Christopher D. Lima

The SUMO pathway: emerging mechanisms that shape specificity, conjugation and recognition

Proteins of the small ubiquitin-related modifier (SUMO) family are conjugated to proteins to regulate such cellular processes as nuclear transport, transcription, chromosome segregation and DNA repair. Recently, numerous insights into regulatory mechanisms of the SUMO modification pathway have emerged. Although SUMO-conjugating enzymes can discriminate between SUMO targets, many substrates possess characteristics that facilitate their modification. Other post-translational modifications also regulate SUMO conjugation, suggesting that SUMO signalling is integrated with other signal transduction pathways. A better understanding of SUMO regulatory mechanisms will lead to improved approaches for analysing the function of SUMO and substrate conjugation in distinct cellular pathways.

Ulp1-SUMO crystal structure and genetic analysis reveal conserved interactions and a regulatory element essential for cell growth in yeast.

Modification of cellular proteins by the ubiquitin-like protein SUMO is essential for nuclear processes and cell cycle progression in yeast. The Ulp1 protease catalyzes two essential functions in the SUMO pathway: (1) processing of full-length SUMO to its mature form and (2) deconjugation of SUMO from targeted proteins. Selective reduction of the proteolytic reaction produced a covalent thiohemiacetal transition state complex between a Ulp1 C-terminal fragment and its cellular substrate Smt3, the yeast SUMO homolog. The Ulp1-Smt3 crystal structure and functional testing of elements within the conserved interface elucidate determinants of SUMO recognition, processing, and deconjugation. Genetic analysis guided by the structure further reveals a regulatory element N-terminal to the proteolytic domain that is required for cell growth in yeast.

Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1.

E2 enzymes catalyze attachment of ubiquitin and ubiquitin-like proteins to lysine residues directly or through E3-mediated reactions. The small ubiquitin-like modifier SUMO regulates nuclear transport, stress response, and signal transduction in eukaryotes and is essential for cell-cycle progression in yeast. In contrast to most ubiquitin conjugation, the SUMO E2 enzyme Ubc9 is sufficient for substrate recognition and lysine modification of known SUMO targets. Crystallographic analysis of a complex between mammalian Ubc9 and a C-terminal domain of RanGAP1 at 2.5 A reveals structural determinants for recognition of consensus SUMO modification sequences found within SUMO-conjugated proteins. Structure-based mutagenesis and biochemical analysis of Ubc9 and RanGAP1 reveal distinct motifs required for substrate binding and SUMO modification of p53, IkappaBalpha, and RanGAP1.

Reconstitution, Activities, and Structure of the Eukaryotic RNA Exosome

Summary The RNA exosome is a multisubunit 3′ to 5′ exoribonuclease complex that participates in degradation and processing of cellular RNA. To determine the activities and structure of the eukaryotic exosome, we report the reconstitution of 9-subunit exosomes from yeast and human and reconstitution of 10- and 11-subunit exosomes from yeast. Comparative biochemical analysis between purified subunits and reconstituted exosomes using AU-rich, polyadenylated (poly[A]), generic, and structured RNA substrates reveals processive phosphorolytic activities for human Rrp41/Rrp45 and the 9-subunit human exosome, processive hydrolytic activities for yeast Rrp44 and the yeast 10-subunit exosome, distributive hydrolytic activities for Rrp6, and processive and distributive hydrolytic activities for the yeast 11-subunit exosome. To elucidate the architecture of a eukaryotic exosome, its conserved surfaces, and the structural basis for RNA decay, we report the X-ray structure determination for the 286 kDa nine-subunit human exosome at 3.35 A.

Insights into E3 ligase activity revealed by a SUMO-RanGAP1-Ubc9-Nup358 complex.

SUMO-1 (for small ubiquitin-related modifier) belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle and responses to stress by altering protein function through changes in activity or cellular localization or by protecting substrates from ubiquitination. Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases. In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO-conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex by means of interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein. Here we describe the 3.0-Å crystal structure of a four-protein complex of Ubc9, a Nup358/RanBP2 E3 ligase domain (IR1-M) and SUMO-1 conjugated to the carboxy-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data obtained with additional substrates, support a model in which Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO–E2-thioester in an optimal orientation to enhance conjugation.

The RNA Exosome Targets the AID Cytidine Deaminase to Both Strands of Transcribed Duplex DNA Substrates

Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) heavy-chain (IgH) class switch recombination (CSR) and Ig variable region somatic hypermutation (SHM) in B lymphocytes by deaminating cytidines on template and nontemplate strands of transcribed DNA substrates. However, the mechanism of AID access to the template DNA strand, particularly when hybridized to a nascent RNA transcript, has been an enigma. We now implicate the RNA exosome, a cellular RNA-processing/degradation complex, in targeting AID to both DNA strands. In B lineage cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions in an AID-dependent fashion, and is required for optimal CSR. Moreover, both the cellular RNA exosome complex and a recombinant RNA exosome core complex impart robust AID- and transcription-dependent DNA deamination of both strands of transcribed SHM substrates in vitro. Our findings reveal a role for noncoding RNA surveillance machinery in generating antibody diversity.

