Protein transport and secretion

Roberto Sitia, Head of Unit

Over the last decade, the lab focused on two tightly related problems, the pathophysiology of B to plasma cell development and redox regulation/signaling in the early secretory pathway. Important traits d’union between the two are ERp44, a multifunctional chaperone we discovered a dozen years ago (Anelli et al., 2002, 2003 EMBO J), the assembly and quality control of polymeric IgM and the biogenesis and homeostatic control of the endoplasmic reticulum and secretory organelles.

Research activities (updated 2015)

B to plasma cell differentiation
Small, metabolically lazy, B lymphocytes can be induced to differentiate into plasma cells, real professionals in antibody secretion. In the process, B cells undergo an impressive architectural and functional metamorphosis: the ER enlarges, hundreds of new proteins replace old ones, cell division ensues, and after a few days of intense Ig production cells die byapoptosis (van Anken et al., 2003 Immunity). Much to our surprise, we noticed that a >50 fold increase in Ig synthesis was matched by a decrease in proteasome abundance (Cenci et al., 2006, EMBO J). My training as a haematologist came into play, as it had become clear that proteasome inhibitors are exquisitely active against multiple myeloma, the tumour of plasma cells. Wesurmised and eventually demonstrated that the unfavourable ratio between load (protein synthesis) and capacity (proteasomal activity) generates proteotoxic signal(s) that limit the lifespan of normal and neoplastic plasma cell (Bianchi et al., 2009, Blood: Cascio et al., 2012, J Leuk Biol). As a connection with the redox aficionados in the lab, inhibitors of glutathione synthesis synergized with proteasome inhibitors (Nerini et al., 2008, Br. J. Haematol.) and plasma cell differentiation entails profound reshaping of the antioxidant defences (Venè et al., 2010, Bertolotti et al. 2010, 2012; Antiox. Redox Sign.). Notably, also autophagy plays key roles in the survival of normal and neoplastic plasma cells (Pengo et al., 2013, Nature Immunology). Plasma cells in which autophagy had been genetically disabled display an enlarged ER and shorter half-life. These findings raise interesting perspectives in the handling of myeloma and systemic amyloidoses (Sitia et al, 2007; Haematologica) as well as endoplasmic reticulum storage disorders (ERSD) (Anelli and Sitia, 2010, Semin Dev. Cell. Biol.).
If cells produce more Ig that they can eliminate (via secretion and/or degradation), dilated cisternae of the early secretory compartment (ESC) are formed where transport-incompetent proteins are condensed, without eliciting obvious ER stress responses. We have developedinducible systems to follow the biogenesis and clearance of Russell bodies (Mattioli et al., 2006, J.Cell Sci; Ronzoni et al., 2010, Traffic). Particularly exciting are recent data in which we could image the growth ofprotein deposits in ESC by virtue of suitably tagged mutant Ig-µ chains (Mossuto et al, 2014, PLosOne) providing a powerful model to dissect the pathogenesis of ERSD. We have also developed metabolomics technologies to describe normal B to plasma cell differentiation (Garcia-Manteiga et al., 2011. J Proteom. Res) and to follow/predict the progression of MGUS to multiple myeloma (Fontana et al, submitted). The latter study offers remarkable clinical applications.

Redox regulation and signalling
Having discovered and characterized Ero1 and ERp44, two key regulators of the ER redox state (Mezghrani et al., 2001, Anelli et al., 2002, 2003 EMBO J), we collaborated with structural biology labs to solve their structures (Wang et al., 2008, EMBO Rep; Inaba et al., 2010; EMBO J). Ero1 flavoproteins promote disulphide bond formation oxidising PDI. Since O2 is the terminal electron acceptor, H2O2 is generated in equimolar amounts to the disulphides formed (Wang et al., 2014, Antiox. Redox Signal.). To limit excessive oxidation and stress, Ero1 forms inhibitory disulfide bonds between conserved cysteines that inhibit interactions with PDI (Masui et al., 2011. J Biol. Chem.). We are also analysing the relationships between Ig biogenesis and redox regulation, H2O2 production in particular, during B to plasma cell differentiation. The work of the Finkel, Rhee, Toledano, Tonks and other labs demonstrated that H2O2 acts as a key secong messenger, inibiting protein tyrosine phosphatases. An exctiting finding is that efficient H2O2 entry into living cells requires protein transporters, including aquaporin 8 (Bertolotti et al, 2010, 2012, 2013 Antiox. Redox Signal). Notably, AQP8 is strongly induced during plasma cell differentiation, and is essential for robust B cell receptor signaling. B cells silenced for AQP8 secrete less IgM upon LPS stimulation, underscoring the importance of redox signalling (Bertolotti et al., in preparation). ERp44 is a multifunctional chaperone involved in thiol-dependent protein quality control and regulating the activity and localization of Ero1 (Anelli et al., 2002, 2003; 2007; EMBO J; 2012; Otsu et al., 2006 Antiox. Redox Signal.). An exciting discovery was that ERp44 is regulated by pH. At neutral pH, as in the ER, the ERp44 carboxy-terminal tail hinders the substrate-binding site and RDEL motif.
At the lower pH of the Golgi, the tail rearranges to simultaneously unmask both the active site and RDEL, allowing the capture of client proteins and their retrieval via KDEL receptors. Acting downstream of calnexin and BiP, the ERp44 cycle allows mammalian cells to couple secretion fidelity and efficiency (Vavassori et al., 2013 Mol. Cell). Recently, we discovered a key role for conserved histidines in regulating ERp44 activity and transport. Moreover, we showed that O-glycosylated ERp44 is differential secreted by endometrial cells during the menstrual cycle, implying unexpected extracellular functions for this multitask ESC resident (Sannino et al., 2014 J. Cell Sci.).

