Research

Research at the Ravid laboratory covers the disciplines of biochemistry, cell biology and genetics, taking advantage of the many research benefits offered by the Baker's yeast Saccharomyces cerevisiae as a model organism. Our area of interest is protein quality control, a universal protective cellular mechanism for ensuring that damaged or aberrant proteins are recognized and repaired or eliminated. Specifically, we are interested in understanding the role of the ubiquitin-proteasome degradation system and cellular chaperones in protein quality control, and the outcome of their deterioration during aging. Our research is driven by findings that link defective ubiquitin conjugation or proteasome function and the concomitant accumulation of cellular protein aggregates, to many human neurodegenerative diseases.

Since joining the Department of Biological Chemistry at the Hebrew University of Jerusalem, in 2007, the Ravid laboratory research aim has been to understand the mechanisms and the fundamental physiological functions of intracellular protein quality control. In keeping with these objectives in mind we have initiated several research projects. You can find more about our research in "Table of Content" to you left.  

Saccharomyces cerevisiae

Introduction

The Ubiquitin-Proteasome System

Intracellular protein degradation has been studied for more than half a century, and it became clear early on that such degradation is highly selective, with individual protein half-lives ranging from minutes to years. Moreover, much of this degradation was found to be energy-dependent, despite the exergonic nature of peptide-bond cleavage. This energy dependence derives from the dual requirements of high substrate specificity and substrate-protein unfolding to make the polypeptide backbone fully accessible for proteolytic cleavage. Most regulated protein degradation in eukaryotes is executed by the ubiquitin–proteasome system. Polyubiquitylation of substrates by specific enzymes provides the major source of selectivity in the system (Figure 1), whereas the 26S proteasome complex performs the protein unfolding that is necessary for processive cleavage of the tagged proteins into short peptides (Figure 2). In addition, ubiquitin ligation can function independently of the proteasome by directing certain proteins (usually membrane proteins) to the lysosome or vacuole for proteolysis. Conversely, proteasomes can degrade some proteins without their prior modification by ubiquitin

Figure 1. Substrate proteins, destined for elimination, are initially attached to polymers of the highly conserved ubiquitin (Ub) protein. This covalent modification of the substrate targets the conjugated protein to a multicatalytic protease complex, the 26S proteasome. The Ub attachment site in substrate proteins is commonly a Lys side chain. A well-defined series of enzymes orchestrates the attachment of mono- and polyubiquitin to proteins (see figure). Ub is first activated in an ATP-consuming reaction by an E1 Ub-activating enzyme, to which it becomes attached by a high-energy thioester bond. Subsequently, the activated Ub is transferred to the active site Cys of a second protein, an E2 ubiquitin-conjugating enzyme. With the aid of a third enzyme, called E3 or ubiquitin-protein ligase, E2 catalyses the transfer of (poly)ubiquitin onto the protein that is destined for degradation.

E3 is the most important enzyme in determining the specificity of substrate ubiquitylation. There are two major classes of mechanistically distinct E3 enzymes, characterized by the RING (or RING-like) and HECT domains. Both types of E3 enzymes are alike in their ability to establish selective substrate binding. The RING finger uses Cys and His residues to coordinate a pair of zinc ions in a characteristic arrangement (not shown). A smaller set of E3 enzymes contain a domain called the U box, which is a degenerate version of the RING-finger that achieves the same general fold without coordinating any metal ions. RING and RING-like E3 enzymes bind to both the E2 enzyme and the substrate, and catalyse the transfer of Ub directly from the E2 enzyme to the substrate. Unlike RING and U-box E3 enzymes, the HECT E3 enzymes have a more direct catalytic role in substrate ubiquitylation. The activated Ub of the Ub–E2 enzyme thioester is transferred to a conserved Cys residue in the HECT domain of the E3 before finally being transferred to a substrate.

