Mutants / Derivatives

SUMO-1 is also known as Sentrin, GMP1, UBL1, PIC1 or SMT3. The physiological consequences of SUMOylation are distinct from that of ubiquitination. SUMO modification of proteins does not appear to target proteins for degradation but has been implicated in cell cycle progression, nuclear import, target subnuclear localization, transcriptional regulation, and the modulation of protein-protein interactions and protein stability. Although a number of SUMO substrates are cytoplasmic (RanGAP1, IκBα, GLUT1, GLUT4), it appears that SUMOylation is predominantly confined to the nucleus. These substrates include transcription factors (p53, c-Jun, c-myc) and proteins involved in DNA regulation (topoisomerase I, PCNA).

There are three different human SUMO proteins or isoforms with distinct functions. SUMO-1 has 50% sequence identity with SUMO-2 (Sentrin-3, SMT3A) and SUMO-3 (Sentrin-2, SMT3B), while SUMO-2 and SUMO-3 genes show 95% sequence identity. SUMO-1 is usually conjugated to proteins as a monomer, while the SUMO-2 and SUMO-3 form high molecular weight polymers on proteins. SUMO proteins only have 10% primary sequence homology to ubiquitin, but they possess the characteristic ubiquitin-fold tertiary structure. They also have an unstructured N-terminal extension which most likely provides an additional interface for protein-protein interactions.

SUMOylation of target proteins is mediated by a pathway analogous to that of ubiquitination, also mediated by similar enzymes (E1, E2, E3) but with important differences. All SUMO proteins are activated by a heterodimeric E1 activating enzyme, with both subunits well conserved from yeast Aos2/Uba2 to human SAE1/SAE2. This ATP-dependent E1 enzyme charges the SUMO by forming a high-energy thiolester intermediate which is transferred to the human UbcH9 conjugating enzyme. UbcH9 is the only known E2 that is able to mediate the conjugation of SUMO (usually in the absence of an E3 ligase) to lysine residues on a variety of cellular targets. Although UbcH9 can directly recognize and modify lysines contained in a SUMOylation motif, E3-like factors most likely facilitate SUMOylation of specific substrates. SUMO modification usually occurs within a conserved consensus motif (ΨXE) found in SUMO-2, SUMO-3 and protein substrates. Within this motif Ψ represents a large hydrophobic amino acid (I, L, or V), K is the lysine that becomes modified, X is any residue and E is glutamic acid. Many known SUMO conjugation sites occur within this consensus sequence, but SUMOylation also occurs on lysine residues located within non-consensus regions. SUMO-2 and SUMO-3 form polymeric SUMO Chains (primarily through K11) and SUMO-1 multimerization in vitro has been shown to occur predominantly via lysines K7, K16 and K17.

SUMO proteins with mutations at the key lysine residues that can participate in chain formation are useful reagents for in vitro studies. These sumo-mutants">SUMO Mutants are designed to implicate given lysine residues in poly-SUMO chain initiation and conjugation events. The lysine (K) to arginine (R) mutants renders Ub unable to form multi-SUMO chains via that specific lysine with other SUMO molecules. However, these proteins can still be linked to the lysine residues on target proteins formed via the remaining SUMO lysine residues that have not been mutated. These mutants are ideal for the reduction in poly-SUMO chain length or conjugation rates and determining if the chains have a specific linkage requirement. Importantly, since the C-terminal residues are intact, these proteins are fully functional for activation and thiolester formation by E1, E2 and E3 conjugating enzymes. These mutants can also be used in binding studies to determine affinities with proteins that contain SUMO recognition domains. These reagents are valuable for the determination of structure-function requirements in chain synthesis, recognition or disassembly.

SUMO precursor processing and deconjugation is catalyzed by a family of cysteine proteases known as SUMO-specific proteases (SENPs) which are distinct from the ubiquitin-specific deubiquitinating enzymes (DUBs). These enzymes are cysteine proteases that contain a conserved C-terminal domain with the characteristic His-Asp-Cys catalytic triad. They function in the maturation of SUMO precursor proteins, the removal of SUMO from modified substrates, and the disassembly of SUMO chains. All SUMO proteins are translated as precursor proteins (pro-forms) with additional C-terminal residues extend beyond the conserved di-glycine motif. These residues are removed specifically by SENPs that expose the di-glycine motif that is characteristic of the mature and active isoforms. SUMO pro-forms can be used as negative controls in in vitro SUMOylation reactions or as a substrate for SENPs. Human SUMO-specific enzymes include SENP1, SENP2 SENP3, SENP5, SENP6 and SENP7 with varying specificities for each SUMO isoform and each substrate type (precursor forms, SUMOylated substrates or SUMO chains). The first four enzymes are most closely related in sequence and function predominantly in precursor processing and SUMO removal from substrates. SENP6 and SENP7 have additional intervening sequences that split the conserved catalytic domain, and they are predominantly involved in SUMO-2/SUMO-3 chain disassembly.

The chemical or enzymatic modification of SUMO proteins at important functional sites results in several useful derivatives. Modifying the C-terminal glycine carboxyl of SUMO to an aldehyde SUMO-H or vinylsulfone SUMO-VS groups results in highly potent inhibitors of SENP enzymes. Alternatively, substrates for the kinetic analysis of SENPs are generated by synthetically conjugating fluorophores to the C-terminus of SUMO SUMO-AMC. SUMO can also be coupled to agarose via its primary amines, leaving the C-terminus free and available to purify ubiquitin binding proteins. The N-terminus or lysine groups of SUMO can be modified by small other haptens (fluorescein/rhodamine or biotin) for the sensitive detection by direct fluorescence or secondary reagents (eg. avidin or streptavidin reagents) respectively. These groups can also be used for the affinity purification of SUMO conjugates or SUMO binding proteins. Since the chemistry of SUMO conjugation involves its C-terminus, these modifications result in a fully functional molecule that is viable in conjugation reactions.