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Regulation of Secretory and Membrane Protein Biogenesis

Ramanujan S. Hegde, MD, PhD
  • Ramanujan S. Hegde, MD, PhD, Head, Protein Biogenesis Section
  • Ajay Sharma, PhD, Staff Scientist
  • Malaiyalam Mariappam, PhD, Visiting Fellow
  • Tara Hessa, PhD, Visiting Fellow
  • Zairong Zhang, PhD, Visiting Fellow
  • Sichen Shao, BS, Graduate Student
  • Erik Gutierrez, BS, Graduate Student
  • Natasha Sinha, BS, Post-Baccalaureate Student
  • Yoseph Abebe, PhD, Animal Care Technician

We seek to understand how newly synthesized secretory and membrane proteins are made, matured, sorted, and metabolized in cells. Such proteins are essential to all intercellular and intracellular communication, and the proteins' precise locations and abundances are tightly regulated to maintain normal cellular and organismal physiology. Indeed, most current drugs target secreted and membrane proteins, underscoring their central role in human biology. Our goal is to develop a molecular-level understanding of the pathways of secretory and membrane protein biosynthesis and metabolism. We are especially interested in the regulatory machinery controlling protein entry and protein insertion into the endoplasmic reticulum (ER), the site at which nearly all secreted and membrane proteins are first made. We use biochemical approaches to purify, identify, and functionally reconstitute the machinery underlying these basic cellular pathways. In parallel, we are analyzing, in cellular and whole-animal models, the physiologic importance of regulating the metabolism of secretory and membrane proteins. We hope that, by developing a greater understanding of the basic cellular pathways, we can gain insight into the ways they are perturbed in various diseases of protein misfolding and processing.

Prion protein cell biology and neurodegeneration

The prion protein (PrP) is a widely expressed and highly conserved cell surface glycoprotein of uncertain function. Aberrant metabolism of PrP is responsible for a variety of neurodegenerative diseases in both humans and animals. These diseases include the transmissible "prion diseases" such as bovine spongiform encephalopathy as well as inheritable neurodegenerative diseases caused by mutations in the PrP gene. In neither case is the pathway (or pathways) leading to cell death and neuronal damage understood. The overall goal of this project is thus to define the pathways of PrP-mediated neurodegeneration. To that end, we are studying the molecular pathways of PrP biosynthesis, intracellular trafficking, metabolism, and degradation. A quantitative analysis of these events is expected to provide insight into how the various inherited mutations in PrP influence its biosynthesis or metabolism in a manner that leads to the cellular dysfunction.

Our analysis suggests that at least two cytotoxic forms of PrP (termed CtmPrP and cyPrP) are made during the initial translocation of PrP into the ER. We created transgenic mice (in collaboration with Lionel Feigenbaum's laboratory) to investigate whether CtmPrP-mediated and cyPrP-mediated neurodegeneration may be averted in vivo by modulating this newly discovered step during PrP biogenesis. To determine whether changes in these pathways are involved in the progression of neurodegeneration, we are also investigating how the various forms of PrP are normally metabolized by the cell. We hypothesize that a combination of defects in biosynthesis and/or clearance of certain forms of PrP collaborate to cause eventual neuronal dysfunction and death. Conversely, manipulation of these events may permit slowing or reversal of the neurodegenerative process in prion diseases.

In parallel, we are performing a systematic analysis of the biosynthesis, trafficking, and metabolism of disease-associated PrP mutants. We aim to identify the precise cellular locale and mechanism of PrP misfolding that initiates the disease process. We discovered that, for a large number of mutants, misfolded PrP is found in a post–ER location, an observation that is notable because it suggests that the misfolded PrP species have escaped the normal cellular quality control mechanisms in the ER. In parallel studies, we are investigating the downstream consequences of PrP misfolding and aggregation to identify the mechanism by which these events lead to cellular dysfunction. We found that the aggregates recruit various cellular factors, thereby depleting the factors' functional availability. We identified one such factor, a protein called Mahogunin, that not only interacts with mislocalized PrP, but whose functional depletion likely contributes to the neurodegeneration seen in certain diseases caused by PrP.

