Maho Niwa
Assistant Professor of Biology, UCSD

e-mail: niwa@ucsd.edu

    In eukaryotic cells, related biochemical processes are often compartmentalized into functionally specialized membrane-bound organelles such as the nucleus, endoplasamic reticulum, mitochondria, and Golgi. The cellular demand for functions provided by individual organelles changes over a cell’s lifetime in response to environmental, developmental and cell cycle specific cues. Our lab is interested in understanding the intracellular signaling pathways used to communicate the need for such changes to 　individual　organelles.　We are further interested to understand the specific pathways and mechanisms used to adjust and coordinate organelle output and organelle abundance to achieve an appropriate response. At present, our studies focus primarily on the signal transduction pathway referred to as the unfolded protein response (UPR), bridging the endoplasmic reticulum (ER) and the cell nucleus.

    What is the UPR? Proteins are synthesized on ribosomes as linear amino acid chains according to the genetic information encoded in RNA transcripts. However, to become functionally useful proteins, these linear chains must first be folded into highly specific, complex three-dimensional structures. Thus, correct folding of nascent proteins is critical to cell function and viability. For secreted proteins such as hormones and for cell surface proteins such as receptors, nascent chain folding and post-translational protein modifications occurs in the lumen of the endoplasmic reticulum (ER) in association with a variety of ER-resident chaperones, protein glycosylases, and other modification enzymes. Only properly folded proteins can exit the ER, while hopelessly misfolded proteins, which are toxic to cells in excess, are eliminated from cells via a proteosome-mediated process termed ER-associated protein degradation (ERAD).

    Periods of increased demand for secreted or membrane proteins require higher than normal levels of ER protein folding capacity. At such times, specific components of the Unfolded Protein Response (UPR) signaling pathway sense this increased demand and initiate downstream events to up-regulate transcription of not only chaperons and other protein folding components, but also of membrane phospholipid synthetic enzymes. Thus, the UPR acts to augment protein folding capacity by increasing both the physical amount of ER well as the levels of available protein folding components. Concurrently, the UPR initiates an overall decrease in the rate of protein synthesis in an effort to alleviate ER protein congestion. Remarkably, if these steps are insufficient to meet at meeting a cell’s protein-folding needs, the UPR activates an apoptotic pathway to eliminate such problematic cells.

Research in our lab focuses on several aspects of UPR signaling. We employ molecular biology, biochemistry, cell biology, and genetic approaches towards a more comprehensive understanding of the UPR. The UPR pathway is conserved among eukaryotes and therefore, one of our labs strengths is our flexibility to experiment in mammalian (primarily human and mouse) systems, as well as in yeast, (primarily S. cerevisiae), depending upon specific questions we are asking. Our flexibility offers students and postdoctoral fellows the opportunity to gain broad experience. Specifically, our work has two major thrusts: 1), to understand the biochemistry of UPR signaling mechanisms and 2), to understand the roles of UPR signaling in cell physiology.

(1) Biochemistry, Molecular and Cell Biology of UPR Components:
Several aspects of UPR signaling components and their modes of action are unconventional. Understanding how they function awaits your great experimental imagination and skill. In mammalian cells, ERprotein folding needs are sensed by at least three molecules: 1) IRE1, a unique ER-transmembrane protein with both kinase and endoribonuclease functions; 2) PERK, an ER-transmembrane kinase that phosphorylates eif2; and 3) ATF6, an ER transmembrane transcription factor that undergoes proteolytic cleavages and nuclear migration. We are focusing on several important questions regarding these components.

(A) Three signaling branches: The UPR initiators IRE1, PERK, and ATF6, sense ER conditions via their (non-homologous) ER luminal domains. Under activating conditions, each initiator activates distinct downstream components that together contribute to the overall UPR response. Recently, we have found that each of these UPR signaling branch may respond differently depending upon the nature of the activating conditions (ER stress). We are investigating the molecular basis of these difference using biochemical and genetic approaches.

(B) NF-κB: A primary cellular event of UPR activation is transcriptional activation of specific genes by UPR-associated transcription factors. Our research has shown that the well studied transcription factor NF-κB is also activated by under UPR activating conditions. However, in response to UPR activating conditions, we found that only a subset of the canonical UPR target genes are activated by NF-κB and further, that NF-κB activation occurs via a non-canonical mechanism. Our goal here is to dissect this UPR-induced activation mechanism as well as to determine the functional importance of NF-κB activation during UPR.

(C) Unusual mRNA splicing: IRE1 is a unique ER transmembrane receptor involved in sensing ER conditions and initiating UPR signaling. Upon activation, IRE1 becomes an active kinase as well as an active RNase. The RNase plays a key role in the non-conventional splicing of pre-mRNA for the UPR-specific transcription factor HAC1 in yeast (XBP1 in mammals) by cleaving a specific intron from this mRNA. Cleavage is then followed by ligation of the HAC1 exon junctions by tRNA ligase to produce the active transcription factor RNA. Because the mechanism of this splicing reaction remains unprecedented, we are studying this mechanism both in vivo and in vitro using purified components. Currently, we are investigating questions including 1), how does IRE1 become an active RNase?; 2), which regions comprise the RNase domain and which residues are required for catalysis;, 3) what is the mechanism of UPR-induced RNA substrate binding; 4), what other RNAs might undergo this unusual splicing reaction; and 5), which RNA elements are recognized by IRE1? These and many more exciting questions are waiting to be answered.