Structures of the SUMO E1 provide mechanistic insights into SUMO activation and E2 recruitment to E1

E1 enzymes facilitate conjugation of ubiquitin and ubiquitin‐like proteins through adenylation, thioester transfer within E1, and thioester transfer from E1 to E2 conjugating proteins. Structures of human heterodimeric Sae1/Sae2‐Mg·ATP and Sae1/Sae2‐SUMO‐1‐Mg·ATP complexes were determined at 2.2 and 2.75 Å resolution, respectively. Despite the presence of Mg·ATP, the Sae1/Sae2‐SUMO‐1‐Mg·ATP structure reveals a substrate complex insomuch as the SUMO C‐terminus remains unmodified within the adenylation site and 35 Å from the catalytic cysteine, suggesting that additional changes within the adenylation site may be required to facilitate chemistry prior to adenylation and thioester transfer. A mechanism for E2 recruitment to E1 is suggested by biochemical and genetic data, each of which supports a direct role for the E1 C‐terminal ubiquitin‐like domain for E2 recruitment during conjugation.

Structure of an mRNA Capping Enzyme Bound to the Phosphorylated Carboxy-Terminal Domain of RNA Polymerase II

The 2.7 A structure of Candida albicans RNA guanylyltransferase Cgt1 cocrystallized with a carboxy-terminal domain (CTD) peptide composed of four Ser5-PO4 YSPTSPS heptad repeats illuminates distinct CTD-docking sites localized to the Cgt1 N-terminal nucleotidyl transferase domain. Tyr1, Pro3, Pro6, and Ser5-PO4 side chains from each of two YSPTSPS repeats contribute to the interface. Comparison to the Pin1-CTD structure shows that the CTD can assume markedly different conformations that are templated by particular binding partners. Structural plasticity combined with remodeling of CTD primary structure by kinases and phosphatases provides a versatile mechanism by which the CTD can recruit structurally dissimilar proteins during transcription. A binding site for the RNA triphosphatase component of the capping apparatus was also uncovered within the Cgt1 OB domain.

Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins

ABSTRACT Modification of proteins by ubiquitin (Ub)-like proteins (UBLs) plays an important role in many cellular processes, including cell cycle progression, nuclear transport, and autophagy. Protein modification occurs via UBL-conjugating and -deconjugating enzymes, which presumably exert a regulatory function by determining the conjugation status of the substrate proteins. To target and identify UBL-modifying enzymes, we produced Nedd8, ISG15, and SUMO-1 in Escherichia coli and equipped them with a C-terminal electrophilic trap (vinyl sulfone [VS]) via an intein-based method. These C-terminally modified UBL probes reacted with purified UBL-activating (E1), -conjugating (E2), and -deconjugating enzymes in a covalent fashion. Modified UBLs were radioiodinated and incubated with cell lysates prepared from mouse cell lines and tissues to allow visualization of polypeptides reactive with individual UBL probes. The cell type- and tissue-specific labeling patterns observed for the UBL probes reflect distinct expression profiles of active enzymes, indicating tissue-specific functions of UBLs. We identify Ub C-terminal hydrolase L1 (UCH-L1) and DEN1/NEDP1/SENP8, in addition to UCH-L3, as proteases with specificity for Nedd8. The Ub-specific protease isopeptidase T/USP5 is shown to react with ISG15-VS. Furthermore, we demonstrate that the desumoylation enzyme SuPr-1 can be modified by SUMO-1-VS, a modification that is dependent on the SuPr-1 active-site cysteine. The UBL probes described here will be valuable tools for the further characterization of the enzymatic pathways that govern modification by UBLs.

Small Ubiquitin-Like Modifier Modulates Abscisic Acid Signaling in Arabidopsis

Post-translational modification of proteins by small polypeptides, such as ubiquitin, has emerged as a common and important mechanism for regulating protein function. Small ubiquitin-like modifier (SUMO) is a small protein that is structurally related to but functionally different from ubiquitin. We report the identification and functional analysis of AtSUMO1, AtSUMO2, and AtSCE1a as components of the SUMO conjugation (sumoylation) pathway in Arabidopsis. In yeast-two hybrid assays, AtSUMO1/2 interacts specifically with a SUMO-conjugating enzyme but not with a ubiquitin-conjugating enzyme. AtSCE1a, the Arabidopsis SUMO-conjugating enzyme ortholog, conjugates SUMO to RanGAP in vitro. AtSUMO1/2 and AtSCE1a colocalize at the nucleus, and AtSUMO1/2 are conjugated to endogenous SUMO targets in vivo. Analysis of transgenic plants showed that overexpression of AtSUMO1/2 does not have any obvious effect in general plant development, but increased sumoylation levels attenuate abscisic acid (ABA)–mediated growth inhibition and amplify the induction of ABA- and stress-responsive genes such as RD29A. Reduction of AtSCE1a expression levels accentuates ABA-mediated growth inhibition. Our results suggest a role for SUMO in the modulation of the ABA signal transduction pathway.