Functional Characterization of Mutations in STUB1, a Protein Involved in the Protein Turnover System, in Patients with Autosomal Recessive Cerebellar Ataxia

Abstract

Abstract

Autosomal recessive cerebellar ataxias (ARCA) are a group of rare monogenic diseases with a polygenic ethology, characterized mainly by the neurodegeneration of the cerebellum that causes the ataxia phenotype. Ataxias as well as many other neurodegenerative diseases are suspected to be caused by alterations in the protein homeostasis that evoke protein accumulation in cells leading to toxicity and cell death.

Using whole exome sequencing in one family, with three siblings affected with ARCA, we identified one homozygous mutation in STUB1 (STIP-1 homology and U-box containing protein) as novel cause of their disorder. Hereinafter, a second family with one affected patient was included in the study: a compound heterozygous with two novel mutations in STUB1. STUB1 encodes CHIP (C-terminus of Hsc70 Interacting Protein), a co-chaperone and an E3-Ubiquitin Ligase that links protein folding and proteasome mediated degradation, binding to heat shock proteins and ubiquitinating their misfolded client proteins to send them to degradation avoiding their aggregation.

In the present study, we functionally characterized the p.Asn65Ser (CHIP-N65S) mutation identified in patient 1 (family 1) and the p.Glu28Lys (CHIP-E28K) mutation identified in patient 2 (family 2), both which are located in the binding domain important for chaperone interactions. The second mutation seen in patient 2 is predicted to lead to a premature stop –codon (CHIP-K144*). We compared their properties with a mutation that was published during our work as the cause of the Gordon-Holmes syndrome - a syndrome characterized by ARCA and hypogonadotropic hypogonadism. We expressed all four CHIP variants as recombinant proteins and performed an in vitro ubiquitination assay to analyse their ability to ubiquitinate Hsc70. The results show that mutated CHIP variants have an impaired capacity to ubiquitinate proteins. Immunoblotting using CHIP specific antibody on extracts from patient primary fibroblast cultures showed reduced protein steady states for all mutated variants. The CHIP-N65S mutant protein from patient 1 was further analysed in a co-immunoprecipitation assay to analyse the binding with Hsc70, as well as an analysis of the conformational state given that it migrates at a different rate in comparison with the wild type protein as seen in SDS-PAGE.

This impaired function of CHIP is suspected to cause aberrant protein aggregation that causes toxicity and cell death, where the neurodegeneration of the cerebellar areas would turn into the ataxia phenotype in the patients.

Abstract

Popular science summary:

Recessive mutations in STUB1 cause cerebellar ataxia

Autosomal recessive cerebellar ataxias (ARCA) constitute a group of rare disorders characterized by ataxia due to cerebellum neurodegeneration. Here we characterize three novel mutations in the STUB1 (STIP1 homology and U-box containing protein) gene as the cause of ARCA in two families. STUB1 encodes CHIP (C-terminus of Hsc70 interacting protein), a protein involved in the protein turnover system of the cell. Aggregation of misfolded proteins seems to cause toxicity and cell death that leads to neurodegeneration of the cerebellum, causing gait impairment.

Many neurodegenerative diseases have protein aggregation as a common feature that leads to
toxicity and cell death. CHIP functions as a link between protein folding and proteasomal mediated degradation. It binds to heat shock proteins (chaperones), such as Hsc70 (Heat shock cognate 70), that are important for stabilising proteins. When CHIP binds Hsc70, it normally adds small molecules called ubiquitin (ubiqutination) to the client proteins of Hsc70.This process facilitates the degradation of misfolded proteins via proteasome (Figure 1). Thus, we hypothesise that impaired CHIP could lead to toxic protein aggregation.

In this study, we present two ARCA families with recessive mutations in the STUB1 gene: one family with a homozygous mutation (CHIP-N65S) and a second family with two different mutations (CHIP-144* and CHIP-E28K, compound heterozygous). We expressed the predicted proteins and analysed their ubiquitination activity to characterize the function of CHIP in the patients. Furthermore, in the first family we performed studies to reveal whether the mutation caused a weaker binding with Hsc70.

Fibroblasts from patients in the two families revealed lower CHIP steady states in comparison with normal fibroblasts. In vitro ubiquitination assay showed impaired function in CHIPN65S from family one (probably due to weaker binding with heat shock proteins) and a complete loss of function of CHIP-144* from family two (encoding a truncated protein), while CHIP-E28K from family two presented similar activity as the wild type protein. These results show that the phenotype of the patients is likely caused by impaired CHIP function: a combination of the inability to ubiquitinate and the lower steady states of the protein in vivo. A consequence could be that some misfolded proteins are less efficiently degraded and begin to form protein aggregates. If these protein aggregates are toxic to the cell they could cause neurodegeneration in the cerebellum, explaining the observed ataxia in these patients.

Advisor/s: Stefan Johansson, Per Knappskog
Master´s Degree Project 60 credits in Molecular Genetics as an Erasmus program exchange at the University of Bergen,
Norway 2013/2014
Department of Biology, Lund University

• author: Sánchez Guixé, Mònica
• supervisor: Stefan Johansson
• organization: Degree Projects in Molecular Biology
• course: MOBT19 20132
• year: 2014
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 4451006
• date added to LUP: 2014-05-27 14:44:17
• date last changed: 2014-05-27 14:44:17