37 research outputs found
ESCRT-II, an Endosome-Associated Complex Required for Protein Sorting Crystal Structure and Interactions with ESCRT-III and Membranes
No full textAbstractESCRT-I, -II, and -III protein complexes are sequentially recruited to endosomal membranes, where they orchestrate protein sorting and MVB biogenesis. In addition, they play a critical role in retrovirus budding. Structural understanding of ESCRT interaction networks is largely lacking. The 3.6 Å structure of the yeast ESCRT-II core presented here reveals a trilobal complex containing two copies of Vps25, one copy of Vps22, and the C-terminal region of Vps36. Unexpectedly, the entire ESCRT-II core consists of eight repeats of a common building block, a “winged helix” domain. Two PPXY-motifs from Vps25 are involved in contacts with Vps22 and Vps36, and their mutation leads to ESCRT-II disruption. We show that purified ESCRT-II binds directly to the Vps20 component of ESCRT-III. Surprisingly, this binding does not require the protruding N-terminal coiled-coil of Vps22. Vps25 is the chief subunit responsible for Vps20 recruitment. This interaction dramatically increases binding of both components to lipid vesicles in vitro
AKTIP interacts with ESCRT I and is needed for the recruitment of ESCRT III subunits to the midbody
No full textTo complete mitosis, the bridge that links the two daughter cells needs to be cleaved. This step is carried out by the endosomal sorting complex required for transport (ESCRT) machinery. AKTIP, a protein discovered to be associated with telomeres and the nuclear membrane in interphase cells, shares sequence similarities with the ESCRT I component TSG101. Here we present evidence that during mitosis AKTIP is part of the ESCRT machinery at the midbody. AKTIP interacts with the ESCRT I subunit VPS28 and forms a circular supra-structure at the midbody, in close proximity with TSG101 and VPS28 and adjacent to the members of the ESCRT III module CHMP2A, CHMP4B and IST1. Mechanistically, the recruitment of AKTIP is dependent on MKLP1 and independent of CEP55. AKTIP and TSG101 are needed together for the recruitment of the ESCRT III subunit CHMP4B and in parallel for the recruitment of IST1. Alone, the reduction of AKTIP impinges on IST1 and causes multinucleation. Our data altogether reveal that AKTIP is a component of the ESCRT I module and functions in the recruitment of ESCRT III components required for abscission
Structural and functional studies of L-PGDS and SMPDL3A, enzymes in lipid signaling family
No full textEnzymes are indispensable in maintaining the biological system. They metabolize complex molecules to supply nutrients, to produce energy, to regulate transcription of gene expression, and to control the concentration of effective signaling molecules in a cell, thus maintaining the homeostasis of biological system. This thesis summarizes the study of the structure and function of two enzymes in lipid signaling family using integrative application of X-ray crystallography, solution NMR spectroscopy, light scattering, ITC and thermal shift assay.Lipocalin prostaglandin D synthase (L-PGDS) is a tissue specific prostaglandin D2 producing enzyme with a lipocalin fold. Apart from its enzymatic role, it is known to act as a lipophilic ligand carrier. Crystal structure of human L-PGDS and substrate analog altogether with NMR spectroscopy experiments revealed binding sites for substrate catalysis and entry. NMR titration experiments with membrane mimetic showed that L-PGDS has intrinsic membrane binding affinity depending on the ligand bound. These results allowed a model of substrate catalysis and product egression to be proposed, hence, converging the enzymatic and transporter role that has been reported in literature previously. Since prostaglandin D2 is a pivotal inflammatory signaling molecule, molecular understanding of L-PGDS is important to facilitate future regulation of the prostaglandin isomerase. The dynamics of substrate-product exchange may guide future design of this lipophilic carrier as vehicle for drug delivery.The second enzyme, human acid sphingomyelinase like 3a (SMPDL3a), belongs to a metallophosphodiesterase family and shares close sequence identity with human acid sphingomyelinase (aSMase). SMPDL3a’s structure is reported for the first time revealing its binuclear catalytic core site bound with Zn metal. Even though it was presumed to be part of the lipid hydrolase family, enzymatic assays showed that it metabolizes nucleotides and modified nucleotides like CDP-choline, CDP- ethanolamine and ADP-ribose. Subsequently, CDP-choline soaked crystal revealed 5’ cytidine monophosphate (CMP) ligand bound in the catalytic site due to spontaneous catalysis. Its α-phosphate forms key interactions with histidine residues in the binuclear center. Based on this CMP-enzyme structure, general catalytic mechanism of aSMase family can be proposed. Besides, SMPDL3a also serves as a template for aSMase catalytic domain homology modeling. Further study on enzymes in the acid sphingomyelinase family can now be guided by the newly available structural information.List of scientific papersI. Sing Mei Lim, Dan Chen, Hsiangling Teo, Annette Roos, Anna Elisabet Jansson, Tomas Nyman, Lionel Trésaugues, Konstantin Pervushin, and Pär Nordlund. Structural and dynamic insights into substrate binding and catalysis of human lipocalin prostaglandin D synthase. The Journal of Lipid Research. Jun 2013; 54(6):1630-1643. https://doi.org/10.1194/jlr.M035410 II. Sing Mei Lim, Kit Yeung, Lionel Trésaugues, Hsiangling Teo, and Pär Nordlund. The structure and mechanism of human sphingomyelin phosphodiesterase like 3A, an acid sphingomyelinase homolog – with novel nucleotide hydrolase activity. [Accepted] https://doi.org/10.1111/febs.13655 </p
Structural insight into the ESCRT-I/-II link and its role in MVB trafficking
