Atrophin-1 Function and Dysfunction in Dentatorubral-Pallidoluysian Atrophy.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a rare, incurable genetic disease that belongs to the group of polyglutamine (polyQ) diseases. DRPLA is the most common in the Japanese population; however, its global prevalence is also increasing due to better clinical recognition. It is characterized by cerebellar ataxia, myoclonus, epilepsy, dementia, and chorea. DRPLA is caused by dynamic mutation of CAG repeat expansion in ATN1 gene encoding the atrophin-1 protein. In the cascade of molecular disturbances, the pathological form of atrophin-1 is the initial factor, which has not been precisely characterized so far. Reports indicate that DRPLA is associated with disrupted protein-protein interactions (in which an expanded polyQ tract plays a crucial role), as well as gene expression deregulation. There is a great need to design efficient therapy that would address the underlying neurodegenerative process and thus prevent or alleviate DRPLA symptoms. An in-depth understanding of the normal atrophin-1 function and mutant atrophin-1 dysfunction is crucial for this purpose. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Fragment-based exploration of the 14-3-3/Amot-p130 interface.

The modulation of protein-protein interactions (PPIs) has developed into a well-established field of drug discovery. Despite the advances achieved in the field, many PPIs are still deemed as 'undruggable' targets and the design of PPIs stabilizers remains a significant challenge. The application of fragment-based methods for the identification of drug leads and to evaluate the 'tractability' of the desired protein target has seen a remarkable development in recent years. In this study, we explore the molecular characteristics of the 14-3-3/Amot-p130 PPI and the conceptual possibility of targeting this interface using X-ray crystallography fragment-based screening. We report the first structural elucidation of the 14-3-3 binding motif of Amot-p130 and the characterization of the binding mode and affinities involved. We made use of fragments to probe the 'ligandability' of the 14-3-3/Amot-p130 composite binding pocket. Here we disclose initial hits with promising stabilizing activity and an early-stage selectivity toward the Amot-p130 motifs over other representatives 14-3-3 partners. Our findings highlight the potential of using fragments to characterize and explore proteins' surfaces and might provide a starting point toward the development of small molecules capable of acting as molecular glues.

1H, 13C, and 15N resonance assignments of the C-terminal lobe of the human HECT E3 ubiquitin ligase ITCH.

ITCH (aka Atrophin-1-interacting protein 4) is a prominent member of the NEDD4 HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family that regulates numerous cellular functions including inflammatory responses through T-cell activation, cell differentiation, and apoptosis. Known intracellular targets of ITCH-dependent ubiquitylation include receptor proteins, signaling molecules, and transcription factors. The HECT C-terminal lobe of ITCH contains the conserved catalytic cysteine required for the covalent attachment of ubiquitin onto a substrate and polyubiquitin chain assembly. We report here the complete experimentally determined 1H, 13C, and 15N backbone and sidechain resonance assignments for the HECT C-terminal lobe of ITCH (residues 784-903) using heteronuclear, multidimensional NMR spectroscopy. These resonance assignments will be used in future NMR-based studies to examine the role of dynamics and conformational flexibility in HECT-dependent ubiquitylation as well as deciphering the structural and biochemical basis for polyubiquitin chain synthesis and specificity by ITCH.

Stall in Canonical Autophagy-Lysosome Pathways Prompts Nucleophagy-Based Nuclear Breakdown in Neurodegeneration.

