PhD Student, University of Alberta
To investigate processes at the MAM to unveil the negative effect of Rab32 in breast cancer
Our data indicate breast cancer patients with high levels of Rab32 have a more aggressive cancer and a statistically significantly lower survival rate. Furthermore, Rab32 is upregulated in almost 25% of breast cancers, making it a relevant potential biomarker for this disease. Therefore, we are investigating how Rab32 mediates this negative prognostic effect in breast cancer. Specifically, my project focuses on investigating how Rab32 regulates the Mitochondria-associated membrane (MAM): a subdomain of the endoplasmic reticulum in close contact with mitochondria. The MAM is a critical cell fate signalling hub since numerous essential processes that directly affect cell death and survival occur there, including apoptosis (programmed cell death), autophagy (degradation of cellular components) and mitochondrial metabolism and dynamics (production of ATP and mitochondrial fission and fusion), to name a few. Unsurprisingly then, the MAM has also been previously linked to cancer. Our lab has identified the GTPase Rab32 as a MAM-enriched protein involved in several of these processes, including calcium signalling, apoptosis onset, mitochondrial dynamics, and autophagy. Our findings demonstrate Rab32 promotes autophagy and leads to the degradation of apoptotic regulators of the Bcl-2 family such as the pro-apoptotic member Bim. We have also shown Rab32 relocalizes Bim, the mitochondrial regulator dynamin-related protein 1 (Drp1), and the autophagy-related protein Atg3 to the MAM, where they undergo different fates. Whereas Bim is targeted for autophagic degradation, Drp1 evades proteosomal degradation, enabling it to promote mitochondrial fission. Furthermore, we have shown Atg3 is a novel autophagy-related binding partner through which Rab32 participates in autophagy. In brief, our work has provided insights into Rab32’s role in regulating cell fate processes occurring at the MAM, including apoptosis and autophagy, in order to understand its negative effects in breast cancer and to lay a foundation for its potential use as a biomarker for this disease.
Abstract: Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed to regulate the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. The same as other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and GTP-bound active form, and the cycle is thought to be regulated by an activating enzyme GEF (guanine nucleotide exchange factor) and an inactivating enzyme GAP (GTPase-activating protein), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has never been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes, and we discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicated that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes.
Pub.: 30 Nov '15, Pinned: 29 Aug '17
Abstract: Rab small GTPases, well-known regulators of membrane trafficking pathways in eukaryotic cells, comprise approximately 60 different members in mammals. During the past decade, our understanding of the functions of mammalian Rab32 subfamily members (Rab32 and Rab38) have deepened, especially on the biogenesis of lysosome-related organelles, such as melanosomes, and the protection mechanisms against several pathogenic microbial infections. Endosome-mediated membrane trafficking by Rab32 subfamily members plays pivotal roles in these events. In this review, we provide an overview of the regulatory mechanisms of mammalian Rab32-family members in endosomal trafficking, especially focusing on their GEF, GAP, and effector molecules, and describe the latest findings on physiological and pathological functions regulated by these molecules.
Pub.: 22 Apr '17, Pinned: 29 Aug '17
Abstract: The flux of Ca(2+) from the endoplasmic reticulum (ER) to mitochondria regulates mitochondria metabolism. Within tumor tissue, mitochondria metabolism is frequently repressed, leading to chemotherapy resistance and increased growth of the tumor mass. Therefore, altered ER-mitochondria Ca(2+) flux could be a cancer hallmark, but only a few regulatory proteins of this mechanism are currently known. One candidate is the redox-sensitive oxidoreductase TMX1 that is enriched on the mitochondria-associated membrane (MAM), the site of ER-mitochondria Ca(2+) flux. Our findings demonstrate that cancer cells with low TMX1 exhibit increased ER Ca(2+), accelerated cytosolic Ca(2+) clearance, and reduced Ca(2+) transfer to mitochondria. Thus, low levels of TMX1 reduce ER-mitochondria contacts, shift bioenergetics away from mitochondria, and accelerate tumor growth. For its role in intracellular ER-mitochondria Ca(2+) flux, TMX1 requires its thioredoxin motif and palmitoylation to target to the MAM. As a thiol-based tumor suppressor, TMX1 increases mitochondrial ATP production and apoptosis progression.
Pub.: 10 Aug '16, Pinned: 29 Aug '17
Abstract: Whereas most of what we know today about the Ras-related small GTPases of the Rab family stems from observations made on Golgi complex, endosome and plasma membrane trafficking, a subset of Rabs localizes in part or predominantly to the ER (endoplasmic reticulum). Here, Rabs such as Rab1, Rab2, Rab6 and Rab33 can regulate the anterograde and retrograde trafficking of vesicles between the Golgi complex, the ERGIC (ER-Golgi intermediate compartment) and the ER itself. However, among the ER-associated Rabs, some Rabs appear to perform roles not directly related to trafficking: these Rabs (e.g. Rab32 or Rab24) could aid proteins of the atlastin and reticulon families in determining the extent and direction of ER tubulation. In so doing, these Rabs regulate not only ER contacts with other organelles such as mitochondria, but also the formation of autophagosomes.
