Brenda A. Schulman - US grants

The proposed research is a response to the National Institute of Aging Program Announcement PAR-02-049, addressing the mechanisms underlying age-related disease changes in sensory and motor processing (problem #11). Parkinson's Disease is a neurodegenerative disorder that affects over 1 million Americans over the age of 50. Insights into the biochemical basis for idiopathic Parkinson's Disease can be gained by studying pathways impaired in genetic forms of the disease. The ubiquitin pathway has long been linked to Parkinson's Disease, because the aggregated inclusions founding patients, called Lewy Bodies, contain ubiquitin. The three recently identified genes mutated in familial Parkinson's Disease begin to provide direct molecular links to the ubiquitin pathway. The PARK5 gene is thought to encode UCH-L1, a ubiquitin C terminal hydrolase. The PARK1 gene encodes alpha-synuclein, which is found along with ubiquitin in Lewy Bodies. The PARK2 gene encodes parkin, which is an E3 ubiquitin ligase. Parkin belongs to the growing family of RING-IBR-RING ubiquitin ligases, which also includes the Dorfin protein that plays a role in another neurodegenerative disorder, amyotrophic lateral sclerosis. Despite the biological importance of RING-IBR-RING ubiquitin ligases in neurodegenerative disorders, little is known about their mechanism. This proposal addresses the structure and function of the parkin ubiquitin ligase. Parkin is found in complex with some alpha-synuclein, suggesting that alpha-synuclein is part of a larger parkin-based ubiquitin ligase complex. We will use our expertise in structural biology of E3 ubiquitin ligases to understand the molecular function of parkin, and its interactions with other proteins including alpha-synuclein, by addressing the following questions. 1) Which of parkin's known interacting proteins, including alpha-synuclein, binds to parkin directly and forms a stoichiometric complex with parkin in vitro. 2) What are the structural domains in parkin, and which ones are involved in parkin's protein-protein interactions? 3) What is the three dimensionalstructure of the RING-IBR-RING E3 ubiquitin ligase motif in parkin?

DESCRIPTION (provided by applicant): Post-translational covalent attachment of ubiquitin and ubiquitin-like proteins has emerged as a predominant cellular regulatory mechanism, with important roles in controlling cell division, signal transduction, embryonic development, endocytic trafficking and the immune response. Indeed, deregulation of the pathways for attaching ubiquitin-like modifications plays a role in a number of diseases, including cancer, birth defects and Parkinson's Disease, and several viruses hijack these pathways during infection. Ubiquitin-like proteins function by remodeling the surface of their target proteins, changing their target' s half-life, enzymatic activity, protein/protein interactions, sub cellular localization or other properties. At least ten different ubiquitin-like modifications exist in mammals, and attachment of different ubiquitin-like proteins to a target leads to different biological consequences. Thus, a key question is how a given ubiquitin-like protein is coordinated with the correct target. Ubiquitin-like proteins are attached via an isopeptide linkage to their targets by the sequential action of El, E2 and in many cases, E3 enzymes. Ubiquitin-like proteins are selected for the pathway by their dedicated El, which coordinates a given ubiquitin-like protein with the right pathway by also selecting the corresponding E2. Despite the wide range of biological processes controlled by ubiquitin-like proteins, the molecular details for how ubiquitin-like proteins are selected by an E1 and coordinated with their E2 remain elusive. Lack of structural data for Els remains a significant limitation in our understanding of ubiquitin-like protein transfer cascades. The proposed research addresses following questions: What are the structures of Els? Which features of the structure are conserved, and which are specific for a particular ubiquitin-like protein family member? How are ubiquitin-like proteins recognized by Els? How are E2s recognized by Els? How do E2s select their particular ubiquitin-like protein? Answering these questions will be of significance to all areas of biology involving regulation by a growing family of ubiquitin-like proteins.

