Membrane Insertion of C-tail Anchored Proteins

The correct localization of proteins is essential for cell viability. In order to achieve correct protein localization to cellular membranes, conserved membrane targeting and translocation mechanisms have evolved. The focus of this work was membrane targeting and translocation of a group of proteins that circumvent the known targeting and translocation mechanisms, the C-tail anchored protein family. Members of this protein family carry out a wide range of functions, from protein translocation and recognition events preceding membrane fusion, to the regulation of programmed cell death. In this work, the mechanisms of membrane insertion and targeting of two C-tail anchored proteins were studied utilizing in vivo and in vitro methods, in yeast and mammalian cell systems. The proteins studied were cytochrome b(5), a well characterized C-tail anchored model protein, and N-Bak, a novel member of the Bcl-2 family of regulators of programmed cell death. Membrane insertion of cytochrome b(5) into the endoplasmic reticulum membrane was found to occur independently of the known protein conducting channels, through which signal peptide-containing polypeptides are translocated. In fact, the membrane insertion process was independent of any protein components and did not require energy. Instead membrane insertion was observed to be dependent on the lipid composition of the membrane. The targeting of N-Bak was found to depend on the cellular context. Either the mitochondrial or endoplasmic reticulum membranes were targeted, which resulted in morphological changes of the target membranes. These findings indicate the existence of a novel membrane insertion mechanism for C-tail anchored proteins, in which membrane integration of the transmembrane domain, and the translocation of C-terminal fragments, appears to be spontaneous. This mode of membrane insertion is regulated by the target membrane fluidity, which depends on the lipid composition of the bilayer, and the hydrophobicity of the transmembrane domain of the C-tail anchored protein, as well as by the availability of the C-tail for membrane integration. Together these mechanisms enable the cell to achieve spatial and temporal regulation of sub-cellular localization of C-tail anchored proteins.Elävissä soluissa valkuaisaineet osallistuvat lähes kaikkiin solua ylläpitäviin toimintoihin. Hallitakseen valkuaisaineiden toimintaa solut ovat kehittäneet erilaisia tapoja säädellä valkuaisaineita, kunnes niitä tarvitaan. Yksi hyvin yleinen tapa on vaikuttaa valkuaisaineiden määrään solussa geenisäätelyn avulla. Useilla valkuaisaineilla on yksilöllisiä toimintoja, joita tarvitaan vain rajoitetusti ja oikeassa paikassa. Esimerkiksi hiivasoluissa puolet valkuaisaineista on kuljetettuina erillisiin soluosastoihin, joissa osa niistä on lisäksi kiinnittyneinä kalvostoissa. Koska valkuaisaineiden esiintyminen väärään aikaan väärässä paikassa voi olla haitallista, on kehittynyt erilaisia hallintamekanismeja. Tällaisia mekanismeja löytyy kaikista elävistä soluista ja ne sisältävät monimutkaisia valkuaisaineista koostuvia ja energiaa kuluttavia porttivahteja . Tässä väitöskirjatyössä tutkin mekanismeja, jolla erään valkuaisaineperheen jäsenet kiinnittyvät erilaisiin solunsisäisiin kalvostoihin. Tämän perheen jäsenet osallistuvat useisiin solulle elintärkeisiin tehtäviin, kuten valkuaisaineitten solunsisäiseen kuljetukseen ja ohjelmoituun solukuolemaan. Väitöskirjatyössäni löysin uudenlaisen mekanismin, jolla valkuaisaineita kiinnitetään solukalvoihin. Yllättäen tämä mekanismi ei näytä tarvitsevan muita avustavia valkuaisaineita eikä energiaa. Sen sijaan, tutkittujen valkuaisaineiden spontaania asettumista solukalvoihin näyttäisi säätelevän kalvon ominaislaatu

Unassisted translocation of large polypeptide domains across phospholipid bilayers