Regulation of protein biogenesis and function by signal sequences

Our discovery that the N-terminal signal sequence of PrP regulates Prp's topology established a new function for this domain independent of its well-studied role in protein targeting. We subsequently developed and used a novel assay for signal-mediated translocon gating to demonstrate that signal sequences display a remarkable degree of variation in initiating nascent chain access to the luminal environment. We found such substrate-specific properties of signals to be evolutionarily conserved, functionally matched to their respective mature domains, and important for the proper biogenesis of some proteins. A recent analysis of several naturally occurring disease-associated mutants in signal sequences revealed that, contrary to previous assumptions, many are altered in their gating rather than targeting function. Thus, we discovered that the long-observed sequence variations in signals do not simply represent functional degeneracy but instead encode differences in translocon gating that are critical to the proper biogenesis of the attached substrate. In a particularly dramatic example, we showed that conditions of ER stress attenuate the translocation of some but not other substrates in a signal sequence–dependent manner. This "pre-emptive" quality control (pQC) pathway is part of the adaptive cellular stress response, and it minimizes protein misfolding in the ER lumen. We are currently investigating whether the cell exploits the substrate-specific properties of signal sequences in other ways in order to regulate the subcellular localization of certain proteins, such as calreticulin, that have been shown to be present in several compartments. Our ongoing studies focus on developing a molecular understanding of the critical interaction between signal sequences and the translocon and delineating the pathway by which translocationally attenuated proteins are routed for degradation.

Cryo-electron microscopy of the protein translocon

In collaboration with Christopher Akey's group, we are using cryo-electronmicroscopy (EM) to compare the structures of ribosome-translocon complexes prepared under various conditions. Conditions include a quiescent translocon, translocons engaged with a translocating substrate, and translocons lacking or containing individual components. To develop an understanding of the architecture of the protein translocation apparatus, the topography of these cryo-EM structures will be fitted with atomic models of individual translocon structures obtained from existing and forthcoming crystal structures. In parallel, we are also analyzing cytosolic ribosome-binding factors, including a protein complex that we recently identified and which is involved in tail-anchored membrane protein insertion.

Degradation machinery for mislocalized secretory and membrane proteins

We are interested in the fate of copies of secretory and membrane proteins that fail to be properly segregated into the ER. We know that they are degraded, but a more detailed delineation of the pathway of their selective recognition, ubiquitination, and degradation is lacking. Because these proteins are often hydrophobic and/or contain unprocessed hydrophobic elements, they are at high risk for aggregation and inappropriate interactions. Thus, the pathway for the selective recognition and degradation of non-translocated secretory and membrane proteins is likely to be of importance not only during pQC but also for normal cellular homeostasis. We are addressing this issue by using an in vitro system in which secretory and membrane proteins may be synthesized in their "non-translocated" state simply by omitting ER-derived rough microsomes from the reaction (or, if they are included, by inhibiting translocation with Cotransin; see below). With either of these manipulations, non-translocated proteins are rapidly ubiquitinated in preparation for their degradation. We are employing both classical fractionation and affinity-purification approaches to identify the machinery for the selective recognition and degradation of these non-translocated proteins.

Small-molecule inhibitors of protein translocation

To develop new methods and probes for protein translocation, we have initiated collaborative studies (with Jack Taunton's group) to develop pharmacologic methods of modulating this process in vivo. We recently synthesized and characterized a novel small-molecule inhibitor of co-translational protein translocation (termed Cotransin) and demonstrated that, both in vitro and in cultured cells, Cotransin inhibits the translocation of some but not other proteins. Remarkably, we found this substrate specificity to be encoded in the signal sequence. Thus, the sensitivity or resistance to Cotransin may be conferred to any protein of interest simply by choice of signal. We identified the likely target of this inhibitor as the Sec61 complex, which is the central component of the protein translocation channel. These tools and findings open the way for selectively and potently modulating the translocation of individual substrates in live cells, an approach that we are using to study the role of protein translocation in various cellular events such as protein aggregation and toxicity, protein degradation, and the cellular response to ER stress. We are now investigating both the mechanistic basis of Cotransin inhibition and searching for other inhibitors that may act with different specificities and/or different mechanisms.

