The Heat Shock Protein B8 (HSPB8) is a chaperone which belongs to the small heat shock protein family. These proteins and the other chaperone members, together with the ubiquitin-proteasome system and the autophagic pathway, are involved in the proteostasis network. In particular, HSPB8 is an actor in a selective type of autophagy, the so called Chaperone Assisted Selective Autophagy (CASA). The CASA complex includes HSPB8, its partner the Bcl-2 associated athanogene 3 BAG3, the heat shock protein HSP70 and the E3 ubiquitin ligase CHIP. This complex was firstly described in muscle cells, where it plays a role in the maintenance of the Z-disc and, in neurons, it promotes the removal of misfolded proteins and protein aggregates. In fact, the overexpression of HSPB8 enhances the clearance of soluble and insoluble form of mutated SOD1, the C-terminal fragments of TDP-43, RAN translated dipeptide repeats, causative of Amyotrophic Lateral Sclerosis. Interestingly, mutations in HSPB8 or BAG3 are found in diseases that affect neurons and muscle cells. Concerning to HSPB8, a single aminoacid substitution (K141E/N) in HSPB8 can be causative of distal hereditary motor neuropathy type II and Charcot Marie Tooth disease type2L. Recently, Dr. Ticozzi N. and colleagues (Istituto Auxologico Italiano) have found two new mutations of HSPB8 in ALS patients, the mutations P41S and S181C. Our preliminary studies aimed to characterize the behaviour of these two new mutants compared to the wild type form. First, we analysed the protein levels and aggregation prone properties of the new mutants on western blot (WB) and filter retardation assay (FTA) in basal conditions and under protein quality control system impairment, using the motoneuronal cell model NSC-34. We also analysed the aggregation prone behaviour and cellular localization of HSPB8 mutants in immunofluorescence (IF). Our results show that there are no differences in insoluble and soluble levels between mutants and the wildtype form in FRA and SDS-PAGE. Conversely, in IF assay we found a different protein localization of mutants, with a predominant membrane localization for the P41S mutant and a predominant nuclear localization for S181C mutant, in respect to the wild type form which mainly localizes in the cytoplasm. Moreover, the S181C mutant is characterized by a different electrophoretic mobility in not reducing SDS-PAGE. WB analysis of autophagic markers p62, LC3-I and its cleaved form LC3-II reported no differences between mutants and the wild type form suggesting that the autophagic pathway is not affected by the mutants. HSPB8 mutants behave differently in respect to the wildtype counterpart in familiar and sporadic cell models of ALS. In addition, models overexpressing the mutated SOD1-G93A, the new mutants P41S and S181C show a detrimental effect in the clearance of mutated SOD1 insoluble species in FTA and IF. In a sporadic model of ALS overexpressing TDP-43, the mutant HSPB8 S181C has a negative impact in the removal of insoluble forms of the protein. All together, these data demonstrate that new HSPB8 mutations may have a detrimental effect or a loss of protective function in ALS disease.
Characterization of two new mutations in the small Heat Shock protein B8 HSPB8 and impact in Amyotrophic Lateral Sclerosis cell models / B. Tedesco, V. Crippa, P. Rusmini, R. Cristofani, M.E. Cicardi, V. Ferrari, G. Vezzoli, M. Meroni, E. Messi, M. Piccolella, R. Galbiati, A. Poletti. ((Intervento presentato al convegno New Perspectives in Neuroscience : Research Results of Young Italian Neuroscientist - National meeting of PhD students In Neuroscience tenutosi a Napoli nel 2018.
|Titolo:||Characterization of two new mutations in the small Heat Shock protein B8 HSPB8 and impact in Amyotrophic Lateral Sclerosis cell models|
|Data di pubblicazione:||23-feb-2018|
|Settore Scientifico Disciplinare:||Settore BIO/13 - Biologia Applicata|
Settore BIO/09 - Fisiologia
|Enti collegati al convegno:||Società Italiana di Neuroscienze|
|Citazione:||Characterization of two new mutations in the small Heat Shock protein B8 HSPB8 and impact in Amyotrophic Lateral Sclerosis cell models / B. Tedesco, V. Crippa, P. Rusmini, R. Cristofani, M.E. Cicardi, V. Ferrari, G. Vezzoli, M. Meroni, E. Messi, M. Piccolella, R. Galbiati, A. Poletti. ((Intervento presentato al convegno New Perspectives in Neuroscience : Research Results of Young Italian Neuroscientist - National meeting of PhD students In Neuroscience tenutosi a Napoli nel 2018.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|