Yeast PIAS-type Ull1/Siz1 is composed of SUMO ligase and regulatory domains

SUMO (small ubiquitin-like modifier)/Smt3 (suppressor of mif two) is a member of the ubiquitin-related protein family and is known to conjugate with many proteins. In the sumoylation pathway, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme), and E3 (SUMO ligase) functions as an adaptor between E2 and each substrate. Yeast Ull1 (ubiquitin-like protein ligase 1)/Siz1, a PIAS (protein inhibitor of activated STAT)-type SUMO ligase, modifies both cytoplasmic and nuclear proteins. In this paper, we performed a domain analysis of Ull1/Siz1 by constructing various deletion mutants. A novel conserved N-terminal domain, called PINIT, as well as the RING-like domain (SP-RING) were required for the SUMO ligase activity in the in vitro conjugation system and for interaction with Smt3 in an in vitro binding assay. The most distal N-terminal region, which contains a putative DNA-binding SAF-A/B, Acinus, and PIAS (SAP) motif, was not required for the ligase activity but was involved in nuclear localization. A strong SUMO-binding motif was identified, which interacted with Smt3 in the two-hybrid system but was not necessary for the ligase activity. The most distal C-terminal domain was important for stable localization at the bud neck region and thereby for the substrate recognition of septins. Furthermore, the C-terminal half conferred protein instability on Ull1/Siz1. Taken together, we conclude that the SP-RING and PINIT of Ull1/Siz1 are core domains of the SUMO ligase, and the other domains are regulatory for protein stability and subcellular localization.     

NMR structure of the N-terminal domain of SUMO ligase PIAS1 and its interaction with tumor suppressor p53 and A/T-rich DNA oligomers

A member of the PIAS (protein inhibitor of activated STAT) family of proteins, PIAS1, have been reported to serve as an E3-type SUMO ligase for tumor suppressor p53 and its own. It also was proposed that the N-terminal domain of PIAS1 interacts with DNA as well as p53. Extensive biochemical studies have been devoted recently to understand sumoylations and its biological implications, whereas the structural aspects of the PIAS family and the mechanism of its interactions with various factors are less well known to date. In this study, the three-dimensional structure of the N-terminal domain (residues 1-65) of SUMO ligase PIAS1 was determined by NMR spectroscopy. The structure revealed a unique four-helix bundle with a topology of an up-down-extended loop-down-up, a part of which the helix-extended loop-helix represented the SAP (SAF-A/B, Acinus, and PIAS) motif. Thus, this N-terminal domain may be referred to as a four-helix SAP domain. The glutathione S-transferase pull-down assay demonstrated that this domain possesses a binding ability to tumor suppressor p53, a target protein for sumoylation by PIAS1, whereas gel mobility assays showed that it has a strong affinity toward A/T-rich DNA. An NMR analysis of the four-helix SAP domain complexed with the 16-bp-long DNA demonstrated that one end of the four-helix bundle is the binding site and may fit into the minor groove of DNA. The three-dimensional structure and its binding duality are discussed in conjunction with the biological functions of PIAS1 as a SUMO ligase.     

Sumoylation of Mdm2 by protein inhibitor of activated STAT (PIAS) and RanBP2 enzymes

Mdm2, a ubiquitin ligase that acts on p53, is regulated by sumoylation. In the current study, we identify the enzymes responsible for the sumoylation of Mdm2. When mammalian cells are co-transfected with cDNAs encoding Mdm2 and PIAS1 or PIASxbeta (protein inhibitor of activated STAT) as sumoylation enzymes, Mdm2 is highly sumoylated. Mdm2 is also sumoylated in an in vitro system containing PIASxbeta, PIAS1, and RanBP2. When several lysine residues of Mdm2 were sequentially mutated to arginine, the K182R mutant was not sumoylated in intact cells; however, in the in vitro system this mutant was sumoylated by PIAS1, PIASxbeta, and RanBP2 as efficiently as the wild-type Mdm2 protein. Lysine residues 182 and 185 map within the nuclear localization signal of Mdm2. A K185R mutant of Mdm2 is sumoylated in intact cells, whereas a K182R protein is not. Only a Mdm2 protein bearing the K182R mutation is localized exclusively in the cytoplasm. Because RanBP2 is a nuclear pore protein and PIAS proteins are localized within the nucleus, our data suggest that Mdm2 is sumoylated during nuclear translocation by RanBP2 and then further sumoylated once in the nucleus by PIASxbeta and PIAS1.     

Identification of a gene in Leishmania infantum encoding a protein that contains a SP-RING/MIZ zinc finger domain

The SP-RING or Miz zinc finger domain that is related to the classical RING-finger motif, defines a class of proteins that can act as E3-like factors in the pathway of small ubiquitin-related modifier (SUMO) conjugation. This family includes the mammalian protein inhibitor of activated STAT (PIAS) proteins and related proteins from lower eukaryotes. Here we report the existence of a gene in Leishmania infantum, present as two identical copies placed upstream of each MAT2 gene copy, and transcribed as a single approximately 2.2 kb mRNA both in the logarithmic and stationary phases of the promastigote stage. This gene encodes a 47 kDa protein that has been named LORIEN. LORIEN is circumscribed to the cell periphery and it is antigenic during L. infantum infection of dogs and hamsters. Strikingly, this novel protein contains a highly conserved SP-RING/Miz zinc finger domain, raising the possibility that a SUMO or ubiquitin-like system may exist in this microorganism.     

PIAS1 enhances SUMO-1 modification and the transactivation activity of the major immediate-early IE2 protein of human cytomegalovirus

The protein inhibitor of activated STAT1 (PIAS1), known to be a small ubiquitin-like modifier (SUMO) E3 ligase, was found to interact with the human cytomegalovirus IE2 protein. We found that the sumoylation of IE2 was markedly enhanced by wild-type PIAS1 but not by a mutant containing a Cys to Ser substitution at position 351 (C351S) within the RING finger-like domain. In target reporter gene assays, wild-type PIAS1, but not the C351S mutant, enhanced the IE2-mediated transactivations of viral polymerase promoter and cellular cyclin E promoter and this augmentation required the intact sumoylation sites of IE2. Our results suggest that PIAS1 acts as a SUMO E3 ligase toward IE2 and that it may regulate the transactivation function of IE2. To our knowledge, IE2 is the first viral target found to be regulated by a SUMO E3 ligase.