(2) Molecular Biology and Genetics for Physiological roles of UPR

(A) Cell Cycle Control: To date, the UPR is most often referred to as a stress response pathway, activated in response to conditions that impose significant challenge to ER protein processing capacity. Activation of the UPR during the development of antibody secreting B-lymphocytes fits neatly in this category. Recently however, we have identified a novel role for the UPR pathway as a facilitator of cytokinesis during mitotic cell division. In S. cerevisiae, UPR deficient cells lacking certain UPR components are defective for cytokinesis, as seen by an accumulation of multi-budded cells. In addition, certain cytokinesis mutants induce UPR signaling, suggesting that UPR signaling plays a role at specific steps in cytokinesis. Our current thinking is that the UPR pathway operates as a “dimmer switch”, allowing the fine-tuning ER functions to effect ER plasticity. These new findings pose many, many new questions, with one primary issue being to learn which UPR components interact with which components involved in cytokinesis.

(B) Hematopoietic Stem Cell Development: In order to facilitate our understanding of the physiological roles of the UPR, we developed fluorescent reporter genes to score the activation status of each of the three UPR signaling branches in in vivo, in single cells. By scoring the activation of these reporters during the development of transplanted hematopoeitic stem cells in mice, we have observed and localized UPR activation to specific lineages and specific stages of stem cell development. In addition to pursuing this line of research, using mouse genetic techniques we are further interested to study UPR activation during the development of other tissues and organs.


(C) Immune Cells: UPR plays a critical role in terminal differentiation of mature B lymphocytes into antibody secreting plasma cells. Under the microscope, the ER membrane size increases as the cells are becoming plasma cells. We are interested in understanding how UPR helps to make such secretory specialized cells.

 


    Why is studying the UPR important? Defective regulation of ER processing capacity is emerging as an important contributor to several human diseases (see below). Due to certain unique aspects of UPR signaling, the UPR may offer attractive targets for disease treatment or prevention. Further, the unique molecular features of certain UPR signaling components, particularly IRE1, offers an opportunity to design highly specific drugs.

    Cancer: Among the many changes that can occur in cells during tumor development is an increased production of proteins that support cell growth and tumor invasiveness such as growth factors, hormones, and membrane receptors. Thus, cellular mechanisms accommodating such changes are likely to play a role in tumorigenesis. Indeed, several transcriptional targets of UPR signaling, including ER chaperons, are elevated in several cancers including breast cancers, glioblastoma, and some leukemias, myelomas and lymphomas. It is interesting that the drug PS-341, developed to treat multiple myeloma, appears to specifically disrupt IRE1 activity, although its exact mechanism of action is not well understood. Thus, a broader and deeper understanding of UPR signaling and regulation may help to uncover molecular mechanisms of tumor development and offer new therapeutic strategies.
    Diabetes: Defects in the mechanisms that regulate the capacity of the endoplasmic reticulum to fold proteins are particularly apparent in cells specialized for protein secretion, including the insulin secreting pancreatic β cells. A strong association between the UPR pathway and diabetes is underscored by the recent finding that mutations inactivating the UPR signaling component PERK are linked to Wolcott-Rallison syndrome (WRS), a neonatal or early onset insulin-dependent (type I) diabetes. In addition, sensitivity of β-cells to nitric oxide is mediated by CHOP/GADD153, a protein potently induced by UPR signaling.
    Heart Disease and Stroke: Several lines of evidence link UPR signaling with ischemic heart disease and stroke. For example, heart reperfusion after ischemic injury is associated with UPR activation due to reperfusion-induced calcium release. Further, elevated serum homocysteine (HC) levels associated with ischemic heart disease and stroke cause UPR activation due to protein disulfide bond inhibition. Recently, in human cells and even in yeast cells, HC was found to be effective for activating IRE1 to cause HAC1 mRNA splicing, demonstrating a direct link between HC and the UPR. As certain UPR signaling components appear unique to the UPR pathway, understanding the molecular mechanisms of HC-induced UPR induction and its consequences may reveal new opportunities for effective treatment.
    Neurodegenerative Diseases and Viral Infection: Several neurodegenerative conditions including Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease are associated with the toxic effects of accumulated misfolded or proteins. Further, a common strategy for virus and pathogen infections involves hijacking ER protein folding capacity. Therefore, understanding of the UPR pathway holds great promise for developing novel therapies against these diseases.

    Maho Niwa received her Ph.D. in Biochemistry from Baylor College of Medicine and was a Jane-Coffin Childs Postdoctoral Fellow in Department of Biochemistry and Biophysics at UCSF. After joining UCSD Division of Biological Sciences, she has been named a Searle Scholar and Hellman Scholar.