Ubiquitylation is reversed by de-ubiquitylating enzymes (DUBs) that remove ubiquitin from proteins and disassemble polyubiquitin chains. DUBs provide additional regulatory control before protein degradation, and they are also fundamental for maintaining a sufficient pool of free ubiquitin molecules in the cell.

Figure 2. Once modified by a polyubiquitin chain of at least four ubiquitins (Ub), the substrate protein is able to bind either directly to intrinsic Ub receptors in the 19S regulatory complex of the 26S proteasome (A) or to adaptor proteins that bear both polyubiquitin-binding and proteasome-binding domains (B). Exactly why certain polyubiquitin-modified substrates must be shuttled to the proteasome by adaptor proteins and others can associate directly with polyubiquitin-binding subunits in the regulatory complex of the proteasome is not fully understood. Binding of the substrate protein to the proteasome is followed by protein unfolding by the half-dozen ATPases that encircle the pore of the proteasome catalytic core, removal of the polyubiquitin chain by proteasome-associated deubiquitylating enzymes (DUBs), and translocation of the unfolded protein into the central proteolytic chamber, where it is cleaved into short peptides (C).

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Ravid T, Hochstrasser MDiversity of degradation signals in the ubiquitin-proteasome system. Nat Rev Mol Cell Biol. 2008;9(9):679-90.

Ubiquitin enzymes mechanisms

We wish to better understand the mechanisms of ubiquitin chain buildup on misfolded substrates at the endoplasmic reticulum, which is a major protein quality control hub in the cell. In yeast, two ubiquitylation complexes are present at the endoplasmic reticulum membrane and they are named by the E3 ligases involved, HRD1 and doa10. Each of the complexes recognize a distinct set of substrates, with minor overlaps. The common E2 enzyme, Ubc7, operates with both E3 complexes Doa10 and Hrd1, while Ubc6 operates only with the Doa10 E3 ligase. How these enzymes co-operate during ubiquitin conjuration is yet unknown but our current studies shade light on the subject. 

We found that the activity of Ubc7, the major conjugating enzyme at the endoplasmic reticulum, requires covalent and non-covalent interactions with ubiquitin. Besides the covalent thioester bond between ubiquitin and Ubc7, interactions between ubiquitin and the α2 helix of Ubc7 are also critical for the enzyme activity (Cohen et al., 2015). Importantly, amino acids that stabilize non-covalent interactions between Ubc7 and ubiquitin are only essential for the degradation of substrates of Doa10 but not of Hrd1. According to the emerging model, non-covalent interactions between helix α2 of Ubc7 and Ub are essential for efficient ubiquitin transfer during the buildup of poly-ubiquitin chains. The differential activation of Ubc7 by its cognate E3 ligases represents a previously unidentified mechanism for regulating protein ubiquitylation.


Another important mechanistic question is why two E2 enzymes are required for ubiquitin conjugation by Doa10? In collaboration with the Thomas Sommer laboratory from the Max Delbruck Center for Molecular Medicine, Berlin, we address the mechanistic basis for this unique requirement for two E2 Ub conjugating enzymes. we find that poly ubiquitylation in the doa10 pathway is accomplished by two sequential steps:

  1. Initially, Ubc6 attaches a single Ub at multiple sites on the protein substrate but cannot synthesize poly-Ub chains (priming).
  2. Ubc7 then forms a poly-Ub chain by successive formation of lysine 48-linked Ub-Ub bonds, thereby generating the signal for degradation by the proteasome (Elongation). 

Besides lysine (K) residues on the substrate, our results implies that Ubc6 can also attach ubiquitin on serine (S) and probably threonine (T) residues via an ester bond. The ability of Ubc6 to prime polyubiquitylation on various amino acid residues may thus serve to increase the target range of quality control substrates available for Doa10 and consequently the degradation capacity of the quality control system.