DESCRIPTION (provided by applicant): Post-translational modification by ubiquitin and ubiquitin-like proteins (ublps) is a predominant cellular regulatory mechanism. Ubiquitination regulates a vast array of biological processes, including cell division, the immune response, and embryonic development. As a result, defects in the ubiquitin pathway are associated with numerous diseases, particularly cancer, and disorders associated with aging, such as neurodegenerative disorders and arid muscle wasting. In addition to ubiquitin, over 10 ublps have been found in higher eukaryotes. Ublps have structures and sequences that closely resemble ubiquitin, but they direct their targets to distinct destinies. Therefore, it is important to understand the molecular bases that define the specificity of the ubiquitination process. Our long-term goal is to understand how ubiquitin and ublps are directed to their particular targets to control processes involved in diseases such as cancers and neurodegenerative disorders. Ubiquitin is ligated to targets by a cascade involving a series of three enzymes in classes known as E1, E2, and E3. Despite the importance of these enzymes, little is known how ubiquitin is selected by ubiquitinating enzymes. We hypothesize that the specificity of ubiquitinating enzymes is dictated by a combination of positive selection for interacting with ubiquitin, and negative selection against the wrong ublp. We are focusing on the E1, E2 and E3 enzymes involved in cell proliferation and involved in regulating tumor suppressor proteins. Because these enzymes are important for cell proliferation, and play roles in pathways that lead to cancers, these enzymes may serve as good targets for anti-mitogenic agents. The recent approval of the proteasome inhibitor Bortezomib (VelcadeTM) for treatment of multiple myeloma underscores the therapeutic potential for targeting enzymes in the ubiquitin, and ublp, pathways, and highlights the importance of understanding the detailed mechanisms and specificities of these enzymes. We plan a multidisciplinary approach that combines biochemical, enzymological and structural analysis of ubiquitinating enzymes, enzymes in ublp conjugation cascades, mutants and complexes, in order to understand the specificity of ubiquitination.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There are more than a dozen Ubiquitin-like proteins (Ubls) in higher eukaryotes (e.g. ubiquitin, NEDD8, SUMO, ISG15) that covalently modify myriad substrates to alter the functions of their targets in different ways. Ubls are covalently attached to targets by parallel, but Ubl-specific enzymatic cascades involving E1, E2, and E3 enzymes. These enzymes generally comprise multi-protein assemblies, and over 700 human proteins have motifs indicating that they are part of E1, E2, or E3 enzymes. Although the physiological functions are known for only a handful of these proteins, in the past few years defects in proteins involved in Ubl conjugation have been associated with diseases such as cancer, neurodegenerative disorders, and viral infections. We are determining the structures of selected E1s, E2s, E3s, complexes with Ubls, and complexes with targets in order to obtain a general molecular picture of catalytic mechanisms underlying Ubl conjugation.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Anaphase Promoting Complex (APC) is a multiprotein E3 ubiquitin ligase that controls cell division by promoting ubiquitin-mediated proteolysis of key cell cycle regulatory proteins. The importance of the folding and assembly of APC subunits is underscored by the fact that many of their genes were identified in classic screens for cell division cycle (Cdc) mutants in S. cerevisiae and S. pombe. The ~13 APC subunits can be broadly divided into subcomplexes, one of which contains a cullin subunit and a RING subunit, making the APC a distal member of the largest family of E3 ubiquitin ligases, the Cullin-RING ligases. Another subcomplex contains the small heat shock protein CDC26 and multiple proteins containing tetratricopeptide (TPR) motifs. Other APC subcomplexes contain fewer motifs to indicate their structure and/or function. We are studying the structure and function of the APC.