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We currently study a number of processes that occur in the endoplasmic reticulum, or ER. In the broadest sense, we are interested in how this key organelle manages the quality and quantity of molecules that are part of, or passing through, the ER compartment. Due to its many functions in protein and membrane biogenesis, the ER is important in many aspects of normal cellular functions and aberrations associated with disease. Much of our research involves yeast or mammalian cells. Here are some of the things that interest us:

Ubiquitin and ER-associated protein degradation (ERAD)

Ubiquitin-mediated protein degradation is widely used in eukaryotes, and appears to be involved in nearly all aspects of eukaryotic biology. Targeting for degradation occurs when proteins are covalently modified with multiple copies of the small (8kD) protein ubiquitin, to form a multiubiquitin chain. This unique structure is recognized by the 26S proteasome, a multi-protein complex dedicated to protein degradation. One of the main ways the ER controls the quantity and quality of its resident or visiting proteins is through targeted degradation by the ubiquitin pathway, referred to as ER-associated degradation, or ERAD. Our work on regulation of sterol synthesis by ERAD led us into this fascinating and broadly important degradation pathway, and numerous avenues of research have been spawned from this starting point.

The HRD pathway- One of the ways that cholesterol synthesis is controlled in eukaryotes is by regulated degradation of the key sterol pathway enzyme HMG-CoA reductase (HMGR). HMGR is an integral membrane, ER resident protein, and it is degraded by ERAD in a feedback-regulated manner (more detail below). Using yeast to discover the mechanism of this conserved degradation pathway, we found a number of HRD genes, required for HMGR Degradation. The encoded Hrd proteins function at different points of this destruction pathway and together define a branch of ERAD. We are currently investigating how the HRD proteins bring about recognition, ubiquitination, extraction from the ER, and proteasomal degradation of the proteins that are degraded by this route, including HMGR and many other substrates. These approaches include molecular analysis of the key proteins, reconstitution of the pathway in vitro, discovery of new HRD genes remaining to be cloned, and analysis of the physiological effects of changing the HRD and related ERAD pathways in yeast and metazoan cells.

Protein quality control: protecting the fold of normal proteins- The HRD pathway, in addition to mediating degradation of HMGR, functions in protein quality control, by which damaged, misfolded and unassembled proteins are degraded. Quality control is an important aspect of cell function represented in all kingdoms of biology, presumably because misfolded proteins can have toxic and harmful effects on normal cell function. Protein quality control pathways apprear to operate all the time in cells. Failures of this scanning and destruction mechanism lie at the heart of many pressing diseases, including Parkinsonism, Huntington's disease, Alzheimer's, cystic fibrosis, retinitis pigmentosa, and many other maladies. There are numerous questions we want to answer about this important and medically relevant protein destruction pathway, including: How does the HRD machinery and other quality control systems detect misfolded proteins? How many quality control pathways are there in the ER? What other quality control pathways function in the cell? What are the physiological effects of diminished quality control? Can drugs be devised that alter a protein's susceptibility to degradation by quality control mechanisms?

Regulation of cholesterol synthesis- HMGR, the rate-limiting enzyme of the cholesterol pathway, undergoes feedback-regulated degradation as part of cellular control of sterol synthesis. When sterol synthesis is high, degradation of HMGR is accelerated, and when sterol synthesis is low, HMGR degradation is slowed. Regulated degradation of HMGR is conserved from yeast to mammals, and is an untapped axis of clinical modulation of cholesterol. How does this regulation occur? We have discovered that a 15 carbon sterol pathway intermediate called farnesol appears to be a key signal controlling HMGR degradation. As mentioned above, we have made the surprising discovery that the HRD quality control pathway mediates the regulated degradation of normally folded HMGR. How does this work? What appears to be going on is that the lipid signal causes HMGR to adopt the features of a quality control substrate, allowing recognition and destruction by the HRD pathway. Thus, we have an example of a protein that is undergoing a regulated, reversible transition to a quality control substrate, mediated by a small molecule signal. The potential of this style of protein regulation, both as a natural mechanism of cellular regulation, and as a new strategy for controlling the proteome with small molecules, is enormous. So we are interested in this axis or regulation both for what it will tell us about cholesterol regulation, and as a new and tractable strategy of protein regulation by small molecules, whether they be divined by evolution or drug discovery.

Lipid signals in baker's yeast and pathogens- Our work indicates that the main signal for HMGR regulation in yeast is a small lipid molecule called farnesol, a hydrocarbon alcohol. This molecule has remarkable effects in a variety of fungi. In S. cerevisiae it serves as a regulator of HMGR stability. In C. albicans, a fungal pathogen that grows in two developmental states depending on course of infection and environment, farnesol is released as a quorum-sensing molecule by which these yeast evaluate their own cell density. We are interested in this lipid both for the specific aspects of its role as a sterol-regulatory signal, and as an extracellular signal that controls developmental decisions in fungal pathogenesis. We want to know how this lipid is generated in yeast, how it gets out of cells, what other functions are sensitive to it in our and other species of fungi, and what the effects of losing this signaling axis are on fungal organisms.

