No abstract

Kinetochore protein CENP-I is regulated by SUMO protease SENP6 and RNF4, a ubiquitin ligase which targets polysumoylated proteins for degradation during mitosis.

Small ubiquitin-related modifier (SUMO) processing and deconjugation are mediated by sentrin-specific proteases/ubiquitin-like proteases (SENP/Ulps). We show that SUMO-specific protease 1 (SUSP1), a mammalian SENP/Ulp, localizes within the nucleoplasm. SUSP1 depletion within cell lines expressing enhanced green fluorescent protein (EGFP) fusions to individual SUMO paralogues caused redistribution of EGFP-SUMO2 and -SUMO3, particularly into promyelocytic leukemia (PML) bodies. Further analysis suggested that this change resulted primarily from a deficit of SUMO2/3-deconjugation activity. Under these circumstances, PML bodies became enlarged and increased in number. We did not observe a comparable redistribution of EGFP-SUMO1. We have investigated the specificity of SUSP1 using vinyl sulfone inhibitors and model substrates. We found that SUSP1 has a strong paralogue bias toward SUMO2/3 and that it acts preferentially on substrates containing three or more SUMO2/3 moieties. Together, our findings argue that SUSP1 may play a specialized role in dismantling highly conjugated SUMO2 and -3 species that is critical for PML body maintenance.

Abbreviations used in this paper: Ni-NTA, nickel -nitrilotriacetic acid; RNAi, RNA interference; rRNA, ribosomal RNA; SUMO, small ubiquitin-like modifi er protein; Ulp/SENP, ubiquitin-like protein/sentrin-specifi c protease; XEE, Xenopus laevis egg extract; xSENP3, Xenopus SENP3.The online version of this article contains supplemental material.

SYNOPSIS The covalent attachment of the small ubiquitin-like protein modifier (SUMO) to target proteins results in modifications in their activity, binding interactions, localization or half-life. The reversal of this modification is catalyzed by SUMO-specific processing proteases (SENPs). Mammals contain four SUMO paralogs and six SENP enzymes. Our studies describe a systematic analysis of human SENPs, integrating estimates of relative selectivity for SUMO1 and SUMO2, and kinetic measurements of recombinant C-terminal SENP catalytic domains (cSENPs). We first characterized the reaction of each endogenous SENP and their catalytic domains (cSENP) with HA-tagged SUMO1 and SUMO2 vinyl sulfones (HA-SUMO-VS), active site-directed irreversible inhibitors of SENPs. We found that all cSENPs and endogenous SENP1 react with both SUMO paralogs, while all other endogeneous SENPs in mammalian cells and tissues display high selectivity for SUMO2-VS. To obtain more quantitative data, the kinetic properties of purified cSENPs were determined using SUMO1 or SUMO2-amidomethyl coumarin (SUMO-AMC) as substrate. All enzymes bind their respective substrates with high affinity. cSENP1 and cSENP2 process either SUMO substrate with similar affinity and catalytic efficiency; cSENP5 and cSENP6 show marked catalytic specificity for SUMO2 as measured by KM and kcat while cSENP7 works only on SUMO2. Compared to cSENPs, recombinant full-length SENP1 and SENP2 show differences in SUMO selectivity indicating that paralog specificity is influenced by the presence of the variable N-terminal domain of each SENP. Our data suggests that SUMO2 metabolism is more dynamic than that of SUMO1 since most SENPs display a marked preference for SUMO2.

We determined that the small, ubiquitin-related modifier–specific isopeptidase, SENP2, is dynamically associated with nuclear pore complexes (NPCs). This association is determined by the activities of three N-terminal signals in SENP2: a high-affinity nuclear localization sequence, an Nup107-160–binding element, and a nuclear export signal. NPC association, and its potential regulation, affects SENP2 accessibility to substrates.

Background:The sumoylation pathway is conserved in Plasmodium falciparum. Results: The small ubiquitin-related modifier (SUMO) E1 and E2 enzymes are not functionally interchangable between humans and the malaria parasite, P. falciparum. Conclusion: P. falciparum E1 and E2 interactions have significantly diverged from humans. Significance: Divergent E1 and E2 interaction could be exploited for the design of parasite specific inhibitors.

Substantial evidence exists in literature to suggest that placental protein 14 (PP14) (recently renamed glycodelin A), exhibits immunosuppressive properties and is an indispensable macromolecule in the maternal system for the establishment, maintenance, and progression of pregnancy. Though there are several reports substantiating the above, the mechanism of its action at the molecular level has not been elucidated as yet. In this paper we provide data that suggest that amniotic fluid PP14 and recombinant PP14 expressed in Pichia pastoris induce apoptosis in human peripheral blood lymphocytes upon activation, independent of monocytes. That PP14 has a direct apoptotic action on T cells but not on monocytes was also demonstrated by utilizing human cell lines. PP14 was shown to induce apoptosis in the human T cell lines, Jurkat and MOLT-4 cells, but not in the human monocytic cell line, U937. Placental protein 14 (PP14)1 or glycodelin A belongs to the family of hydrophobic molecule transporter proteins known as lipocalins. PP14, a 162-amino acid glycosylated protein, is secreted by the late secretory phase endometrium during the menstrual cycle (1) and has been proposed as a biochemical marker of endometrial function in women (2). The gene encoding the protein consists of three putative progesterone/ corticosterone response elements (3). Consistent with this is the finding that PP14 concentrations in the endometrial tissue, as well as in circulation, are highest in the late luteal phase and lowest in the periovulatory phase of the menstrual cycle (4 -6) in concert with progesterone levels (7). During pregnancy, PP14 concentration rises, peaking at ϳ10 -12 weeks, being high during the first and second trimesters of pregnancy, and declining thereafter (8 -10).PP14 has been shown to inhibit sperm-zona interaction (11) and is associated with endometrial preparation for blastocyst implantation (12, 13). Apart from this, PP14 has been shown to have immunosuppressive properties in that it inhibits both phytohemagglutinin (14) and anti-CD3 antibody-induced lymphocyte proliferation (15). PP14 also inhibits Natural Killer cell activity (16). That the protein is essential for normal pregnancy progression has been established by the association of low levels of PP14 with habitual abortion (17, 18), unexplained infertility (19), and establishment of pregnancy in women during those cycles in which normal concentrations of PP14 were detected (20). Despite the growing literature on the multifunctional role of PP14, the mechanism of action of this protein has not been delineated at the molecular level. Our studies demonstrate that the immunosuppressive effect of PP14 is the result of induction of apoptosis in T cells. The protein also induced apoptosis in the human T cell lines Jurkat and MOLT-4 but not in the monocyte cell line U937. EXPERIMENTAL PROCEDURES Cells and Cell Lines-Peripheral blood mononuclear cells (PBMCs)were isolated using Histopaque (Sigma) from freshly drawn blood of normal healthy donors (male and female; age 25-...