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Microcins are modified peptides that inhibit growth of competing Gram-negative bacteria such as Escherichia, Salmonella, and Enterobacter. Microcin C7 (MccC7) is produced by E. coli to eradicate competitors through a "Trojan horse" mechanism. After import into target cells, MccC7 is cleaved by nonspecific peptidases to release a toxic adenylated-aspartic-acid mimic that targets aspartyl-tRNA synthetase. MccC7 is generated by modification of a precursor heptapeptide, MccA (sequence MRTGNAN). Posttranslational steps in MccC7 biosynthesis involve conversion of the C-terminal Asn7 to an Asp amide with the nitrogen phosphoramidate-linked to AMP, followed by aminopropylation of a phosphate oxygen. Migration of the Asn7 carboxamido nitrogen and the N-P bond-forming step are catalyzed by the enzyme MccB. In a conventional acyl-adenylation reaction that consumes one ATP, MccB catalyzes adenylation of the MccA C-terminus. After an intramolecular rearrangement, an MccA peptidyl-succinimide intermediate is formed. Next, MccB catalyzes an unusual adenylation of the succinimide: the succinimidyl nitrogen attacks the [unreadable]-phosphate of a second ATP molecule, linking AMP to the peptide terminus via an N-P bond. The succinimide ring is hydrolyzed by regiospecific water-mediated opening to yield to the peptidyl-acyl-N-P-Adenosine group that is the Trojan horse reagent. MccB's N-terminal ~90 residue region is not detectably homologous to known structures. MccB's C-terminal ~260 residues share homology with a portion of ubiquitin-like protein (UBL) activating enzymes, also called E1s, which initiate UBL conjugation. This sequence similarity raises several questions about common and divergent aspects of MccB and E1 mechanisms. First, how does MccB recognize a heptapeptide substrate, whereas other family members have a e8 kDa UBL substrate? Second, why does MccB catalyze adenylation of a peptide C-terminal Asn or succinimide, rather than the Gly-Gly sequence found at UBL C-termini? Third, how can MccB catalyze two successive adenylation reactions? To address these questions, we analyzed MccB-MccA-nucleotide interactions.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The best understood Cullin-RING E3 ubiquitin ligase (CRL) is the SCF ubiquitin ligase, composed of Cul1, Skp1 and a member of the F-box family of proteins. Skp1 serves as an adaptor that simultaneously binds sequences near the N-terminus of Cul1 and the F-box motif of an F-box protein. In turn, F-box proteins contain additional protein interaction domains that recruit the substrate into a Cul1-Skp1-F-box protein complex, thereby facilitating ubiquitination of the target via the catalytic core assembled on the C-terminus of Cul1. In contrast, Cul3 employs BTB proteins as substrate specific adaptor. "BTB" is a protein interaction/dimerization domain that is structurally homologous to the cullin-binding region of Skp1, and that binds Cul3 via motifs analogous to those in the Skp1-Cul1 complex. Many BTB-domain proteins also contain additional protein interaction domains, some of which have been shown to recruit ubiquitination targets. Thus, BTB proteins are thought to merge the functional properties of Skp1or EloC and their F-box or SOCS-box partners into a single polypeptide chain, without an intervening F- or SOCS- box. The human genome encodes more than 150 proteins with recognizable BTB domains, often in combination with MATH, Kelch, or other interaction domains. BTB proteins containing MATH and Kelch domains have been linked to substrate targeting by Cul3, although it is unclear precisely how many BTB proteins engage Cul3 in vivo. Also, little is known about how MATH domains select targets for ubiquitination by CRLs. The MATH domain, present in numerous diverse proteins, is most frequently found linked to a C-terminal BTB domain. Indeed, the MATH-BTB module is the 10th most abundant of 2-domain combinations encoded by 131 genomes.

