A Chain of Events: Regulating Target Proteins by SUMO Polymers.

Small ubiquitin-like modifiers (SUMOs) regulate virtually all nuclear processes. The fate of the target protein is determined by the architecture of the attached SUMO protein, which can be of polymeric nature. Here, we highlight the multifunctional aspects of dynamic signal transduction by SUMO polymers. The SUMO-targeted ubiquitin ligases (STUbLs) RING-finger protein 4 (RNF4) and RNF111 recognize SUMO polymers in a chain-architecture-dependent manner, leading to the formation of hybrid chains, which could enable proteasomal destruction of proteins. Recent publications have highlighted essential roles for SUMO chain disassembly by the mammalian SUMO proteases SENP6 and SENP7 and the yeast SUMO protease Ulp2. SENP6 is particularly important for centromere assembly. These recent findings demonstrate the diversity of SUMO polymer signal transduction for proteolytic and nonproteolytic purposes.

Exome sequencing links the SUMO protease SENP7 with fatal arthrogryposis multiplex congenita, early respiratory failure and neutropenia.

BACKGROUND: SUMOylation involves the attachment of small ubiquitin-like modifier (SUMO) proteins to specific lysine residues on thousands of substrates with target-specific effects on protein function. Sentrin-specific proteases (SENPs) are proteins involved in the maturation and deconjugation of SUMO. Specifically, SENP7 is responsible for processing polySUMO chains on targeted substrates including the heterochromatin protein 1α (HP1α). METHODS: We performed exome sequencing and segregation studies in a family with several infants presenting with an unidentified syndrome. RNA and protein expression studies were performed in fibroblasts available from one subject. RESULTS: We identified a kindred with four affected subjects presenting with a spectrum of findings including congenital arthrogryposis, no achievement of developmental milestones, early respiratory failure, neutropenia and recurrent infections. All died within four months after birth. Exome sequencing identified a homozygous stop gain variant in SENP7 c.1474C>T; p.(Gln492*) as the probable aetiology. The proband's fibroblasts demonstrated decreased mRNA expression. Protein expression studies showed significant protein dysregulation in total cell lysates and in the chromatin fraction. We found that HP1α levels as well as different histones and H3K9me3 were reduced in patient fibroblasts. These results support previous studies showing interaction between SENP7 and HP1α, and suggest loss of SENP7 leads to reduced heterochromatin condensation and subsequent aberrant gene expression. CONCLUSION: Our results suggest a critical role for SENP7 in nervous system development, haematopoiesis and immune function in humans.

SUMO-activated target traps (SATTs) enable the identification of a comprehensive E3-specific SUMO proteome.

Ubiquitin and ubiquitin-like conjugation cascades consist of dedicated E1, E2, and E3 enzymes with E3s providing substrate specificity. Mass spectrometry-based approaches have enabled the identification of more than 6500 SUMO2/3 target proteins. The limited number of SUMO E3s provides the unique opportunity to systematically study E3 substrate wiring. We developed SUMO-activated target traps (SATTs) and systematically identified substrates for eight different SUMO E3s, PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, ZNF451, LAZSUL (ZNF451-3), and ZMIZ2. SATTs enabled us to identify 427 SUMO1 and 961 SUMO2/3 targets in an E3-specific manner. We found pronounced E3 substrate preference. Quantitative proteomics enabled us to measure substrate specificity of E3s, quantified using the SATT index. Furthermore, we developed the Polar SATTs web-based tool to browse the dataset in an interactive manner. Overall, we uncover E3-to-target wiring of 1388 SUMO substrates, highlighting unique and overlapping sets of substrates for eight different SUMO E3 ligases.

The poly-SUMO2/3 protease SENP6 enables assembly of the constitutive centromere-associated network by group deSUMOylation.

In contrast to our extensive knowledge on ubiquitin polymer signaling, we are severely limited in our understanding of poly-SUMO signaling. We set out to identify substrates conjugated to SUMO polymers, using knockdown of the poly-SUMO2/3 protease SENP6. We identify over 180 SENP6 regulated proteins that represent highly interconnected functional groups of proteins including the constitutive centromere-associated network (CCAN), the CENP-A loading factors Mis18BP1 and Mis18A and DNA damage response factors. Our results indicate a striking protein group de-modification by SENP6. SENP6 deficient cells are severely compromised for proliferation, accumulate in G2/M and frequently form micronuclei. Accumulation of CENP-T, CENP-W and CENP-A to centromeres is impaired in the absence of SENP6. Surprisingly, the increase of SUMO chains does not lead to ubiquitin-dependent proteasomal degradation of the CCAN subunits. Our results indicate that SUMO polymers can act in a proteolysis-independent manner and consequently, have a more diverse signaling function than previously expected.

The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctions.

Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell-cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissue.