Christopher Nicchitta - US grants

Protein translocation in the rough endoplasmic reticulum (RER) represents the point of entry of secretory and membrane protein precursors into the cellular protein trafficking pathways and comprises both the transfer of secretory precursors from the cytoplasm to the RER lumen and the assembly of membrane protein precursors into the RER bilayer. Protein translocation in the RER requires protein factors acting on the cis-, or cytoplasmic, and trans-, or lumenal, sides of the RER membrane. Cis-side factors target precursors to the RER membrane and maintain the protein in an unstructured state. The function of trans-side proteins in translocation has not been defined and is the focus of the proposed studies. In addition to our interest in translocation as an essential cellular process, recent studies on the assembly and sorting of membrane protein precursors indicate a pressing health interest in the elucidation of the mechanism of translocation. In the case of the visual pigment rhodopsin, for example, defects in assembly of various rhodopsin mutants in the RER have been demonstrated to result in autosomal dominant retinitis pigmentosa, a progressive blinding disease. The experimental analyses will exploit a well-characterized in vitro cell-free system which accurately mimics in vivo protein translocation. The role of lumenal proteins in translocation will be assessed using a series of reporter proteins; the secretory precursor, preprolactin, the type I integral membrane protein VSV-G (vesicular stomatitis viral glycoprotein), the type II integral membrane protein HNA (hemagglutinin/neuraminidase protein of Newcastle virus) and the polytopic membrane protein opsin, in experiments involving the depletion and biochemical reconstitution of wild-type and mutant forms of the lumenal proteins in isolated RER derived microsomal vesicles. Chemical cross-linking studies will be performed to identify associations of lumenal proteins with precursors at different stages of translocation. These studies will be elaborated in experiments designed to determine the mechanism of lumenal protein function and will include studies on the role of domain specific interactions between precursors and individual lumenal proteins. Such studies will allow characterization of the structural determinants of the precursor necessary for efficient translocation in the RER and further the mechanistic description of protein translocation.

The PI proposes to identify the functional, structural and regulatory properties of GRP94 which define it as a (poly) peptide binding protein. GRP94, the endoplasmic reticulum (ER) homolog of hsp90, is an abundant resident molecular chaperone of the ER lumen. In addition to its chaperone role, studies in animal carcinogenesis models have established that GRP94 can function as a tumor-specific vaccine. Vaccine activity is know to require the uptake of GRP94-peptide complexes by antigen-presenting cells, and the transfer of GRP94- bound peptides to nascent MHC class I molecules in the ER. The molecular signals governing the finding the release of the GRP94 associated peptides are currently unknown, but are certain to prove significant to the identification of the molecular mechanism of GRP94 function and to the development of GRP94 as an immunotherapy agent. To achieve this goal, the PI proposes 1) to define the kinetics and regulation of (poly) peptide binding to GRP94. To this end, the hypothesis that peptide binding by GRP94 is regulated by ribonucleotides will be tested and assays will be developed to identify potential regulatory contributions of resident ER integral and lumenal proteins to the GRP94 peptide binding and release reactions. 2) To perform structural analyses of GRP94 bound peptides and identify peptide binding motifs. Having developed procedures for the purification of native GRP94, a large scale, mass spectrometry based, bound peptide sequencing study will be initiated to test the hypothesis that GRP94 recognizes specific peptide structural motifs. 3) To identify the GRP94 peptide binding site(s) through techniques including chemical and photo-crosslinking, proteolytic domain structure studies, and in vitro binding studies with purified expression constructs and isolated structural domains. 4) To determine the role of GRP94 dimerization in the regulation of peptide binding activity and in vivo function. To test whether dimerization is necessary for function, mutations in the assembly domain that block dimerization will be assessed by in vitro studies of peptide binding and in vivo studies of protein assembly and secretion.

The investigator proposes a reexamination of current models and assumptions concerning the nature and role of the association of ribosomes and the nascent chain with the ER membrane.