Although transmembrane proteins generally require membrane-embedded machinery for integration, a few can insert spontaneously into liposomes. Previously, we established that the tail-anchored (TA) protein cytochrome b(5) (b5) can posttranslationally translocate 28 residues downstream to its transmembrane domain (TMD) across protein-free bilayers (Brambillasca, S., M. Yabal, P. Soffientini, S. Stefanovic, M. Makarow, R.S. Hegde, and N. Borgese. 2005. EMBO J. 24:2533–2542). In the present study, we investigated the limits of this unassisted translocation and report that surprisingly long (85 residues) domains of different sequence and charge placed downstream of b5's TMD can posttranslationally translocate into mammalian microsomes and liposomes at nanomolar nucleotide concentrations. Furthermore, integration of these constructs occurred in vivo in translocon-defective yeast strains. Unassisted translocation was not unique to b5 but was also observed for another TA protein (protein tyrosine phosphatase 1B) whose TMD, like the one of b5, is only moderately hydrophobic. In contrast, more hydrophobic TMDs, like synaptobrevin's, were incapable of supporting unassisted integration, possibly because of their tendency to aggregate in aqueous solution. Our data resolve long-standing discrepancies on TA protein insertion and are relevant to membrane evolution, biogenesis, and physiology

Correction: TNFR2 induced priming of the inflammasome leads to a RIPK1-dependent cell death in the absence of XIAP.

The original version of this article contained an error in the name of one of the co-authors (Erika Owsley). This has been corrected in the PDF and HTML versions

TNFR2 induced priming of the inflammasome leads to a RIPK1-dependent cell death in the absence of XIAP.

The pediatric immune deficiency X-linked proliferative disease-2 (XLP-2) is a unique disease, with patients presenting with either hemophagocytic lymphohistiocytosis (HLH) or intestinal bowel disease (IBD). Interestingly, XLP-2 patients display high levels of IL-18 in the serum even while in stable condition, presumably through spontaneous inflammasome activation. Recent data suggests that LPS stimulation can trigger inflammasome activation through a TNFR2/TNF/TNFR1 mediated loop in xiap-/- macrophages. Yet, the direct role TNFR2-specific activation plays in the absence of XIAP is unknown. We found TNFR2-specific activation leads to cell death in xiap-/- myeloid cells, particularly in the absence of the RING domain. RIPK1 kinase activity downstream of TNFR2 resulted in a TNF/TNFR1 cell death, independent of necroptosis. TNFR2-specific activation leads to a similar inflammatory NF-kB driven transcriptional profile as TNFR1 activation with the exception of upregulation of NLRP3 and caspase-11. Activation and upregulation of the canonical inflammasome upon loss of XIAP was mediated by RIPK1 kinase activity and ROS production. While both the inhibition of RIPK1 kinase activity and ROS production reduced cell death, as well as release of IL-1β, the release of IL-18 was not reduced to basal levels. This study supports targeting TNFR2 specifically to reduce IL-18 release in XLP-2 patients and to reduce priming of the inflammasome components

TNFR2 induced priming of the inflammasome leads to a RIPK1-dependent cell death in the absence of XIAP

The pediatric immune deficiency X-linked proliferative disease-2 (XLP-2) is a unique disease, with patients presenting with either hemophagocytic lymphohistiocytosis (HLH) or intestinal bowel disease (IBD). Interestingly, XLP-2 patients display high levels of IL-18 in the serum even while in stable condition, presumably through spontaneous inflammasome activation. Recent data suggests that LPS stimulation can trigger inflammasome activation through a TNFR2/TNF/TNFR1 mediated loop in xiap$^{-/-}$ macrophages. Yet, the direct role TNFR2-specific activation plays in the absence of XIAP is unknown. We found TNFR2-specific activation leads to cell death in xiap$^{-/-}$ myeloid cells, particularly in the absence of the RING domain. RIPK1 kinase activity downstream of TNFR2 resulted in a TNF/TNFR1 cell death, independent of necroptosis. TNFR2-specific activation leads to a similar inflammatory NF-kB driven transcriptional profile as TNFR1 activation with the exception of upregulation of NLRP3 and caspase-11. Activation and upregulation of the canonical inflammasome upon loss of XIAP was mediated by RIPK1 kinase activity and ROS production. While both the inhibition of RIPK1 kinase activity and ROS production reduced cell death, as well as release of IL-1β, the release of IL-18 was not reduced to basal levels. This study supports targeting TNFR2 specifically to reduce IL-18 release in XLP-2 patients and to reduce priming of the inflammasome components

Correction: TNFR2 induced priming of the inflammasome leads to a RIPK1-dependent cell death in the absence of XIAP