The terminal stages of neuronal degeneration and death in neurodegenerative diseases remain elusive. Autophagy is an essential catabolic process frequently failing in neurodegeneration. Selective autophagy routes have recently emerged, including nucleophagy, defined as degradation of nuclear components by autophagy. Here, we show that, in a mouse model for the polyglutamine disease dentatorubral-pallidoluysian atrophy (DRPLA), progressive acquirement of an ataxic phenotype is linked to severe cerebellar cellular pathology, characterized by nuclear degeneration through nucleophagy-based LaminB1 degradation and excretion. We find that canonical autophagy is stalled in DRPLA mice and in human fibroblasts from patients of DRPLA. This is evidenced by accumulation of p62 and downregulation of LC3-I/II conversion as well as reduced Tfeb expression. Chronic autophagy blockage in several conditions, including DRPLA and Vici syndrome, an early-onset autolysosomal pathology, leads to the activation of alternative clearance pathways including Golgi membrane-associated and nucleophagy-based LaminB1 degradation and excretion. The combination of these alternative pathways and canonical autophagy blockade, results in dramatic nuclear pathology with disruption of the nuclear organization, bringing about terminal cell atrophy and degeneration. Thus, our findings identify a novel progressive mechanism for the terminal phases of neuronal cell degeneration and death in human neurodegenerative diseases and provide a link between autophagy block, activation of alternative pathways for degradation, and excretion of cellular components.

Studying polyglutamine diseases in Drosophila.

Polyglutamine (polyQ) diseases are a family of dominantly transmitted neurodegenerative disorders caused by an abnormal expansion of CAG trinucleotide repeats in the protein-coding regions of the respective disease-causing genes. Despite their simple genetic basis, the etiology of these diseases is far from clear. Over the past two decades, Drosophila has proven to be successful in modeling this family of neurodegenerative disorders, including the faithful recapitulation of pathological features such as polyQ length-dependent formation of protein aggregates and progressive neuronal degeneration. Additionally, it has been valuable in probing the pathogenic mechanisms, in identifying and evaluating disease modifiers, and in helping elucidate the normal functions of disease-causing genes. Knowledge learned from this simple invertebrate organism has had a large impact on our understanding of these devastating brain diseases.

Increased aggregation of polyleucine compared with that of polyglutamine in dentatorubral-pallidoluysian atrophy protein.

Polyglutamine (polyQ) diseases result from expansion of CAG trinucleotide repeats in their responsible genes. Although gene products with polyQ expansions undergo conformational changes to aggregate in neurons, the relationship between inclusions and neurotoxicity remains unclear. Dentatorubral-pallidoluysian atrophy (DRPLA) is a polyQ disease, and DRPLA protein, also known as atrophin-1 (ATN1), carries an expanded polyQ tract. To investigate how an expanded polyQ tract influences ATN1 aggregation and localization, we compared the aggregation of ATN1 with a polyQ tract to that of ATN1 with a polyleucine (polyL) tract. In COS-7 cells, polyL-ATN1 triggered more aggregation than polyQ-ATN1 of similar repeat sizes. Immunocytochemical and biochemical studies revealed that replacement of the polyQ tract with polyL alters ATN1 localization, leading to retention of polyL-ATN1 in the cytoplasm. Despite this change in localization, polyL-ATN1 and polyQ-ATN1 demonstrate comparable repeat length dependent toxicity. These results suggest that expanded polyQ repeats in ATN1 may contribute to neurodegeneration via alterations in both protein aggregation and intracellular localization.

Pathological accumulation of atrophin-1 in dentatorubralpallidoluysian atrophy.

Dentatorubral-pallidoluysian atrophy (DRPLA) is caused by the expansion of polyglutamine (polyQ) in atrophin-1 (ATN1), also known as DRPLA protein. ATN1 is ubiquitously expressed in the central nervous system (CNS), although selective regions of CNS are degenerated in DRPLA, and this selective neuronal damage gives rise to the specific clinical features of DRPLA. Accumulation of mutant ATN1 that carries an expanded polyQ tract seems to be the primary cause of DRPLA neurodegeneration, but it is still unclear how the accumulation of ATN1 leads to neu-rodegeneration. Recently, cleaved fragments of ATN1 were shown to accumulate in the disease models and the brain tissues of patients with DRPLA. Furthermore, proteolytic processing of ATN1 may regulate the intracellular localization of ATN1 and its fragments. Therefore, proteolytic processing of ATN1 may provide clues to disease pathogenesis and hopefully aid in the determination of molecular targets for effective therapeutic approaches for DRPLA.