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Elimination of Misfolded and Aggregated Proteins

Research Summary

Pedro Carvalho studies the mechanisms by which potentially toxic secretory proteins, such as misfolded and aggregated proteins, are eliminated from cells.

Intracellular folding of proteins is inherently an inefficient process. As a consequence, a significant portion of the newly synthesized proteins never acquire a native conformation and end up terminally misfolded. Cells have evolved quality control mechanisms that detect and selectively eliminate these aberrant, potentially toxic proteins. Defects in protein quality control are in many instances linked to pathologies such as Parkinson's and other protein aggregation diseases. My laboratory studies the molecular mechanisms by which misfolded proteins are detected and eliminated. We focus on the quality control of secretory and membrane proteins, whose biogenesis occurs in a dedicated compartment, the endoplasmic reticulum (ER).

The spectrum of misfolded proteins present in the ER is quite broad and includes species as diverse as proteins with minor conformation instability up to protein aggregates. Moreover, for membrane proteins containing portions in the ER lumen as well as in the cytoplasm, detection of misfolded domains on each side of the membrane occurs independently. Therefore, ER protein quality control includes a collection of pathways that have evolved to deal with a wide range of substrates. These pathways are thought to work in parallel and the relative contribution of each one in the elimination of misfolded proteins will likely depend on a number of factors, such as cell type and physiological status of the cell. My lab's goal is to dissect mechanistically each of these pathways to gain a comprehensive, integrated understanding of the processes that handle misfolded proteins in the ER and that are critical in maintaining ER homeostasis.

ER-Associated Protein Degradation
The first defense line to eliminate misfolded proteins from the ER is mediated by a pathway called ER-associated protein degradation or ERAD. This pathway consists of different steps that initiate with the recognition of a protein as being misfolded. Substrates are then moved across the ER membrane back into the cytoplasm, a process known as retrotranslocation. On the cytosolic side of the ER, substrates are ubiquitinated by specific, membrane-bound ubiquitin ligases. An ATPase complex facilitates the release of ubiquitinated substrates from the ER membrane into the cytosol where they are eventually degraded by the proteasome.

A few years ago, as a first step toward a mechanistic understanding of the pathway, we used Saccharomyces cerevisiae to systematically map the interactions between ERAD components. This work showed that Doa10p and Hrd1p, the yeast ubiquitin ligases previously implicated in ERAD by genetic studies, are core components of two distinct protein complexes required for the degradation of different classes of substrates: ER proteins with a misfolded domain in the cytosol (ERAD-C substrates) are targeted to a Doa10 complex while proteins with a misfolded luminal domain (ERAD-L substrates) are targeted to an Hrd1 complex. A subset of the components of the Hrd1 complex is required for degradation of proteins with misfolded intramembrane domains (ERAD-M substrates). Interestingly, each of the ubiquitin ligase complexes contained components involved in substrate recognition, ubiquitination, and membrane extraction, a solution that might facilitate coordination of the complex series of events occurring on opposite sides of the ER membrane. This finding offered a novel framework for the organization and cooperation among ERAD components that appear to be conserved among eukaryotes.

How are ERAD substrates moved across the ER membrane into the cytosol where they will eventually be degraded by the proteasome? The translocation of newly synthesized proteins into the ER is very well characterized and is known to occur through a protein conducting channel, the translocon. Therefore, it has been postulated that the retrotranslocation step in ERAD is also mediated by a protein conducting channel; however, the identity of this channel is not known. To start addressing this issue we developed a protein cross-linking strategy to identify the binding partners of an ERAD-L substrate (luminal misfolded protein) that is undergoing retrotranslocation. This work identified the ubiquitin ligase Hrd1p as the main membrane component in retrotranslocation of this model substrate. Future studies are now required to understand the mechanism by which Hrd1p and probably other membrane-bound ubiquitin ligases involved in ERAD, like Doa10p, promote substrate retrotranslocation.

Most ERAD components have been identified and a general idea of the organization of the pathway is now available. Therefore, the current challenge is to reveal the mechanisms of ERAD. Toward this goal, my lab is developing biochemical tools to recapitulate individual steps of the ERAD pathway in vitro, which will allow us to address mechanistically critical issues, such as determining how substrates are recognized by ERAD components or dissecting the driving force for ERAD substrate retrotranslocation.

Certain proteins fail to engage in ERAD, such as misfolded proteins that have a propensity to aggregate. They are predominantly targeted by ER-phagy, a form of selective autophagy in which certain protein aggregates in the ER are engulfed in autophagosomes and delivered to the lysosome for degradation. It is known that ER-phagy is stimulated when levels of misfolded proteins in the ER are abnormally high and the folding capacity of this organelle is exceeded, a condition known as ER stress, which is common in many diseases. Other aspects of ER-phagy are mysterious. For example, how is accumulation of misfolded proteins in the ER communicated to the autophagy machinery in the cytosol? Do the ER-containing autophagosomes form at specific ER subdomains that contain the misfolded proteins or do they form at random ER sites? We are taking a genetic approach to search for components of the ER-phagy pathway in S. cerevisiae. The identified components will provide a molecular handle for future mechanistic studies of ER-phagy.

Partition of ER Proteins During Cell Division
Asymmetric partition of cytosolic misfolded proteins during cell division is a well-documented phenomenon in eukaryotes and prokaryotes. This process is invariably linked to the generation and maintenance of long-lived cellular lineages that need to be damage-free—for example, stem cells. There are some recent indications that misfolded ER proteins might also partition asymmetrically during cell division; however, the biological significance of this observation is not yet known. Some questions we are interested in understanding are, for example, Do certain physiological conditions (like stress) affect the partition of ER proteins? Is asymmetric partitioning of ER proteins important to establish different cell fates? Do all misfolded ER proteins segregate asymmetrically or are only certain classes involved (e.g., aggregated versus nonaggregated)? We are using a strategy to survey the segregation of proteins during mitosis that will allow us to address these and related issues.

Center for Genomic Regulation internal funds and a grant from the Spanish Ministry of Science and Innovation provided partial support for these projects.

As of January 17, 2012

Scientist Profile

International Early Career Scientist
Center for Genomic Regulation
Biochemistry, Cell Biology