The pediatric immune deficiency X-linked proliferative disease-2 (XLP-2) is a unique disease, with patients presenting with either hemophagocytic lymphohistiocytosis (HLH) or intestinal bowel disease (IBD). Interestingly, XLP-2 patients display high levels of IL-18 in the serum even while in stable condition, presumably through spontaneous inflammasome activation. Recent data suggests that LPS stimulation can trigger inflammasome activation through a TNFR2/TNF/TNFR1 mediated loop in xiap$^{-/-}$ macrophages. Yet, the direct role TNFR2-specific activation plays in the absence of XIAP is unknown. We found TNFR2-specific activation leads to cell death in xiap$^{-/-}$ myeloid cells, particularly in the absence of the RING domain. RIPK1 kinase activity downstream of TNFR2 resulted in a TNF/TNFR1 cell death, independent of necroptosis. TNFR2-specific activation leads to a similar inflammatory NF-kB driven transcriptional profile as TNFR1 activation with the exception of upregulation of NLRP3 and caspase-11. Activation and upregulation of the canonical inflammasome upon loss of XIAP was mediated by RIPK1 kinase activity and ROS production. While both the inhibition of RIPK1 kinase activity and ROS production reduced cell death, as well as release of IL-1β, the release of IL-18 was not reduced to basal levels. This study supports targeting TNFR2 specifically to reduce IL-18 release in XLP-2 patients and to reduce priming of the inflammasome components

SPATA2 restricts OTULIN-dependent LUBAC activity independently of CYLD

SPATA2 mediates the recruitment of CYLD to immune receptor complexes by bridging the interaction of CYLD with the linear ubiquitylation assembly complex (LUBAC) component HOIP. Whether SPATA2 exhibits functions independently of CYLD is unclear. Here, we show that, while Cyld−/− and Spata2−/− mice are viable, double mutants exhibit highly penetrant perinatal lethality, indicating independent functions of SPATA2 and CYLD. Cyld−/−Spata2−/− fibroblasts show increased M1-linked TNFR1-SC ubiquitylation and, similar to Cyld−/−Spata2−/− macrophages and intestinal epithelial cells, elevated pro-inflammatory gene expression compared with Cyld−/− or Spata2−/− cells. We show that SPATA2 competes with OTULIN for binding to HOIP via its PUB-interacting motif (PIM) and its zinc finger domain, thereby promoting autoubiquitylation of LUBAC. Consistently, increased pro-inflammatory signaling in Cyld−/−Spata2−/− cells depends on the presence of OTULIN. Our data therefore indicate that SPATA2 counteracts, independently of CYLD, the deubiquitylation of LUBAC by OTULIN and thereby attenuates LUBAC activity and pro-inflammatory signaling

Disease-causing mutations in the XIAP BIR2 domain impair NOD2-dependent immune signalling.

X-linked Inhibitor of Apoptosis (XIAP) is an essential ubiquitin ligase for pro-inflammatory signalling downstream of the nucleotide-binding oligomerization domain containing (NOD)-1 and -2 pattern recognition receptors. Mutations in XIAP cause X-linked lymphoproliferative syndrome type-2 (XLP2), an immunodeficiency associated with a potentially fatal deregulation of the immune system, whose aetiology is not well understood. Here, we identify the XIAP baculovirus IAP repeat (BIR)2 domain as a hotspot for missense mutations in XLP2. We demonstrate that XLP2-BIR2 mutations severely impair NOD1/2-dependent immune signalling in primary cells from XLP2 patients and in reconstituted XIAP-deficient cell lines. XLP2-BIR2 mutations abolish the XIAP-RIPK2 interaction resulting in impaired ubiquitylation of RIPK2 and recruitment of linear ubiquitin chain assembly complex (LUBAC) to the NOD2-complex. We show that the RIPK2 binding site in XIAP overlaps with the BIR2 IBM-binding pocket and find that a bivalent Smac mimetic compound (SMC) potently antagonises XIAP function downstream of NOD2 to limit signalling. These findings suggest that impaired immune signalling in response to NOD1/2 stimulation is a general defect in XLP2 and demonstrate that the XIAP BIR2-RIPK2 interaction may be targeted pharmacologically to modulate inflammatory signalling