Pub.: 28 Nov '12, Pinned: 29 Aug '17
Abstract: More than a billion years ago, bacterial precursors of mitochondria became endosymbionts in what we call eukaryotic cells today. The true significance of the word "endosymbiont" has only become clear to cell biologists with the discovery that the endoplasmic reticulum (ER) superorganelle dedicates a special domain for the metabolic interaction with mitochondria. This domain, identified in all eukaryotic cell systems from yeast to man and called the mitochondria-associated membrane (MAM), has a distinct proteome, specific tethers on the cytosolic face and regulatory proteins in the ER lumen of the ER. The MAM has distinct biochemical properties and appears as ER tubules closely apposed to mitochondria on electron micrographs. The functions of the MAM range from lipid metabolism and calcium signaling to inflammasome formation. Consistent with these functions, the MAM is enriched in lipid metabolism enzymes and calcium handling proteins. During cellular stress situations, like an altered cellular redox state, the MAM alters its set of regulatory proteins and thus alters MAM functions. Notably, this set prominently comprises ER chaperones and oxidoreductases that connect protein synthesis and folding inside the ER to mitochondrial metabolism. Moreover, ER membranes associated with mitochondria also accommodate parts of the machinery that determines mitochondrial membrane dynamics and connect mitochondria to the cytoskeleton. Together, these exciting findings demonstrate that the physiological interactions between the ER and mitochondria are so bilateral that we are tempted to compare their relationship to the one of a married couple: distinct, but inseparable and certainly dependent on each other. In this paradigm, the MAM stands for the intracellular location where the two organelles tie the knot. Resembling "real life", the happy marriage between the two organelles prevents the onset of diseases that are characterized by disrupted metabolism and decreased lifespan, including neurodegeneration and cancer. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.
Pub.: 12 May '12, Pinned: 29 Aug '17
Abstract: The mitochondria-associated membrane (MAM) is an endoplasmic reticulum (ER) domain that forms contacts with mitochondria and accommodates Ca(2+) transfer between the two organelles. The GTPase Rab32 regulates this function of the MAM via determining the localization of the Ca(2+) regulatory transmembrane protein calnexin to the MAM. Another function of the MAM is the regulation of mitochondrial dynamics mediated by GTPases such as dynamin-related protein 1 (Drp1). Consistent with the importance of the MAM for mitochondrial dynamics and the role of Rab32 in MAM enrichment, the inactivation of Rab32 leads to mitochondrial collapse around the nucleus. However, Rab32 and related Rabs also perform intracellular functions at locations other than the MAM including melanosomal trafficking, autophagosome formation and maturation, and retrograde trafficking to the trans-Golgi network (TGN). This plethora of functions raises questions concerning the original cellular role of Rab32 in the last common ancestor of animals and its possible role in the last eukaryotic common ancestor (LECA). Our results now shed light on this conundrum and identify a role in Drp1-mediated mitochondrial dynamics as one common denominator of this group of Rabs, which includes the paralogues Rab32A and Rab32B, as well as the more recently derived Rab29 and Rab38 proteins. Moreover, we provide evidence that this mitochondrial function is dictated by the extent of ER-association of Rab32 family proteins.
Pub.: 15 Mar '15, Pinned: 29 Aug '17
Abstract: The past decade has seen dramatic progress in our understanding of membrane contact sites (MCS). Important examples of these are endoplasmic reticulum (ER)-mitochondria contact sites. ER-mitochondria contacts have originally been discovered in mammalian tissue, where they have been designated as mitochondria-associated membranes (MAMs). It is also in this model system, where the first critical MAM proteins have been identified, including MAM tethering regulators such as phospho-furin acidic cluster sorting protein 2 (PACS-2) and mitofusin-2. However, the past decade has seen the discovery of the MAM also in the powerful yeast model system Saccharomyces cerevisiae. This has led to the discovery of novel MAM tethers such as the yeast ER-mitochondria encounter structure (ERMES), absent in the mammalian system, but whose regulators Gem1 and Lam6 are conserved. While MAMs, sometimes referred to as mitochondria-ER contacts (MERCs), regulate lipid metabolism, Ca(2+) signaling, bioenergetics, inflammation, autophagy and apoptosis, not all of these functions exist in both systems or operate differently. This biological difference has led to puzzling discrepancies on findings obtained in yeast or mammalian cells at the moment. Our review aims to shed some light onto mechanistic differences between yeast and mammalian MAM and their underlying causes.This article was reviewed by Paola Pizzo (nominated by Luca Pellegrini), Maya Schuldiner and György Szabadkai (nominated by Luca Pellegrini).
Pub.: 27 Jan '17, Pinned: 29 Aug '17
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