Suresh Subramani - US grants

Project Summary This study is designed to develop novel sensors for mammalian Autophagy-related protein 8 (mAtg8). Autophagy is an evolutionarily conserved, lysosomal, degradation pathway in which various cytoplasmic components are selectively or non-selectively sequestered by double-membrane vesicle called autophagosomes and transported into lysosomes. This process requires a unique set of proteins called autophagy-related (Atg) proteins, such as Atg8, which play central roles in forming the autophagosomal membrane and recruiting cargo receptors. Malfunction of autophagy has been linked to a wide range of human pathologies, including cancer, different neurodegenerative diseases, immunological disorders, aging, diabetes and heart diseases. Before improvements are enabled for the treatment for diseases caused by autophagy malfunction, extensive efforts must be dedicated to develop robust and reliable methods to monitor, analyze and understand this process. This research proposal will develop original molecular sensors, which recognize Atg8-positive puncta, surpassing current limitations of similar tools available and providing insight into the functions of mAtg8s. The basis of the proposed approach is to engineer short peptides that can detect endogenous mAtg8 proteins without using an Atg8 interaction motif (AIM) or its equivalent, the LC3 interaction region (LIR). In preliminary work, a novel sensor archetype that binds mAtg8s in an AIM/LIR-independent manner has already been generated by us and shown to co-localize with one of the members of mAtg8-protein family. The specific aims of the proposed work are: (1) to modify and optimize the molecular structure architecture of the sensor archetype followed by the verification of accuracy of the subcellular localization in vivo of the newly generated sensors; and (2) to evaluate the effects of sensor(s) generated in Aim 1 on the autophagy pathway from autophagosome formation to its maturation and degradation. Successful completion of the objectives of this research will result in the development of a novel type of molecular sensor for mAtg8-positive puncta (such as autophagosomes) and the function of mAtg8s. The possibility of using an AIM/LIR-independent fluorescent probe for labeling of mAtg8s will open up an exciting opportunity to monitor populations of endogenous mAtg8s in vivo in a time-dependent manner and without having the sensor compete for the binding of important AIM/LIR-containing proteins. Future research efforts will focus on using the sensors for detecting where endogenous mAtg8s reside in addition to autophagosomal membranes, identifying the uncharacterized protein partners of mAtg8s that bind through their AIM/LIR motif at these cellular locations, and developing a peptide-based fluorescent probe for mAtg8 proteins.

Peroxisomes are essential subcellular organelles that play crucial roles in the oxidation of fatty-acids and homeostasis of glutathione, as well as reactive oxygen and nitrogen species (ROS and RNS, respectively). They play critical roles in the regulation of intracellular redox states and antiviral signaling, as well as cellular differentiation and metabolism, and their impairment causes many debilitating, and often fatal, human peroxisome biogenesis disorders (PBDs). Their biogenesis is orchestrated by 36 PEX genes, encoding peroxins, involved in the biogenesis of peroxisomal membrane and matrix proteins, as well as in the control of organelle size, number and inheritance. Their biogenesis has been studied in many organisms from yeast to plants and mammals, and more than 15 peroxins and their modes of action are conserved from yeast to man. While much has been learned about the biogenesis of peroxisomal matrix and membrane proteins to pre-existing peroxisomes, far less is known about how this organelle is generated de novo from other endogenous membranes. Such an ability to generate new peroxisomes de novo is obviously relevant under conditions where peroxisome biogenesis is impaired (e.g. human PBDs), or under conditions of stress (e.g. ROS) when peroxisomes are turned over by autophagy (pexophagy). Indeed, any disorders associated with imbalanced peroxisome homeostasis can be corrected, in principle, by manipulating either peroxisome biogenesis or its turnover, as we have shown. Such a global understanding of the mechanisms involved in peroxisome homeostasis is the long-term interest of my lab. Over almost 3 decades, we exploited the yeast, Pichia pastoris, to provide many major insights into our knowledge of peroxisome biogenesis and turnover. This proposal focuses on gleaning a deeper understanding of the proteins involved in the intra-ER sorting and budding of peroxisomal membrane proteins (PMPs) to a pre- peroxisomal exit site on the ER (pER) from where at least two type of pre-peroxisomal vesicles (ppVs) bud to ultimately generate peroxisomes, either by fusion with pre-existing peroxisomes or anew when peroxisomes are absent. Budding of ppVs is conserved between yeast and mammals and several proteins we will study have counterparts involved in human health. There are also reports of ppVs derived from mitochondria contributing to peroxisome biogenesis. Our approach is based on the use of novel genetic and biochemical strategies, including ppV purification and characterization, in vitro budding reactions and the use of innovative techniques to follow what these novel proteins do, where they act, who they interact with and how they function. The Aims are: Aim 1 - Isolation and characterization of the ATPase/s and other proteins involved in ppV budding. Aim 2 ? How do Pex25 and Pex36 act in stimulating intra-ER sorting and budding of Pex2 and other RING- domain peroxins? Aim 3 ? Does ppV budding occur from yeast mitochondria and what is its physiological relevance?!