The nucleoporin RanBP2 has SUMO1 E3 ligase activity.

Posttranslational modification with SUMO1 regulates protein/protein interactions, localization, and stability. SUMOylation requires the E1 enzyme Aos1/Uba2 and the E2 enzyme Ubc9. A family of E3-like factors, PIAS proteins, was discovered recently. Here we show that the nucleoporin RanBP2/Nup358 also has SUMO1 E3-like activity. RanBP2 directly interacts with the E2 enzyme Ubc9 and strongly enhances SUMO1-transfer from Ubc9 to the SUMO1 target Sp100. The E3-like activity is contained within a 33 kDa domain of RanBP2 that lacks RING finger motifs and does not resemble PIAS family proteins. Our findings place SUMOylation at the cytoplasmic filaments of the NPC and suggest that, at least for some substrates, modification and nuclear import are linked events.     

Identification of putative in vivo substrates of calpain 3 by comparative proteomics of overexpressing transgenic and nontransgenic mice

Calpain 3 (CAPN3) is a calcium-dependent protease, mutations in which cause limb girdle muscular dystrophy type 2A. To explore the physiological function of CAPN3, we compared the proteomes of transgenic mice that overexpress CAPN3 (CAPN3 Tg) and their nontransgenic (non-Tg) counterparts. We first examined known muscular dystrophy-related proteins to determine if overexpression of CAPN3 results in a change in their distribution or concentration. This analysis did not identify any known muscular dystrophy proteins as substrates of CAPN3. Next, we used a proteomic approach to compare and identify differentially represented proteins in 2-DE of CAPN3 Tg and non-Tg mice. LC-MS/MS analysis led to the identification of ten possible substrates for CAPN3, classified into two major functional categories: metabolic and myofibrillar. Myosin light chain 1 (MLC1) was focused upon because our previous studies suggested a role for CAPN3 in sarcomere remodeling. In this study, CAPN3 was shown to proteolyze MLC1 in vitro. These studies are the first to identify possible substrates for CAPN3 in an in vivo system and support a role for CAPN3 in sarcomere remodeling by cleavage of myofibrillar proteins such as MLC1. In addition, these data also suggest a role for CAPN3 in mitochondrial protein turnover.     

Pli1PIAS1 SUMO Ligase Protected by the Nuclear Pore-associated SUMO Protease Ulp1SENP1/2

Covalent modification of the proteome by SUMO is critical for genetic stability and cell growth. Equally crucial to these processes is the removal of SUMO from its targets by the Ulp1 (HuSENP1/2) family of SUMO proteases. Ulp1 activity is normally spatially restricted, because it is localized to the nuclear periphery via interactions with the nuclear pore. Delocalization of Ulp1 causes DNA damage and cell cycle defects, phenotypes thought to be caused by inappropriate desumoylation of nucleoplasmic targets that are normally spatially protected from Ulp1. Here, we define a novel consequence of Ulp1 deregulation, with a major impact on SUMO pathway function. In fission yeast lacking Nup132 (Sc/HuNUP133), Ulp1 is delocalized and can no longer antagonize sumoylation of the PIAS family SUMO E3 ligase, Pli1. Consequently, SUMO chain-modified Pli1 is targeted for proteasomal degradation by the concerted action of a SUMO-targeted ubiquitin ligase (STUbL) and Cdc48-Ufd1-Npl4. Pli1 degradation causes the profound SUMO pathway defects and associated centromere dysfunction in cells lacking Nup132. Thus, perhaps counterintuitively, Ulp1-mediated desumoylation can promote SUMO modification by stabilizing a SUMO E3 ligase.     

SUMO assay with peptide arrays on solid support: insights into SUMO target sites

The modification of proteins by SUMO (small ubiquitin-like modifier) regulates various cellular processes. Sumoylation often occurs on a specific lysine residue within the consensus motif psiKxE/D. However, little is known about the specificity and selectivity of SUMO target sites. We describe here a SUMO assay with peptide array on solid support for the simultaneous characterization of hundreds of different SUMO target sites. This approach was used to characterize known SUMO substrates. The position of the motif within the peptide and the amino acids flanking the acceptor site affected the efficiency of SUMO modification. Interestingly, a sequence of only four amino acids, corresponding to the SUMO consensus motif without flanking amino acids, was a bona fide target site. Analysis of a peptide library for all variants of the psiKxE/D consensus motif revealed that the first and third positions in the tetrapeptide preferably contain aromatic amino acid residues. Furthermore, by adding the SUMO E3 ligase PIAS1 to the reaction mixture, we show specific enhancement of the modification of a PIAS1-dependent SUMO substrate in this system. Overall, our results demonstrate that the sumoylation assay with peptide array on solid support can be used for the high-throughput characterization of SUMO target sites, and provide new insights into the composition, selectivity and specificity of SUMO target sites.     