No full textESCRT (endosomal sorting complex required for trans-port) complexes orchestrate efficient sorting of ubiquiti-nated transmembrane receptors to lysosomes via multivesicular bodies (MVBs). Yeast ESCRT-I and ESCRT-II interact directly in vitro; however, this association is not detected in yeast cytosol. To gain understanding of the molecular mechanisms of this link, we have characterised the ESCRT-I/-II supercomplex and determined the crystal structure of its interface. The link is formed by the vacuo-lar protein sorting (Vps)28 C-terminus (ESCRT-I) binding with nanomolar affinity to the Vps36-NZF-N zinc-finger domain (ESCRT-II). A hydrophobic patch on the Vps28-CT four-helix bundle contacts the hydrophobic knuckles of Vps36-NZF-N. Mutation of the ESCRT-I/-II link results in a cargo-sorting defect in yeast. Interestingly, the two Vps36 NZF domains, NZF-N and NZF-C, despite having the same core fold, use distinct surfaces to bind ESCRT-I or ubiqui-tinated cargo. We also show that a new component of ESCRT-I, Mvb12 (YGR206W), engages ESCRT-I directly with nanomolar affinity to form a 1:1:1:1 heterotetramer. Mvb12 does not affect the affinity of ESCRT-I for ESCRT-II in vitro. Our data suggest a complex regulatory mechan-ism for the ESCRT-I/-II link in yeast
ESCRT-I Core and ESCRT-II GLUE Domain Structures Reveal Role for GLUE in Linking to ESCRT-I and Membranes
No full textSummaryESCRT complexes form the main machinery driving protein sorting from endosomes to lysosomes. Currently, the picture regarding assembly of ESCRTs on endosomes is incomplete. The structure of the conserved heterotrimeric ESCRT-I core presented here shows a fan-like arrangement of three helical hairpins, each corresponding to a different subunit. Vps23/Tsg101 is the central hairpin sandwiched between the other subunits, explaining the critical role of its “steadiness box” in the stability of ESCRT-I. We show that yeast ESCRT-I links directly to ESCRT-II, through a tight interaction of Vps28 (ESCRT-I) with the yeast-specific zinc-finger insertion within the GLUE domain of Vps36 (ESCRT-II). The crystal structure of the GLUE domain missing this insertion reveals it is a split PH domain, with a noncanonical lipid binding pocket that binds PtdIns3P. The simultaneous and reinforcing interactions of ESCRT-II GLUE domain with membranes, ESCRT-I, and ubiquitin are critical for ubiquitinated cargo progression from early to late endosomes
A role for IkappaB kinase 2 in bipolar spindle assembly.
No full textIkappaB kinase 2 (IKK2 or IKKbeta) is a component of the IKK complex that coordinates the cellular response to a diverse set of extracellular stimuli, including cytokines, microbial infection, and stress. In response to an external stimulus, the complex is activated, resulting in the phosphorylation and subsequent proteasome-mediated degradation of IkappaB proteins. This event triggers the nuclear import of the NF-kappaB transcription factor, which activates the transcription of genes that regulate a variety of fundamental biological processes, including immune response, cell survival, and development. Here, we define an essential role for IKK2 in normal mitotic progression and the maintenance of spindle bipolarity. Chemical and genetic perturbation of IKK2 promotes the formation of multipolar spindles and chromosome missegregation. Depletion of IKK2 results in the deregulation of Aurora A protein stability and coincident hyperactivation of a putative Aurora A substrate, the mitotic motor KIF11. These data support a function for IKK2 as an antagonist of Aurora A signaling during mitosis. Additionally, our results indicate a direct role for IKK2 in the maintenance of genome stability and underscore the potential for oncogenic consequences in targeting this kinase for therapeutic intervention
WIP1 phosphatase is a negative regulator of NF-κB signalling
No full text10.1038/ncb1873Nature Cell Biology115659-666NCBI
Telomere-independent Rap1 is an IKK adaptor and regulates NF-κB-dependent gene expression
No full textProduction and functional characterization of ALIX and VPS28 depleted HeLa cells.
No full text(A-B) Representative images and quantification of sictr and siTSG101 transiently transfected HeLa cells stained for TSG101 (red) and α-tubulin (green). (C-D) Representative images and relative quantification of shctr and shAKTIP HeLa cells stained for ALIX (red) and α-tubulin (green) showing that ALIX is present at the midbody in shAKTIP cells. (E-F) Representative images and relative quantification of sictr and siALIX HeLa cells stained for AKTIP (red) and α-tubulin (green) showing that AKTIP is present at the midbody in siALIX cells. (G-H) Representative images and quantification of sictr and siALIX transiently transfected HeLa cells stained for ALIX (red) and α-tubulin (green). (I-J) Representative images and quantification of sictr and siVPS28 transiently transfected HeLa cells stained for VPS28 (red) and α-tubulin (green). Scale bars, 5μm. For results in (B, D, F, H and J) at least 80 midbodies per condition were counted. (TIF)</p
Schematic representation of AKTIP structural and functional association with the ESCRT machinery.
No full text(A) AKTIP localizes at the midbody in proximity with the annular structure formed by MKLP1 and with that formed by the ESCRT I subunit TSG101 and VPS28. AKTIP super-structure is flanked by the rings formed by the ESCRT III subunits CHMP4B, CHMP2A and IST1. (B) AKTIP is recruited to the midbody in a MKLP1 dependent, CEP55 independent way. AKTIP and TSG101 cooperate in the recruitment of CHMP4B to the midbody through independent routes, likely involving the ESCRT I subunit VPS28, that interacts with both TSG101 and AKTIP. AKTIP and TSG101 are needed for the recruitment of IST1 to the midbody, and act in a common pathway leading to abscission.</p
