a carregar...

UC.PT

IBILI

Protein quality control in health and disease

Henrique Girão, Group Leader

Objectives

Many of the degenerative and age-related diseases are associated with endothelial dysfunction and in many cases with abnormal angiogeneis (neovascularization), such as diabetes retinopahty, heart ischemia and cancer. Proliferation of endothelial cells and the formation of new blood vessels are, in part, regulated by the transcription factor Hypoxia Inducible Factor (HIF). One of our major objectives is to understand the molecular mechanisms and signalling events that may interfere with proteasome-dependent degradation of HIF-1α, leading to abnormal retinal vessel proliferation. For example we have provided evidence to shown that inactivation of critical components of the ubiquitin-proteasome pathway in retinal pigmented epithelial cells (RPE) may contribute for cellular and organ alterations associated with AMD, including the neovascular component (wet form of AMD) and the inflammatory component.

It has been suggested that aging affects the cells ability to protect protein integrity, which is essential for cellular homeostasis, maintained by a complex system of refolding or degradation, such as ubiquitin-proteasome pathway (UPP) and lysosome pathway (autophagy). The carboxyl terminus of Hsp70-interacting protein (CHIP) interacts with Hsp70 and Hsp90 via three tandem tetratricopeptide repeat motifs, whereas it’s carboxyl-terminal U-box domain associates with ubiquitin-conjugating enzymes. A major objective in this area is to elucidate the role of CHIP in the protein quality control in a number of diseases such as cataract, using an animal model that overexpresses CHIP in the lens epithelial cells, examine the lens transparency and correlate with changes in lens proteins and degradation markers.

AMD is also characterized by the accumulation of subretinal deposits and drusens. Even though the association between proteasomal disruption and accumulation of drusen-like deposits is not obvious, it was recently suggested that an age-dependent increase in the autophagic flux, as a result of proteasome inhibition, would overload the lysosomal system and subsequently lead to accumulation of subcellular deposits containing unprocessed material in the form of drusens. We are currently using an animal model of AMD, with impaired protein degradation through the proteasome, as a tool to investigate the biological determinants of age-related proteolytic stress and its impact on proteostasis in the retina and presumably in other tissues affected by age-related diseases.

It is now widely accepted that ubiquitin-conjugation not only helps destroy damaged proteins in the proteasome but is also involved in downregulation and lysosomal degradation of membrane proteins. One of the objectives of this proposal is to study the role of ubiquitin in the regulation of internalization and intra- cellular trafficking of membrane proteins. We are particulary interested in elucidating the mechanisms and molecular players involved in the degradation of the gap junction protein (GJ) Cx43. GJ are specialized cell–cell contacts that provide direct intercellular communication (IC) between eukaryotic cells, playing a vital role in regulating cell homeostasis, proliferation and differentiation and maintain homogeneous tissue function. GJIC can be regulated at various levels, including the number of channels localized at the plasma membrane, being ubiquitin-dependent internalization and degradation of GJ one of the mechanisms involved in this regulation. Defects in GJIC are often associated with disease, such as cataract, diabetic retinopathy, neurological disorders, cancer and cardiomyopathies. A major objective of our group is to investigate how Cx43 degradation may contribute to cancer and heart diseases, in particular how ubiquitin signal determines the degradation pathway followed by Cx43, either through the endocytic or the autophagic pathway.

    Achievements

    • A major objective of our research is to understand the molecular mechanisms and signalling events that may interfere with regulation of cell response to hypoxia leading vascular dysfunction. Our studies identified a novel molecular mechanism involved in HIF-1α proteasomal degradation in diabetes, that involves the recruitment of molecular chaperones and the E3 ligase CHIP, even under hypoxia (Diabetologia 2011;PlosOne 2010; PlosOne 2010; Exp Physiol 2010). Furthermore, we demonstrated that CHIP-mediated ubiquitination of HIF-1α can also signal forchaperone-mediated autophagy (CMA), through a mechanism that depends on the chaperone Hsc70 (minor revision Dev Cell). This makes HIF-1α good molecular model to evaluate a putative crosstalk between UPS and CMA, and CHIP a good candidate in the shuttling of HIF-1α between both pathways.
    • To evaluate the implications of age-related decline in proteasome activity, we have generated an animal model of AMD, with impaired proteasomal degradation in the RPE. First data showed that inhibition of the proteasome recapitulates some of the main features of AMD, including angiogenesis and accumulation of subretinal deposits, that is likely caused by the increased load of the lysosome with non-degraded proteasome substrates. This animal might constitute a privileged model to investigate the putative crosstalk between proteasomal andlysossomal degradation.
    • Changes in gap junction (GJ) intercellular communication induced, for example, by deregulation of connexin degradation mechanisms, are often associated to cancer, cardiopathies and neurological diseases. We showed that incorporation of ubiquitinated Cx43 into GJ induces the internalization and degradation of the channels through a mechanism that requires the recruitment of Esp15, and is independent of the canonical endocytic Tyr-sorting signal present in Cx43 (Biochem J 2011). We further demonstrated that GJ plaques are degraded by autophagy, through a mechanism that is regulated by Cx43 ubiquitination. Importantly, we show that the endocytic adaptor Eps15 is required for Cx43degradation by autophagy, thus ascribing, for the first time, a role for an endocytic adaptor in signalling degradation by autophagy (Mol Biol Cell, 2012).

    Group Members

    Email

    Américo Figueiredo

    PhD - Investigator

    Email

    Carla Marques
    PhD - Investigator

    Email

    Filipe Silva 

    PhD - Investigator

    Email

    Paulo Pereira
    PhD - Investigator

    Email

    Lino Gonçalves
    PhD - Investigator

    Email

    João Ferreira
    PhD - Investigator

    Email

    Rui Travasso
    PhD - Investigator

    Email

    Steve Catarino
    PhD - Investigator

    Email

    Ana Pinheiro
    MSc - Student

    Email

    Filipa Marques
    PhD - Student

    Email

    Inês Mahu
    MSc - Student

    Email

    Hugo Fôfo
    Phd - Student

    João Carlos Ribeiro MD - PhD Student
    Rui Batista MD - PhD Student
    Email

    Sara Ramos MD - PhD Student

    Email

    Teresa Rodrigues
    MSc - Student

    Email

    Ana Paula Vieira

    MSc - Research Fellow

    Email

    Ana Soares
    MSc - Research Fellow

    Email

    Tânia Marques

    BSc - Research Fellow