ER plasticity and proliferation: controlling organelle size and content

Almost nothing is known about how membranes and organelles are created and destroyed to meet changing cellular demands for such structures. In this regard, the ER presents an opportunity to understand many aspects of this very general and important aspect of cell biology. The endoplasmic reticulum is a highly plastic organelle. That is, it is an organelle that can undergo dramatic changes in size and shape depending on cellular conditions. The classic examples of this plasticity are when cells become "professional secretors", developing the capacity to produce large amounts of secreted proteins, such as insulin-producing pancreatic beta cells or antibody-producing plasma B cells. In such cases, the ER is massively expanded to accommodate the high secretory demands of the cell. The changes to ER structure in these examples are accompanied by complex changes in gene expression that program development of these professional secretors. In contrast, there are cases where sufficient expression of a single ER-resident protein will trigger dramatic changes in ER size and content. Such molecules are called "ER proliferants" due to their ability to proliferate this organelle. These simpler cases allow detailed and precise analysis of ER plasticity by a variety of means.

It turns out that HMGR is in this category of "ER proliferants". The HMGR molecule consists of a soluble C-terminal domain responsible for catalytic activity connected to a multispanning N-terminal region that keeps the protein in the ER membrane. When HMGR expression levels are high, the ER membrane content increases, apparently to accommodate this increased burden of resident protein. Although this idea makes sense, we know very little, almost nothing, about how elevated levels of a transmembrane protein are coupled to the membrane dynamics of the ER. One thing we do know is that the enzymatic activity of the HMGR molecule is not involved. Rather, the cell can sense some feature(s) of the HMGR transmembrane region and alter the size and amount of ER membrane.

Why do we want to understand this version of ER proliferation? The ability of the cell to alter the size and shape of its membrane compartments is a fundamental feature of being a eukaryote. HMGR-induced ER proliferation in particular is important in both normal biology and a variety of clinical circumstances. Many sterol-synthesizing tissues produce sufficient HMGR to cause massive proliferation of the smooth ER. This effect of ER proliferation is also important in the action of widely used class of drugs, the statins, which are specific inhibitors of HMGR activity. These drugs are used to keep serum cholesterol levels at acceptable values, and about 50 million people worldwide currently benefit from use of these inhibitors. However, the inhibition of HMGR by these drugs causes feedback upregulation of the protein, resulting in ER expansion in the liver and other sterol synthesizing cells of experimental animals, and in all likelihood in the millions of people on this regimen. Finally, HMGR-induced ER proliferation, which is quite amenable to study, presents a model for a number of related membrane dynamic processes in both prokaryotes and eukaryotes.

HMGR-induced ER proliferation is conserved in eukaryotes occurring in both mammalian cells and yeast when the transmembrane region of the protein is produced in abundance. We are using yeast to discover both the molecules and processes that underlie this version of membrane plasticity. Some of the specific lines of inquiry are described below.

PIE genes required for ER proliferation- None of the proteins required the for ER proliferation are known. Possibilities could include enzymes or regulators of lipid synthesis, proteins needed for transport or asymmetry of membrane components, ER factors needed for processing and folding of new resident proteins, signaling components that herald an increase of proliferant levels, or proteins involved in the targeting of ER membranes to destructive pathways, to name a few. Genetic analysis is a proven strategy for discovery of such components, and baker's yeast provides the genetic platform for this approach. We are currently involved in a variety of classical and "reverse genetic" strategies to isolate such genes, which we have named PIE genes, for Protein Induced Endoplasmic reticulum. There are two broad categories of such PIE genes. We imagine that there could be genes that are needed for production of correctly made proliferations, so that the loss of function of such PIE genes would result in aberrant looking or absent proliferations when HMGR or other proliferants are produced. We also imagine that there could be genes that are needed for the cell to remain healthy when the demand for ER proliferation is placed on it. Accordingly we are currently searching for both categories and have candidate pie mutants from each class of these ongoing genetic screens.

A novel stress pathway- When ER proliferants are elevated by natural or molecular biological means, the cell responds by altering the composition and size of the ER. One way to view this is as a response to the stress of elevated membrane proteins. Accordingly we are investigating the role of new or novel stress pathways in ER proliferation. One well-known stress pathway that mediates changes in ER composition in response to increased production of nascent or misfolded proteins is called the unfolded protein response, or UPR. Surprisingly, the UPR is not needed for ER proliferation to occur, nor is it activated by sudden induction of proliferants such as HMGR, despite the drastic effects on ER structure that this molecule causes. Using a sensitive assay devised in the lab we have found that proliferants such as HMGR do cause a measurable stress on the cells, although it is something different from the UPR. What is this stress and what signaling pathways are induced when it occurs? How are increased levels of ER proliferants indicated in the cell, and what cellular processes are affected when the expansion of the ER is demanded?

Dynamic aspects of the ER membrane- ER proliferation is caused by increased levels of trigger proteins such as HMGR, resulting in the expansion of the membrane. However, studies in a variety of cells show that when expression of the inducing proteins is shut off or diminished, the resulting membranes disappear soon thereafter. Thus, using regulated expression of a proliferant such as HMGR allows examination of membrane dynamics (their rate and mechanisms of appearance and disappearance) in ways that have not been previously exploited. We are interested in the molecular processes that are employed to create and destroy the proliferated ER. Furthermore, we want to know the changes in lipid, protein and RNA composition that accompany the production and destruction of the ER membrane. In this way we will learn about the molecular underpinnings of these broadly relevant processes that control membrane content and structure.

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