 

Degradation signals

Identification and characterization of degradation signals

A failure to maintain cell homeostasis is associated with the pathogenesis of many human diseases, such as malignancies and neurodegenerative disorders. The ubiquitin-proteasome system plays a critical role in maintaining the cellular environment by removing misfolded proteins from the cell. One of our research interests is understanding how misfolded substrates are recognized for degradation by the ubiquitin system. In general, every protein has the potential to undergo misfolding and therefore to become a substrate for ubiquitin-mediated degradation. However, what is recognized as “misfolded” by the ubiquitin system is largely unknown.

In order to better understand how misfolded proteins are recognized for degradation we adopted a misfolded substrate model in yeast, termed Ndc10. We found that the C-terminal region of the protein contains a hidden degron, termed DegAB, composed of two amphipathic helices, followed by an unstructured tail (Furth et al., 2011)(See below). 

We further show that the hydrophobic regions of the helices provide the signal for the ubiquitin system while the unstructured tail is essential for degradation by the proteasome (Alfassy et al., 2013). Importantly, we find that mild structural changes in the degradation signal that exposes the hydrophobic surface of the amphipathic helices are sufficient to trigger degradation, suggesting that the recognition by the ubiquitin system takes place at early stages of protein misfolding when the protein secondary structure is still intact.

Mutation that disrupt the hydrophobic interactions in DegA expose a hydrophobic surface, thereby triggering degradation

Currently we perform a new study for large scale identification of degrons in yeast. Using growth assays, combined with deep sequencing, we measure a quantitative degradation potency for thousands of polypeptides in yeast simultaneously (Novel methodology – See methods section). We utilize this assay for the identification of degradation signals involved in protein quality control. The new research system we developed will enable researchers to examine the stability of the proteome of multiple organisms, from yeast to human.  On a broader perspective, we anticipate this work to be a first step toward a complete assessment of the general and specific degradation requirements for each and every protein of interest. 

Chaperones and degradation

The role of molecular chaperones in misfolded protein degradation

Molecular chaperones assist proteins to fold to their natural state. The levels of various chaperones increase during stress such as heat shock and therefore they are also known as Heat Shock Proteins or Hsp(s). Studies over the last decade demonstrated that the same Hsp40 kDa and Hsp70 kDa chaperones that assist folding are also involved in the aggregation of misfolded proteins and in the degradation by the ubiquitin system. Clearly there is a quality control stage in which the fate of the protein must be decided: to fold, to aggregate or to be eliminated (Figure 1). How precisely this triage decision is made is currently unknown and we address this question using a model substrate in yeast cells.

Figure 1. Misfolded protein quality control: making the correct triage decision. Preferentially, the misfolded protein is refolded, keeping the active protein pool constant. However, if the protein cannot refold to its native conformation, it can be removed by Ub-dependent proteasomal degradation. If the chaperone and/or the degradation machineries become rate limiting, misfolded proteins are likely to aggregate. Both degradation and aggregation reduce the active pool of the protein. The proteasome cartoon was adapted from Nickell et al.  

Genetic studies in our laboratory indicate that the yeast Hsp70 chaperone Ssa1 and the Hsp40 chaperone sis1 are essential for the degradation of a model misfolded substrate harboring a protein quality control degrons (Furth et al., 2011; Shiber et al., 2013). However, these chaperones play different roles in substrate proteolysis. Whereas the Hsp40 Sis1 is absolutely required for ubiquitin conjugation, the Hsp70 Ssa1 is not. Our finding localizes Sis1 function to the early stage of substrate targeting to the ubiquitin system while Ssa1 likely functions downstream to the ubiquitin system, probably by escorting the ubiquitylated substrate to the proteasome.

Agregation example, a gif file



Interestingly, in the absence of Ssa1 the misfolded model substrate form cytosolic aggregates (see movie). This protein forms foci only when ubiquitylated, suggesting that besides triggering proteasomal degradation, poly-ubiquitin can also serve as a signal for aggregation in the absence of  Hsp70 in people suffering from misfolded protein diseases (also called neurodegenerative diseases), such as Alzheimer’s, Huntington’s and Parkinson’s contain ubiquitin molecules. Understanding the mechanism of ubiquitin-mediated aggregate formation in yeast may provide important information about the role of ubiquitin in the aggregates associated with neurodegenerative diseases (Shiber et al., 2013Shiber and Ravid, 2014). 