DESCRIPTION (provided by applicant): Covalent attachment of ubiquitin-like proteins (UBLs) such as ubiquitin (Ub), NEDD8, and SUMO is a predominant form of eukaryotic protein regulation. UBLs modify a vast number of proteins, altering their functions in a variety of ways. UBL modifications can affect the target's half-life, subcellular localization, enzymatic activity, or ability to interact with protein or DNA partners. As a result, UBLs regulate numerous biological processes, such as the cell cycle, signal transduction, apoptosis, the immune response, autophagy, and development. Defects in UBL pathways are widely associated with diseases, including cancers, developmental disorders, high blood pressure, neurodegenerative disorders, and cachexia. We propose to extend our expertise on UBL conjugation to the two largest E3 families: RING (Really Interesting New Gene - 570 predicted in humans) and HECT (Homologous to E6AP C-Terminus - 28 predicted in humans). Among the RING E3s, the largest class consists of the modular, multisubunit Cullin-RING (CRL) family. CRLs function sequentially with distinct E2s to modify distinct targets: first the RING domain binds a NEDD8 E2, and the cullin subunit is activated by self-modification with NEDD8. Then a CRL binds a Ub-loaded E2, which is the source of Ub to be transferred to a target. HECT E3s utilize a distinct mechanism, in which a HECT domain catalytic Cys participates directly via a thioester-linked intermediate. First, the HECT domain binds a thioester-linked E2~Ub complex, and Ub is transferred from the E2 Cys to the HECT domain catalytic Cys. Ultimately Ub is transferred from the HECT E3 Cys to a target or Ub Lys. We propose a research plan focused on structural biology and biochemistry to understand mechanisms underlying functions of CRL E3s (Aim 1) and HECT E3s (Aim 2).

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cullin RING ligases (CRLs) comprise the largest subfamily of E3 ubiquitin ligases. In humans, six cullins (CUL1, 2, 3, 4A, 4B, and 5), two RBX-family RING proteins (RBX1 and 2), and hundreds of substrate receptors assemble into distinct CRLs that mediate ubiquitination of thousands of targets to regulate a vast array of cellular processes. CRL function is regulated by attachment of the ubiquitin-like protein (UBL) NEDD8 to a conserved Lys in a cullin's C-terminal domain. NEDD8 both enhances intrinsic CRL ubiquitination activity, and prevents CRL binding to the inhibitor CAND1. In humans, the NEDD8 cascade is known to contain a single E1 (NAE1-UBA3), and two E2s (Ubc12 and UBE2F). In yeast, only a single E2, Ubc12, has been found to work with the NEDD8 ortholog, Rub1. Despite the importance of CRL activation by NEDD8/Rub1, mechanisms underlying cullin ligation to NEDD8/Rub1 remain incompletely understood. A detailed mechanistic view is lacking, in part because two different proteins have been reported as being the E3 for NEDD8/Rub1 ligation to Cul1 or its yeast ortholog, Cdc53. One candidate E3 is Rbx1, which binds Cul1/Cdc53 and has a RING domain. However, Dcn1 was also identified as a Rub1 E3. The Dcn1 crystal structure revealed two domains, a UBA domain, and a "potentiating neddylation" (PONY) domain. The PONY domain alone was reported to bind Ubc12 and Cdc53, and is sufficient to enhance Cdc53~Rub1 levels in vivo and in lysates. Upon this discovery, it was suggested that Rbx1 may play a passive structural role in cullin modification by Rub1. However, Cdc53 can be modified in vitro without Dcn1, raising questions as to Dcn1's function as an E3. Thus, we are dissecting mechanisms underlying NEDD8/Rub1 ligation to Cul1/Cdc53.