GRP94, the endoplasmic reticulum Hsp90 chaperone, has been identified as a potent tumor rejection antigen, capable of eliciting vigorous immune responses against primary and distant metastases of its tumor of origin. The immunogenic activity of GRP94 is thought to derive from an as yet uncharacterized peptide binding activity. In eliciting an immune response, GRP94-peptide complexes are internalized by antigen presenting cells (APC) and the GRP94-bound peptides re-presented on the APC class I molecules for avtivation of peptide-specific cytotoxic T lymphocytes. The immunogenic properties of GRP94 are well established, and tumor-derived GRP94 is now in multi-center Phase III clinical trials as an immunotherapy for renal cell carcinoma. In addition to its activity as a tumor antigen, there now exists strong evidence that GRP94 functions as a protein second messenger in the communication of pathological cell death to the immune system. This conclusion extends from recent work demonstrating that GRP94 is released from cells in response to lethal viral infection and can subsequently be processed by APCs to elicit virus-specific immune responses. In pursuing research into the biological basis of GRP94 function, two primary long term objectives are proposed: 1) Define the structural and regulatory basis for GRP94's function as a peptide binding protein; 2) Define the mechanism of GRP94 uptake into, and subcellular fate within, antigen presenting cells. The proposed studies will draw on a variety of biophysical techniques, for the analysis of protein structure; biochemical techniques, to study peptide-GRP94 interactions; and cell biological/immunological techniques, to define the cellular basis for GRP94-dependent immune responses. The broad scope of techniques used in these studies is intended to allow a detailed molecular description of the structural and functional basis for the immunogenic activity of GRP94. Insights obtained in achieving the primary objectives of this proposal are expected to contribute significantly to the development of molecular chaperone proteins as immunotherapeutic agents for the treatment of cancer and infectious disease. In addition, results obtained in studies defining a role for GRP94 as a second messenger for pathological cell death will benefit the fields of human and animal vaccin4 development and the study of the genesis and therapeutic resolution of autoimmunity.

DESCRIPTION (provided by applicant): Vaccination with tumor-derived GRP94/gp96, the endoplasmic reticulum Hsp90 molecular chaperone, can elicit suppression of tumor growth and metastasis. The substantial therapeutic efficacy of GRP94/gp96 in prophylactic and therapeutic vaccination of mice has prompted its clinical evaluation as an immunotherapeutic for treatment of cancer in humans. Currently, GRP94/gp96 is undergoing Phase III trials for kidney cancer and melanoma. Although GRP94/gp96 has generated substantial interest as a novel immunotherapeutic, its mechanism of action remains unknown. The broad, long-term objective of the proposed research is to identify the mechanism of GRP94/gp96-mediated anti-tumor immune responses. This objective will be accomplished in the following specific aims: 1. Define the immunological basis for a gp96-secreting, cell-based tumor vaccine strategy; 2. Determine the role of gp96 and gp96 NTD in the regulation of innate immune responses. 3. Examine the role of gp96 and gp96 NTD in early immune modulation of retrovirus-induced disease. 4. Identify the effector cells and effector cell receptor(s) functioning in gp96/gp96NTD recognition and cell activation. The proposed studies will utilize a recently developed novel immunization strategy wherein animals are vaccinated with cells expressing a secretory form of GRP94/gp96. Using this experimental approach, we have demonstrated that GRP94/gp96-mediated tumor rejection is independent of the tissue of origin and thusGRP94/gp96-elicited anti-tumor immune responses can be elicited using GRP94/gp96 of non-tumor origin. Given the absence of tumor-restriction identified in these experiments, we hypothesize that GRP94/gp96functions through activation of innate immune responses. Such responses are a necessary prerequisite to robust anti-cancer adaptive immune responses. In executing the Specific Aims presented above, we will define the efficacy of the gp96-secreting, cell-based tumor vaccine strategy in multiple tumor models, using prophylactic and therapeutic immunization strategies. Detailed studies of the interactions of GRP94/gp96 with effector cells of the innate immune system will be performed. These studies will emphasize in vivo models and analyses of in vivo cellular responses. To better define the mechanism of GRP94/gp96 interaction with the innate immune system, we propose to extend analyses to the study of retrovirus-induced disease, in a neonate model, and to identification of signaling pathways accessed by GRP94/gp96.