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Nickell, S., Beck, F., Scheres, S.H., Korinek, A., Forster, F., Lasker, K., Mihalache, O., Sun, N., Nagy, I., Sali, A., et al. (2009). Insights into the molecular architecture of the 26S proteasome. Proc Natl Acad Sci U S A 106, 11943-11947.

 

Methodologies:

To advance our research capabilities we constantly invest efforts in developing as well as in adapting relevant state of the art technologies. In-house developed research tools so far include the establishment of:

  1. An assay and related software for automatic calculation of yeast growth rate from multiple samples.
  2. A high throughput screening method for the systematic identification of multiple degradation signals expressed in the yeast proteome.
  3. A flow cytometry-based assay for the quantification of cellular content of protein aggregate.

 

Cell growth and protein degradation

Correlating yeast growth under selective conditions to protein degradation rates  

The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). In a uracil-deficient medium growth is proportional to the relative levels of Ura3. Since the reporter protein is designed so that its synthesis rate is constant, its cellular level is solely determined by its degradation rate. Thus, this method accurately recapitulates reporter protein intracellular degradation kinetics. The method has so far been applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of degradation signals. Application of the degron-URA3-based system constitutes a powerful method that can be generally adapted in any investigation aimed at monitoring changes of protein levels associated with functions of other cellular pathways. (Link to the video describing this method)

Measuring the degradation of a reporter substrate of the Doa10 pathway in various yeast strains. The indicated strains, expressing the reporter are incubated in SD-complete or SD-URA media and OD600 is measured every 15 min. The Minimal Doubling Time (MDT) for each strain is calculated using MDTcalc (Cohen et al., 2014).

High throughput screen

Identification of novel degradation signals in yeast

The coupling of yeast growth and stability of a selection marker allows isolation of surviving population whose plasmid DNA is then subjected to deep sequencing. In this way we are able to identify simultaneously the degradation rates of thousands of peptides. The method is currently employed to identify degradation signals involved in protein quality control but can also be applied to investigating proteome stability in multiple organisms, from yeast to human. 

Degron search: Method overview. A. Method premise - The presence of a degron will lead to lower levels of Ura3p, and therefore for faster growth in the presence of 5FOA. B. Mid-log yeast mRNA are purified, reverse transcribed to cDNA and cleaved using a 4-cutter. The resulting fragments are cloned into the c-terminus of the reporter. C. Plasmids from are transformed into yeast and kept in plasmid-selective conditions. Yeast samples are drawn at indicated time points and plasmid DNA was amplified using specific primers followed by high-throughput sequencing. D. An illustration of the expected competition and how it shapes the abundance profiles of the different clones. These profiles can be used to estimate the growth rate of each individual clone.

Measurement of aggregates

Flow cytometric-based measurement of intracellular protein aggregates

Accumulation of misfolded proteins into aggregates is an integral pathway of the protein quality control network that becomes particularly prominent during proteotoxic stress and in various pathologies. We have developed a flow cytometry-based method for quantitative measurement of fluorescently-labeled protein aggregates in detergent soluble cell extracts. The assay is used to quantify the number of fluorescent aggregates and to monitor changes in the cellular content and properties of aggregates, produced under various conditions. In addition, the assay also enables identification of aggregate components other than the primary aggregation substrate such as chaperones. The potential of this method may be extended by fluorescence-activated sorting which will facilitate the isolation of distinct protein aggregates, including those harboring proteins associated with conformation disorders. 

Determination of relative aggregate content in cells. This figure shows the fluorescence distribution of aggregates in cells expressing no reporter, Hsp104-GFP, Hsp42-mCherry, or both fluorescent reporters. The proportion of cells expressing the fluorescent reporters was determined by flow cytometry analysis (Shiber et al., 2014).