DESCRIPTION (provided by applicant): Autophagy is an indispensable process mediating bulk protein degradation and organelle turnover in eukaryotic cells. During autophagy, cytoplasmic organelles and proteins are engulfed into a double-lipid bilayer "autophagosome" to be degraded in bulk upon autophagosome fusion with a lysosome. In addition to numerous proteins regulating autophagy, at least 15 distinct so-called "Atg" proteins are core components for autophagic membrane formation common to many forms of autophagy. Among these key core components are two families of ubiquitin-like proteins (Atg8 and Atg12), and their noncanonical conjugation systems [a noncanonical E1 enzyme (Atg7), two noncanonical E2 enzymes (Atg3 and Atg10), and a noncanonical E3 enzyme partially composed of a UBL (the Atg12~Atg5 conjugate, here ~ refers to a covalent bond)]. Despite the essential roles of these UBL conjugation cascades in the process of autophagy, and the association of defects in these pathways with numerous disease processes, our knowledge of the detailed enzymatic bases for UBL conjugation in autophagy remains relatively rudimentary. We propose to apply our expertise in UBL conjugation cascades to the mechanisms and specificities of noncanonical enzymes that conjugate UBLs during autophagy. Our research plan will utilize structural biology and biochemistry to understand mechanisms underlying Atg7-mediated initiation of autophagy UBL cascades (Aim 1) and ligation of autophagy UBLs to their targets (Aim 2). PUBLIC HEALTH RELEVANCE: Autophagy is required to remove non-functional organelles and maintain protein homeostasis, and has been connected to numerous diseases, including cancers, diabetes, metabolic disorders, infections, and numerous debilitating processes associated with aging such as neurodegenerative disorders. Therefore, it is important to understand the molecular mechanisms underlying UBL conjugation in autophagy, both to provide insights into how defects in this pathway can lead to diseases, and because enzymes mediating UBL conjugation in autophagy are likely to be excellent targets for therapeutic agents.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A key question in understanding ubiquitin (Ub) conjugation is how Ub is transferred between enzymes in E1-E2-E3 cascades. For E3s in the HECT (Homologous to E6AP C-Terminus) class, the ~40 kDa C-terminal HECT domain binds a reactive thioester-linked E2~Ub (here "~" refers to thioester or thioester-like covalent linkage). Then a transthiolation reaction ensues, whereby Ub is transferred from the E2 catalytic Cys to the HECT domain catalytic Cys. Thus, the Ub C-terminus and the active sites of the E2 and HECT domain must all be juxtaposed for E2-to-E3 Ub transfer. In humans, nearly 30 HECT E3s become charged by selective interactions with distinct E2s, and subsequently catalyze target ubiquitination. For example, the HECT E3 NEDD4L has been shown to bind and receive Ub from a subset of E2s including UbcH5B and Ube2E3. A well-recognized downstream function of NEDD4L is regulation of blood pressure through ubiquitination of the Epithelial Sodium Channel (ENaC). Despite important physiological roles of NEDD4L and other HECT E3s, their fundamental enzymatic mechanisms remain poorly understood. A particularly vexing question is how a HECT domain and a specific Ub-loaded E2 interact to promote Ub transfer. Prior studies showed that HECT domains have two structural "lobes" tethered by a flexible linker. The N-terminal "N-lobe" binds part of an E2 distal from the E2 catalytic Cys. The C-terminal "C-lobe" contains the HECT catalytic Cys, which receives Ub from the E2 to form a thioester-linked E3~Ub complex. In the only crystal structure of an E2-HECT domain complex, containing UbcH7 and the HECT domain of E6AP, a 41 [unreadable] gap separates the E2 and E3 cysteines. Thus, we are studying structures of HECT E3 complexes in complexes with E2~ubiquitin in order to understand fundamental mechanisms of ubiquitin transfer.