[unreadable] DESCRIPTION (provided by applicant): In contemporary models, eukaryotic cells segregate the synthesis of different classes of proteins; secretory/integral membrane proteins are made on the endoplasmic reticulum (ER) and soluble proteins in the cytosol. Recent studies of mRNA partitioning between the cytosol and ER compartments have, however, identified a prominent role for the ER in the synthesis of both soluble and secretory/membane proteins alike. In addition to describing new functions for the ER in global protein synthesis, these findings identify a significant gap in our understanding of how eukaryotic cells partition mRNAs between the two compartments and in turn regulate the synthesis of these two broad classes of proteins. To address this gap, we are focusing our research efforts to 1) understand how eukaryotic cells partition mRNAs between the cytosol and the endoplasmic reticulum (ER) and 2) determine how eukaryotic cells regulate the protein synthesis activity of the cytosol and ER compartments during homeostasis and cell stress. This research is expected to provide significant insights into the regulatory mechanisms governing protein synthesis in health and disease. In addition, this research will serve as a significant contribution to our understanding of the mRNA localization and protein synthesis pathways that are essential to eukaryotic life. Three specific aims are proposed: i) Define the in vivo role of the SRP pathway in mRNA partitioning to the ER; ii) Identify the mRNA localization signals that confer non-canonical mRNA partitioning to the ER and iii) Define the global role of the ER compartment in cytosolic protein syntehsis. To address these specific aims, we will use mammalian tissue culture cell models and will emphasize established biochemical techniques of cell fractionation, cell biological analysis of reporter mRNA localization in situ and molecular biological analysis of the structure/function elements of mRNAs that confer ER localization. Many prominent human diseases, including obesity, diabetes and stroke activate cell stress responses that profoundly alter the types and amounts of proteins cells synthesize and consequently, cell viability. By understanding how cells decide which and how much of each protein to make, in health and disease, we will understand the basic mechanisms used by cells to respond to pathological stress. By understanding these mechanisms, we hope to identify new targets for therapeutic intervention. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Understanding the molecular principles that regulate endomembrane organelle maintenance and biogenesis stands as a principle question in cell biology. Research to date has established the protein/membrane trafficking pathways that support endomembrane organelle maintenance and through these studies, the central importance of the ER as the site of resident endomembrane protein synthesis has been identified. The molecular basis for endomembrane organelle biogenesis remains, however, mysterious. The objective of the proposed research is to define the role of ER-directed mRNA localization in endomembrane organelle biogenesis. It is well established that the protein trafficking pathways of eukaryotic cells also direct the partitioning of the mRNA transcriptome between the two primary protein synthesis compartments in the cell, the cytoplasm and the endoplasmic reticulum (ER). Thus, mRNAs encoding secretory/integral membrane proteins are localized to the ER via the Signal Recognition Particle (SRP) pathway and mRNAs encoding cytosolic/nucleoplasmic proteins, which lack encoded signal sequences, retain default localization in the cytosol. Although this positive selection model is well established, we have recently reported that i) mRNAs encoding resident proteins of the endomembrane organelles (mRNAendo) can be localized to the ER via a SRP- and translation-independent pathway; ii) the mRNAendo cohort is bound to the ER via direct, ribosome-independent interactions; and iii) the mRNAendo cohort is distinguished by its exceptionally high ER enrichment. These findings suggest that mRNA localization serves an integral, though unexplored, role in organelle biogenesis. We hypothesize that the autonomous localization of endomembrane resident protein-encoding mRNAs to the ER, and their direct, ribosome-independent association with the ER membrane, represents a self-organization mechanism functioning in organelle biogenesis. This hypothesis will be tested in the following specific aims: 1) Establish, at a genome-wide scale, the subcellular partitioning patterns of endomembrane resident protein-encoding mRNAs; 2) Identify the molecular signals that direct endomembrane resident protein-encoding mRNAs to the ER; 3) Identify the RNA binding proteins and ER resident proteins that mediate ribosome-independent association of mRNAs with the ER membrane; 4) Determine if ribosome-independent binding of endomembrane protein encoding mRNAs to the ER is essential for organelle biogenesis. Genome-wide studies of mRNA partitioning in eukaryotic cells have revealed an unexpected complexity in the subcellular patterns of mRNA localization. In this proposal, we propose to extend recent discoveries of mRNA cohort-specific patterns of RNA localization to the ER to the question of organelle biogenesis. If successful, these studies will provide fundamental insights into the molecular principles of organelle biogenesis and support the development of therapeutic approaches to diseases of organelle dysfunction. PUBLIC HEALTH RELEVANCE: The organelles of eukaryotic cells perform numerous functions that are essential for cell viability. Indeed, numerous inherited metabolic diseases as well as diseases of ageing arise through disruptions in organelle function. The goal of this research is to identify the basic principles of how organelles are created and repaired, so that organelle-based diseases can be better understood and new therapies for organelle- based disease identified.