Mechanism of tail-anchored membrane protein insertion

Insertion of proteins into biological membranes is a fundamental process vital to all organisms. Most membrane proteins use the classical co-translational translocation pathway. The essential and universally conserved machinery for substrate recognition, targeting, and insertion by this pathway is well established and extensively characterized. By contrast, little is known about post-translational membrane protein insertion pathways. The main clients for this pathway are tail-anchored (TA) membrane proteins defined by a cytosolic-facing N-terminal domain followed by a single C-terminal transmembrane domain (TMD). Examples of TA proteins are found on essentially all cellular membranes in every organism; the proteins have diverse functions ranging from intracellular trafficking to regulation of cell death. Despite the proteins' widespread importance, the machinery and mechanisms underlying the recognition, targeting, and insertion of TA proteins into the correct organellar membrane remain largely unknown. Recently, we discovered a cytosolic TMD recognition complex (TRC) that selectively interacts with TA proteins destined for the ER membrane. We identified a central component of the TRC as a highly conserved 40 kD ATPase that represents the first molecular factor in this widely used membrane protein insertion pathway. Still unknown are other components that collaborate with TRC40 to mediate selective recognition, targeting, and insertion of TA proteins into the ER. We are now taking various approaches to identify these additional factors. Along these lines, we recently identified a novel chaperone complex (the Bat3 complex) that captures TA proteins at the ribosome and transfers then to TRC40 for insertion. Further studies are aimed at reconstituting the targeting reaction for TA proteins with purified components. Achieving these goals will permit us to define the core machinery and functions for a fundamental protein trafficking pathway and will pave the way for future mechanistic and structural analyses. In parallel collaborative studies (with Robert Keenan's laboratory), we are undertaking a structural analysis of TRC40 and other key factors in this pathway. We recently determined the X-ray structure of the TRC40 homolog Get3 in two conformations that helps explain how it recognizes TA substrates.

Additional Funding

  • Intramural AIDS Targeted Antiviral Program (IATAP)

Publications

  • Mariappan, M., Li, X., Sharma, A., Mateja, A., Keenan, R.J., and Hegde, R.S. A ribosome-associating factor chaperones tail-anchored membrane proteins. Nature 2010 466:1120-1124
  • Rane, N.S., Chakrabarti, O., Feigenbaum, L., and Hegde, R.S. Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein. J Cell Biol. 2010 188:515-526
  • Mateja A, Szlachcic A, Downing ME, Dobosz M, Mariappan M, Hegde RS, Keenan RJ. The structural basis of tail-anchored membrane protein recognition by Get3. Nature 2009 461:361-366
  • Chakrabarti O, Hegde RS. Functional depletion of Mahogunin by cytosolically exposed prion protein contributes to neurodegeneration. Cell 2009 137:1136-1147
  • Ashok A, Hegde RS. Selective processing and metabolism of disease-causing mutant prion proteins. PLoS Pathogens 2009 5:e1000479

Collaborators

  • Christopher Akey, PhD, Boston University School of Medicine, Boston, MA
  • Robert Keenan, PhD, University of Chicago, Chicago, IL
  • Jennifer Lippincott-Schwartz, PhD, Cell Biology and Metabolism Program, NICHD, Bethesda, MD
  • Jack Taunton, PhD, Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California

Contact

For further information, contact hegder@mail.nih.gov or visit cbmp.nichd.nih.gov/upb.

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