Ongoing Projects

  1. Physiology of post-ER quality control
    A fundamental question in cell biology is how compartmental homeostasis is maintained and adapted during differentiation or environmental changes. Professional secretory cells develop the endoplasmic reticulum (ER) and downstream organelles of the exocytic pathway to sustain massive proteostatic challenges and guarantee secretion efficiency and fidelity. Suitable checkpoint systems are needed along the secretory route to adapt flux and further refine ER production. We have recently uncovered a post-ER quality control system that patrols complex oligomeric proteins, in which ERp44 is pivotal. ERp44 is a multitask sentinel protein that also senses secretory fluxes regulating KDEL receptor-, redox- and Ca2+- dependent signalling at the ER-Golgi interface. Moreover, it is selectively secreted during endometrial cell differentiation. Different cells, particularly under stress conditions, may release other ER-resident enzymes. These findings raise the tantalising possibility that ERp44 and some of its interactors (e.g. Ero1 oxidases) act as transcellular signals in physiological and pathological processes such as differentiation and inflammation.
    Merging continuity and innovation, we will investigate the mechanisms and regulation of post-ER quality control/signalling at the molecular, cellular and organismal levels, ERp44 being our port of entry. How can some proteins, designed to reside in the early secretory compartment, be secreted? What are the systemic consequences of their selective release? Can we manipulate these pathways to improve production of man-made proteins and alleviate diseases caused by altered proteostasis or chronic inflammation? We will address these questions exploiting robust in vitro and in vivo cellular and animal models, and provide mechanistic information on the pathophysiology of intracellular and transcellular secretory proteostasis, with wide implications in biotechnology and medicine.
  2. Redox homeostasis and signaling in and between cells
    Reactive oxygen species (ROS), particularly H2O2, play both positive and negative roles in cell proliferation and survival. This dual nature is exploited by cancer cells to promote growth and genomic instability. Repeated shifts towards oxidising conditions can cause stress and promote proinflammatory responses, that may favor tumor progression. Since strongeroxidising shifts can trigger apoptosis, cancer cells enhance anti-oxidant defenses, increasing their resistance and adaptive capabilities. In addition, H2O2 can act as an intercellular signaling molecule promoting migration, a hallmark of tumour progression. Stimulation of cell surface receptors generates H2O2, which causes reversible cysteine modifications. These molecular switches are controlled by an elaborate enzyme network, recalling protein (de)phosphorylation. H2O2 transiently inactivates PTEN and other phosphatases by sulfenylation, thus amplifying kinase signaling. Hence, the generation, transport and diffusion of H2O2 are key issues, as the enzymes and transporters involved may become suitable targets against cancer.
    Over the last couple of years, we have developed a wide panel of ratiometric H2O2 sensors targeted into different organelles. These reagents allowed us to analyse H2O2 fluxes into and inside living cells. Ourdata indicate that H2O2 transport across the plasma membrane requires aquaporin 8 (AQP8) and possibly other protein channels. In fact, AQP8 silencing severely inhibits EGF-induced intracellular H2O2 spikes and downstream tyrosine phosphorylation. We have also observed that certain cellular stress conditions reversibly inhibit AQP8-mediated H2O2 entry into cells, impacting signaling.
    We surmise that (dis)regulation of H2O2 transport in cells could be a hallmark of, and potentially a target, in cancer. Modulating redox signaling may open novel possibilities of intervention to control inflammation and cancer cell migration and apoptosis.Therefore, we are aiming to:
    1. Identify AQP8 interactors involved in H2O2 transport and study their regulation in normal and neoplastic cells
    2. Characterize stress-induced AQP8 modification(s) and the effector pathways involved
    3. Analyze the pathophysiological role of AQP8 and other transporters in intra- and inter-cellular redox signaling

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