DESCRIPTION (provided by applicant): E1-E2-E3 enzyme-mediated covalent attachment of ubiquitin (Ub) or ubiquitin-like proteins (Ubls) such NEDD8 is a predominant form of eukaryotic protein regulation. Ubls modify a vast number of proteins and alter their functions in a variety of ways. For example, Ub/Ubl modifications can affect the target protein's half-life, subcellular localization, enzymatic activity, or ability to interact with protein or DNA partners. As a result, Ub/Ubls regulate numerous biological processes, such as the cell cycle, signal transduction, apoptosis, the immune response, autophagy, and development. Defects in Ub/Ubl pathways are widely associated with diseases, including cancers, developmental disorders, high blood pressure, neurodegenerative disorders, and cachexia. We propose to extend our expertise on Ub/Ubl pathways to mechanisms of ligation by the three largest E3 families: HECT (Homologous to E6AP C-Terminus - 28 predicted in humans), RING (Really Interesting New Gene - 570 predicted in humans) and RBR (RING1-IBR-RING2 - 13 predicted in humans). HECT and RBR E3s participate directly in catalysis, with a catalytic cysteine that forms a covalent intermediate through thioester-linkage with Ub's C-terminus via a 2-step reaction. First, the HECT or RBR E3 binds a thioester-linked E2~Ub intermediate, and Ub is transferred from the E2 catalytic cysteine to an E3 catalytic cysteine. Second, Ub is transferred from the E3 Cys to a target lysine. By contrast, RING E3s promote transfer of Ub or a Ubl from from an E2~Ub/Ubl intermediate to an associated substrate. Among the RING E3s, the largest class consists of the modular, multisubunit Cullin-RING (CRL) family. CRLs function sequentially with distinct E2s to modify distinct targets: first the RING domain binds an E2~NEDD8 intermediate, and the cullin subunit is activated by self-modification with NEDD8. Then a NEDD8-modifed CRL binds a Ub-loaded E2, which is the source of Ub to be transferred to a target. Ultimately, for all three classes of E3, repeated cycles through their enzymatic cascades can lead to polyubiquitination, with specific enzymes selectively linking a donor Ub's C-terminus to distinctive lysines on the acceptor Ub's surface. We propose a research plan focused on structural biology and biochemistry to understand mechanisms underlying Ub/Ubl ligation, determining target specificity, and modulating functions of HECT (Aim 1), CRL (Aim 2), and RBR E3s (Aim 3).

Abstract The long-term goal is to generate and use complimentary chemical and biological probes to study the cullin RING ubiquitin ligases (CRL?s) and understand their activation controlled by the interaction of by the Defective in Cullin Neddylation 1 (DCN1) and UBE2M proteins. Because the CRL?s ultimately control ubiquitination of many diverse proteins, thus regulating their stability, intracellular localization, and function, having spatiotemporal control over DCN-mediated CRL activity has the potential to unravel the mechanism regulating key cellular signaling networks and driving disease progression. The health relatedness of this project lies in two facts: 1) DCN1 is an oncoprotein, amplified in squamous cell carcinomas, that drives a highly malignant phenotype, and 2) CRL driven ubiquitination is a validated target in multiple diseases, particularly cancer and immune dysfunction. Therefore, inhibitors of the DCN1-UB2M interaction that are potent, selective, and bioavailable have the potential to be developed as antitumor drugs and possibly for other diseases. Ubiquitination is regulated by a highly complex, dynamic, and redundant network. Inhibitors of DCN1-UB2M will allow direct interrogation of the function of sub-portions of the network and are likely to unveil fundamental principles of the regulation ubiquitination. The generation of complementary cellular and mouse genetic models will enable independent verification of hypotheses. Finally, the DCN1-UBE2M interaction requires N-terminal acetylation of UBE2M, a common posttranslational modification controlling protein interactions. Therefore, a strategy for targeting N- terminal acetylation dependent protein interactions could be widely applicable. The research design and methods for achieving these goals involves the integrated and recursive use of structure-driven, hypothesis- based medicinal chemistry; in vitro biochemical measures of affinity and inhibitory potency; in vivo measures of compound efficacy and pharmacodynamic responses; and in vitro and in vivo measures of compound bioavailability, distribution, metabolism, excretion, and toxicity. The overall goal is to develop new complimentary chemical and biological tools to understand the regulation of the ubiquitin-like protein NEDD8, and uncover the specific role of DCN-mediated neddylation in Cullin-RING ligase substrate receptor exchange, growth factor signaling, and driving tumor progression. Our aims are: Aim 1: Improve the potency and oral exposure of our current DCN1/2 inhibitors and generate new chemical probes with sufficient potency and selectivity to enable ourselves and others to study the consequences of inhibiting this E2-E3 interaction in cells and animals. Aim 2. Investigate how the composition of cellular CUL1 and CUL3 ligases dynamically responds to environmental perturbations and the importance of DCN1/2 in this process. Aim 3: Use genetic and pharmacological approaches to study the effects of inhibiting DCN1 activity in animal tumor models.