Solution structures and DNA binding properties of the N-terminal SAP domains of SUMO E3 ligases from Saccharomyces cerevisiae and Oryza sativa

SUMO E3 ligase of the Siz/PIAS family that promotes sumoylation of target proteins contains SAP motif in its N-terminal region. The SAP motif with a consensus sequence of 35 residues was first proposed to be as a new DNA binding motif found in diverse nuclear proteins involved in chromosomal organization. We have determined solution structures of the SAP domains of SUMO ligases Siz1 from yeast and rice by NMR spectroscopy, showing that the structure of the SAP domain (residues 2-105) of rice Siz1 is a four-helix bundle with an up-down-extended loop-down-up topology, whereas the SAP domain (residues 1-111) of yeast Siz1 is comprised of five helices where the fifth helix alpha5 causes a significant change in the alignment of the four-helix bundle characteristic to the SAP domains of the Siz/PIAS family. We have also demonstrated that both SAP domains have binding ability to an A/T-rich DNA, but that binding affinity of yeast Siz1 SAP is at least by an order of magnitude higher than that of rice Siz1 SAP. Our NMR titration experiments clearly showed that yeast Siz1 SAP uses alpha2-helix for DNA binding more effectively than rice Siz1 SAP, which would result from the dislocation of this helix due to the existence of the extra helix alpha5. In addition, based on the structures of the SAP domains determined here and registered in Protein Data Bank, general features of structures of the SAP domains are discussed in conjunction with equivocal nature of their DNA binding.     

The SUMO (Small Ubiquitin-like Modifier) Ligase PIAS3 Primes ATR for Checkpoint Activation

The maintenance of genomic stability relies on the concerted action of DNA repair and DNA damage signaling pathways. The PIAS (protein inhibitor of activated STAT) family of SUMO (small ubiquitin-like modifier) ligases has been implicated in DNA repair, but whether it plays a role in DNA damage signaling is still unclear. Here, we show that the PIAS3 SUMO ligase is important for activation of the ATR (ataxia telangiectasia and Rad3 related)-regulated DNA damage signaling pathway. PIAS3 is the only member of the PIAS family that is indispensable for ATR activation. In response to different types of DNA damage and replication stress, PIAS3 plays multiple roles in ATR activation. In cells treated with camptothecin (CPT), PIAS3 contributes to formation of DNA double-stranded breaks. In UV (ultraviolet light)- or HU (hydroxyurea)-treated cells, PIAS3 is required for efficient ATR autophosphorylation, one of the earliest events during ATR activation. Although PIAS3 is dispensable for ATRIP (ATR-interacting protein) SUMOylation and the ATR-ATRIP interaction, it is required for maintaining the basal kinase activity of ATR prior to DNA damage. In the absence of PIAS3, ATR fails to display normal kinase activity after DNA damage, which accompanies with reduced phosphorylation of ATR substrates. Together, these results suggest that PIAS3 primes ATR for checkpoint activation by sustaining its basal kinase activity, revealing a new function of the PIAS family in DNA damage signaling.     

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.     

Impact of genetic insights into calpain biology

Calpain has long been an enigmatic enzyme, although it is involved in a variety of biological phenomena. Recent progress in calpain genetics has highlighted numerous physiological contexts in which the functions of calpain are of great significance. This review focuses on recent findings in the field of calpain genetics and the importance of calpain function. Calpain is an intracellular Ca(2+)-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02) found in almost all eukaryotes. It is also present in a few bacteria, but not in archaebacteria. Calpain has limited proteolytic activity; rather, it transforms or modulates the structure and/or activity of its substrates. It is, therefore, referred to as a 'modulator protease'. Within the human genome, 15 genes (CAPN1-3, CAPN5-16) encode a calpain-like protease (CysPc) domain along with several different functional domains. Thus, calpains can be regarded as a distinct family of versatile enzymes that fulfil numerous tasks in vivo. Genetic studies show that a variety of defects in many different organisms, including lethality, muscular dystrophies and gastropathy, actually stem from calpain deficiencies. The cause-effect relationships identified by these studies form the basis for ongoing and future studies regarding the physiological role of calpains.