RNA localization, a ubiquitous cellular strategy for regulating the subcellular site of mRNA translation, operates via a common, staged mechanism. First, a cis-encoded localization sequence (?zipcode?) is recognized by RNA-binding proteins and the mRNA assembled into a translationally-silenced RNP transport complex. The RNP complex is then localized to the appropriate subcellular destination, either by diffusion or by active transport, and anchored. Lastly, translation of the mRNA is derepressed and local protein synthesis ensues. Although substantial progress has been made in identifying zipcode signals, trans-acting RNA binding proteins, molecular motors and transport mechanisms, very little is known regarding molecular mechanisms of mRNA anchoring, which is critical to the maintence of localized protein synthesis. In our research into mechanisms of mRNA localization and anchoring on the endoplasmic reticulum (ER), we discovered that organelle protein-encoding mRNAs are directly anchored to the ER membrane. In contrast, secretory protein-encoding mRNAs, which also localize to the ER, and are anchored indirectly, via translation on ER-bound ribosomes. We hypothesize that a direct RNA anchoring mechanism acts to spatially coordinate the synthesis of functionally related genes. To identify the mechanism of direct mRNA anchoring to the ER, we performed proteomic interactor screens of ER-bound polyribosomes and identified candidate ER integral membrane RNA anchoring proteins. In a first aim, functional validation studies of candidate RNA anchoring proteins will be performed. mRNA identities, cis-ER anchoring motifs, and RNA binding domains for candidate interactors will be identified via photocrosslinking and immunoprecipitation/RNA- Seq (CLIP-Seq) and PAR-CLIP approaches. Candidate ER-RNA anchoring protein function will be further validated through assays of target mRNA translation and localization, using siRNA knockdown and where available, knockout animal models, to determine roles for direct ER-mRNA anchoring in gene expression. The finding that mRNAs can be directly anchored to the ER suggests a novel mechanism of ribosome trafficking to the ER, where membrane-anchored mRNAs directly recruit ribosomes for de novo translation. In support of this model, we reported previously that ER-bound ribosomes function in de novo translation initiation and remain ER-associated following translation termination. Extending from these observations, we hypothesize that translation on the ER is functionally compartmentalized from cytosolic translation. A primary prediction of this model is that the ER translation cycle operates without an obligatory exchange of ribosomal subunits with a cytosolic pool. We propose to test this hypothesis in a second aim, where we will determine the subcellular site(s) of de novo translation initiation and the role of translation in the regulation of ribosome exchange on the ER. We expect that the proposed research will reveal new paradigms for the subcellular organization of mRNA translation and its regulation in health and disease.

