluorescent amyloid-tracer readout that overcomes some limitations of existing HT methods. It allows addressing diverse aspects of aggregation modulation that are critical for pathomechanistic studies, such as the specificity of compounds toward various amyloids and their effects on aggregation kinetics, as well as the co-assembly propensity of distinct amyloids and the influence of prion-like seeding on self-assembly. Furthermore, SynAggreg is the first HT technology that integrates tailored methodology to systematically identify synergistic compound combinations-an emerging strategy to improve fatal amyloidoses by targeting multiple steps of the aggregation pathway. To this end, we apply analytical combinatorial scores to rank the inhibition efficiency of couples of compounds and to readily detect synergism. Finally, the SynAggreg platform should be suited for the characterization of a broad class of amyloids, whether of interest for drug development purposes, for fundamental research on amyloid functions, or for biotechnological applications.

Structure of a single-chain Fv bound to the 17 N-terminal residues of huntingtin provides insights into pathogenic amyloid formation and suppression.

Huntington's disease is triggered by misfolding of fragments of mutant forms of the huntingtin protein (mHTT) with aberrant polyglutamine expansions. The C4 single-chain Fv antibody (scFv) binds to the first 17 residues of huntingtin [HTT(1-17)] and generates substantial protection against multiple phenotypic pathologies in situ and in vivo. We show in this paper that C4 scFv inhibits amyloid formation by exon1 fragments of huntingtin in vitro and elucidate the structural basis for this inhibition and protection by determining the crystal structure of the complex of C4 scFv and HTT(1-17). The peptide binds with residues 3-11 forming an amphipathic helix that makes contact with the antibody fragment in such a way that the hydrophobic face of this helix is shielded from the solvent. Residues 12-17 of the peptide are in an extended conformation and interact with the same region of another C4 scFv:HTT(1-17) complex in the asymmetric unit, resulting in a ß-sheet interface within a dimeric C4 scFv:HTT(1-17) complex. The nature of this scFv-peptide complex was further explored in solution by high-resolution NMR and physicochemical analysis of species in solution. The results provide insights into the manner in which C4 scFv inhibits the aggregation of HTT, and hence into its therapeutic potential, and suggests a structural basis for the initial interactions that underlie the formation of disease-associated amyloid fibrils by HTT.

Biochemical and NMR mapping of the interface between CREB-binding protein and ligand binding domains of nuclear receptor: beyond the LXXLL motif.

CBP, cAMP-response element-binding protein (CREB)-binding protein, plays an important role as a general cointegrator of various signaling pathways and interacts with a large number of transcription factors. Interactions of CBP with ligand binding domains (LBDs) of nuclear receptors are mediated by LXXLL motifs, as are those of p160 proteins, although the number, distribution, and precise sequences of the motifs differ. We used a large N-terminal fragment of murine CBP to map by biochemical methods and NMR spectroscopy the interaction domain of CBP with the LBDs of several nuclear receptors. We show that distinct zones of that fragment are involved in the interactions: a 20-residue segment containing the LXXLL motif (residues 61-80) is implicated in the interaction with all three domains tested (peroxisome proliferator-activated receptor gamma-LBD, retinoid X receptor alpha-LBD, and estrogen-related receptor gamma-LBD), whereas a second N-terminal well conserved block of around 25 residues centered on a consensus L(40)PDEL(44) motif constitutes a secondary motif of interaction with peroxisome proliferator-activated receptor gamma-LBD. Sequence analysis reveals that both zones are well conserved in all vertebrate p300/CBP proteins, suggesting their functional importance. Interactions of p300/CBP coactivators with the LBDs of nuclear receptors are not limited to the canonical LXXLL motifs, involving both a longer contiguous segment around the motif and, for certain domains, an additional zone.

Ataxin-7 is a subunit of GCN5 histone acetyltransferase-containing complexes.

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder caused by a CAG repeat expansion in the SCA7 gene leading to elongation of a polyglutamine tract in ataxin-7, a protein of unknown function. A putative ataxin-7 yeast orthologue (SGF73) has been identified recently as a new component of the SAGA (Spt/Ada/Gcn5 acetylase) multisubunit complex, a coactivator required for transcription of a subset of RNA polymerase II-dependent genes. We show here that ataxin-7 is an integral component of the mammalian SAGA-like complexes, the TATA-binding protein-free TAF-containing complex (TFTC) and the SPT3/TAF9/GCN5 acetyltransferase complex (STAGA). In agreement, immunoprecipitation of ataxin-7 retained a histone acetyltransferase activity, characteristic for TFTC-like complexes. We further identified a minimal domain in ataxin-7 that is required for interaction with TFTC/STAGA subunits and is conserved highly through evolution, allowing the identification of a SCA7 gene family. We showed that this domain contains a conserved Cys(3)His motif that binds zinc, forming a new zinc-binding domain. Finally, polyglutamine expansion in ataxin-7 did not affect its incorporation into TFTC/STAGA complexes purified from SCA7 patient cells. We demonstrate here that ataxin-7 is the human orthologue of the yeast SAGA SGF73 subunit and is a bona fide subunit of the human TFTC-like transcriptional complexes.