Motoneuron diseases (MNDs), such as Amyotrophic Lateral Sclerosis (ALS) or Spinal and Bulbar Muscular Atrophy (SBMA), are caused by mutations or aberrant behaviour of proteins characterized by a misfolded conformation. Misfolded proteins alter proteostasis by impairing the protein degradative systems, and accumulate into insoluble species and aggregates in motoneurons (MN). Proteins are degraded by autophagy and the ubiquitin proteasome system (UPS), which belong to the protein quality control (PQC) system. Other components of the PQC system are chaperones, proteins that recognize misfolded or unfolded substrates and refold or route them to degradation. The small Heat Shock Protein B8 (HSPB8) is a chaperone highly expressed in MN and muscles of patients affected by MNDs and mutation in HSPB8 are associated with Charcot-Marie-Tooth disease type 2L and distal Hereditary Motor Neuropathy type IIA. The overexpression of HSPB8 is associated with an increased clearance of proteins involved in ALS and SBMA, such as SOD1-G93A, the N- and the C-terminal fragments of TDP-43 and mutated androgen receptor (ARpolyQ). The hypothesized molecular mechanism consists in the recognition of misfolded substrate by HSPB8, followed by the interaction with its partner BAG3 in a oligomeric complex with HSP70 and the E3 ubiquitin ligase CHIP, that permits the ubiquitination and routing of the misfolded substrate to autophagosomes. In this way, HSPB8 acts as an autophagy facilitator and its induction could be protective in MNDs, by restoring proteostasis. Recently, new HSPB8 mutants (P41S and S181C) have been found in two familial ALS patients by Dr. Nicola Ticozzi and his team (Istituto Auxologico Italiano). Our results show that these new mutants are not aggregate-prone, as supported by immunofluorescence (IF), western blotting (WB) and filter retardation assays (FRA), but differently localize in respect to HSPB8 WT. In fact, immunofluorescence assay show that HSPB8 P41S mainly localizes in the plasmatic membrane similar to HSPB8 K141E mutant, while HSPB8 S181C prefers a nuclear localization. Moreover, HSPB8 WT and K141E are mainly degraded via UPS, while P41S and S181C mutants are mainly degraded by the autophagic machinery. The mutants do not alter expression and behaviour of the main autophagic markers even when the PQC system is impaired. In ALS cell models overexpressing SOD1 G93A, P41S and S181C mutants show a detrimental effect on mutant SOD1 aggregation and turnover, while the prodegradative and antiaggregating role of HSPB8 WT and the partial loss of function of HSPB8 K141E are confirmed. The detrimental role of HSPB8 S181C is confirmed in a sporadic ALS model, obtained by overexpression of TDP-43, while P41S mutant show a slight, but not significant decrease in the antiaggregating and prodegradative activity. These data proved that new mutants of HSPB8 are characterized by a different behaviour in respect to the WT form, affecting the clearance of misfolded proteins involved in MNDs.

Effects of mutations of the pro-autophagic protein Heat Shock Protein B8 in motoneuron disease / 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 2. convegno IFOM Symposium : Advanced Lecture Course : Ubiquitin-assisted autophagy from mechanisms to pathology tenutosi a Milano nel 2017.

Effects of mutations of the pro-autophagic protein Heat Shock Protein B8 in motoneuron disease

tedesco barbara;crippa valeria;rusmini paola;cristofani riccardo;cicardi maria elena;ferrari veronica;meroni marco;messi elio;piccolella margherita;galbiati rita;poletti angelo
2017-10

Abstract

Motoneuron diseases (MNDs), such as Amyotrophic Lateral Sclerosis (ALS) or Spinal and Bulbar Muscular Atrophy (SBMA), are caused by mutations or aberrant behaviour of proteins characterized by a misfolded conformation. Misfolded proteins alter proteostasis by impairing the protein degradative systems, and accumulate into insoluble species and aggregates in motoneurons (MN). Proteins are degraded by autophagy and the ubiquitin proteasome system (UPS), which belong to the protein quality control (PQC) system. Other components of the PQC system are chaperones, proteins that recognize misfolded or unfolded substrates and refold or route them to degradation. The small Heat Shock Protein B8 (HSPB8) is a chaperone highly expressed in MN and muscles of patients affected by MNDs and mutation in HSPB8 are associated with Charcot-Marie-Tooth disease type 2L and distal Hereditary Motor Neuropathy type IIA. The overexpression of HSPB8 is associated with an increased clearance of proteins involved in ALS and SBMA, such as SOD1-G93A, the N- and the C-terminal fragments of TDP-43 and mutated androgen receptor (ARpolyQ). The hypothesized molecular mechanism consists in the recognition of misfolded substrate by HSPB8, followed by the interaction with its partner BAG3 in a oligomeric complex with HSP70 and the E3 ubiquitin ligase CHIP, that permits the ubiquitination and routing of the misfolded substrate to autophagosomes. In this way, HSPB8 acts as an autophagy facilitator and its induction could be protective in MNDs, by restoring proteostasis. Recently, new HSPB8 mutants (P41S and S181C) have been found in two familial ALS patients by Dr. Nicola Ticozzi and his team (Istituto Auxologico Italiano). Our results show that these new mutants are not aggregate-prone, as supported by immunofluorescence (IF), western blotting (WB) and filter retardation assays (FRA), but differently localize in respect to HSPB8 WT. In fact, immunofluorescence assay show that HSPB8 P41S mainly localizes in the plasmatic membrane similar to HSPB8 K141E mutant, while HSPB8 S181C prefers a nuclear localization. Moreover, HSPB8 WT and K141E are mainly degraded via UPS, while P41S and S181C mutants are mainly degraded by the autophagic machinery. The mutants do not alter expression and behaviour of the main autophagic markers even when the PQC system is impaired. In ALS cell models overexpressing SOD1 G93A, P41S and S181C mutants show a detrimental effect on mutant SOD1 aggregation and turnover, while the prodegradative and antiaggregating role of HSPB8 WT and the partial loss of function of HSPB8 K141E are confirmed. The detrimental role of HSPB8 S181C is confirmed in a sporadic ALS model, obtained by overexpression of TDP-43, while P41S mutant show a slight, but not significant decrease in the antiaggregating and prodegradative activity. These data proved that new mutants of HSPB8 are characterized by a different behaviour in respect to the WT form, affecting the clearance of misfolded proteins involved in MNDs.
Settore BIO/13 - Biologia Applicata
Settore BIO/09 - Fisiologia
Istituto FIRC di Oncologia Molecolare (IFOM)
Fondazione Italiana per la Ricerca sul Cancro-FIRC
Effects of mutations of the pro-autophagic protein Heat Shock Protein B8 in motoneuron disease / 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 2. convegno IFOM Symposium : Advanced Lecture Course : Ubiquitin-assisted autophagy from mechanisms to pathology tenutosi a Milano nel 2017.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/813962
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