Project Summary: Eukaryotic cells partition ribosomes and mRNAs between the cytosol and endoplasmic reticulum (ER) as a mechanism for compartmentalizing secretory and membrane protein synthesis to the ER. In contrast to prominently studied examples of RNA localization, where localized RNAs traffic to specialized regions of the cell periphery as translationally-silenced ribonucleoprotein particles (RNPs) and undergo translation once anchored at their distal destination, RNA localization to the ER occurs via the translation-dependent signal recognition particle (SRP) pathway. In the SRP pathway, all mRNAs initiate translation in the cytosol, with signal sequence-encoding mRNAs undergoing co-translational trafficking to the endoplasmic reticulum (ER). Upon termination, ER-bound ribosomes return to the cytosol, thereby completing a cycle of selective mRNA localization and ribosome exchange. With the benefit of robust in vitro biochemical assays and atomic structures of pathway components, the mechanism of SRP selection is well understood. The question of SRP pathway function in vivo has, however, remained largely unexplored. Two sets of observations suggest that the in vivo mechanism of mRNA localization to the ER is divergent from current models. One, analyses of the mRNA compositions of cytosolic and ER-bound ribosomes in mammalian tissues and cells, yeast, and fly demonstrate that cytosolic protein- encoding mRNAs are broadly represented on the ER, though they lack encoded signal sequences. Two, genetic inactivation of SRP or SRP receptor in yeast is tolerated, as is stable knockdown of SRP and SRP receptor expression in mammalian cells, findings which suggest a selective, rather then general, role for the SRP pathway in RNA localization to the ER. These observations point to significant gaps in our understanding the mechanisms of translational compartmentalization in vivo. Here we propose three aims to investigate this fundamental question. In the first aim, we will generate SRP, SRP receptor-? and SRP receptor-? CRISPR- Cas9 knock-out and inducible shRNA knock-down human cell lines, as model systems to study translational compartmentalization. In a second aim, RNA-Seq/Ribo-Seq studies of parental and SRP pathway-mutant cell lines will be performed to define SRP, SRP receptor-?, and SRP receptor-? function in mRNA and ribosome localization to the ER, and to identify candidate mechanisms that compensate for SRP pathway inactivation. In a third aim, we will use 4-thiouridine pulse-labeling, SRP pathway mutant cell lines, and pharmacological translation inhibitors to define the subcellular site(s) of translation initiation and the role of the SRP pathway in directing the pioneer localization step pathway for newly exported mRNAs. These studies are expected to provide critical new insights into mechanisms of mRNA and ribosome compartmentalization in eukaryotic cells and thereby accelerate our understanding of translational regulation in homeostasis and disease.

The endoplasmic reticulum (ER) is the subcellular site of secretory and membrane protein synthesis and performs critical functions in secretory/membrane protein biogenesis and cellular proteostasis. In addition its established role in secretory/membrane protein synthesis, recent studies examining the mRNA transcriptomes of cytosolic and ER-bound ribosomes reveal that cytosolic protein transcripts are broadly represented on the ER, with ribosome footprinting analyses demonstrating translation of cytosolic protein mRNAs on ER-associated ribosomes. These findings identify an unexpected mRNA transcriptome-wide function for the ER in proteome expression and reopen fundamental questions regarding the mechanisms regulating mRNA localization and translation on the ER. Principally, where current models posit that mRNA localization to the ER is co-translational and signal sequence-dependent, the abundant presence and translation of cytosolic protein mRNAs on the ER indicates that either alternative and/or multiple pathways mediate mRNA localization to the ER. As well, and although SRP pathway function in protein translocation is well established, the question of SRP pathway function in mRNA localization to the ER remains largely unexplored. Also of significance, the recent findings that a number of translocon-associated proteins, including Sec61?,?, TRAP?, ribophorin I, and p180, are mRNA binding proteins (RBPs) suggest previously unappreciated roles for ER resident RBPs in the biology of RNA localization and translation on the ER. This proposal merges three primary research themes of our laboratory; SRP pathway function in mRNA and ribosome localization to the ER; ii) ER-localized translation initiation as a mechanism of localized protein synthesis, and iii) RNA binding protein function in RNA localization and translational regulation, to address new questions regarding cellular mechanisms of mRNA and ribosome localization to the ER. Building on the past decade and a half of our research into RNA localization and translational regulation on the ER, including founding evidence identifying an mRNA transcriptome-wide role for the ER in cellular proteome expression, the proposed research will utilize mammalian tissue culture cell systems, gene editing and silencing approaches, RNA-seq and Ribo-seq transcriptome analyses, HITS-CLiP and PAR-CLIP studies of ER RNA binding proteins and their RNA interactomes, and biochemical analyses of the subcellular organization of the translation machinery, to obtain new insights into the cellular organization and regulation of proteome expression. This research is expected to advance understanding into the systems and pathways governing post-transcriptional gene expression in the cell. In emphasizing in vivo analyses and native biosynthetic approaches to the study of RNA and ribosome trafficking dynamics, this research is significant in its efforts to rigorously test existing paradigms and advance understanding of cellular mechanisms of localized protein synthesis.