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Featured researches published by Gunes Bozkurt.


Proceedings of the National Academy of Sciences of the United States of America | 2010

FlhA provides the adaptor for coordinated delivery of late flagella building blocks to the type III secretion system

Gert Bange; Nico Kümmerer; Christoph Engel; Gunes Bozkurt; Klemens Wild; Irmgard Sinning

Flagella are the bacterial organelles of motility and can play important roles in pathogenesis. Flagella biosynthesis requires the coordinated export of huge protein amounts from the cytosol to the nascent flagellar structure at the cell surface and employs a type III secretion system (T3SS). Here we show that the integral membrane protein FlhA from the gram-positive bacterium Bacillus subtilis acts as an adaptor for late export substrates at the T3SS. The major filament protein (flagellin) and the filament-cap protein (FliD) bind to the FlhA cytoplasmic domain (FlhA-C) only in complex with their cognate chaperones (FliS and FliT). To understand the molecular details of these interactions we determined the FlhA-C crystal structure at 2.3 Å resolution. FlhA-C consists of an N-terminal linker region, three subdomains with a novel fold, and a disordered region essential for the adaptor function. We show that the export protein FliJ associates with the linker region and modulates the binding properties of FlhA-C. While the interaction of FliD/FliT is enhanced, flagellin/FliS is not affected. FliJ also keeps FliT associated with FlhA-C and excess of FliT inhibits binding of FliD/FliT, suggesting that empty FliT chaperones stay associated with FliJ after export of FliD. Taken together, these results allow to propose a model that explains how the T3SS may switch from the stoichiometric export of FliD to the high-throughput secretion of flagellin.


Proceedings of the National Academy of Sciences of the United States of America | 2009

Structural insights into tail-anchored protein binding and membrane insertion by Get3

Gunes Bozkurt; Goran Stjepanovic; Fabio Vilardi; Stefan Amlacher; Klemens Wild; Gert Bange; Vincenzo Favaloro; Karsten Rippe; Ed Hurt; Bernhard Dobberstein; Irmgard Sinning

Tail-anchored (TA) membrane proteins are involved in a variety of important cellular functions, including membrane fusion, protein translocation, and apoptosis. The ATPase Get3 (Asna1, TRC40) was identified recently as the endoplasmic reticulum targeting factor of TA proteins. Get3 consists of an ATPase and α-helical subdomain enriched in methionine and glycine residues. We present structural and biochemical analyses of Get3 alone as well as in complex with a TA protein, ribosome-associated membrane protein 4 (Ramp4). The ATPase domains form an extensive dimer interface that encloses 2 nucleotides in a head-to-head orientation and a zinc ion. Amide proton exchange mass spectrometry shows that the α-helical subdomain of Get3 displays considerable flexibility in solution and maps the TA protein-binding site to the α-helical subdomain. The non-hydrolyzable ATP analogue AMPPNP-Mg2+- and ADP-Mg2+-bound crystal structures representing the pre- and posthydrolysis states are both in a closed form. In the absence of a TA protein cargo, ATP hydrolysis does not seem to be possible. Comparison with the ADP·AlF4−-bound structure representing the transition state (Mateja A, et al. (2009) Nature 461:361–366) indicates how the presence of a TA protein is communicated to the ATP-binding site. In vitro membrane insertion studies show that recombinant Get3 inserts Ramp4 in a nucleotide- and receptor-dependent manner. Although ATP hydrolysis is not required for Ramp4 insertion per se, it seems to be required for efficient insertion. We postulate that ATP hydrolysis is needed to release Get3 from its receptor. Taken together, our results provide mechanistic insights into posttranslational targeting of TA membrane proteins by Get3.


Nature Medicine | 2015

Active Pin1 is a key target of all- trans retinoic acid in acute promyelocytic leukemia and breast cancer

Shuo Wei; Shingo Kozono; Lev Kats; Morris Nechama; Wenzong Li; Jlenia Guarnerio; Manli Luo; Mi Hyeon You; Yandan Yao; Asami Kondo; Hai Hu; Gunes Bozkurt; Nathan J. Moerke; Shugeng Cao; Markus Reschke; Chun Hau Chen; Eduardo M. Rego; Francesco Lo-Coco; Lewis C. Cantley; Tae Ho Lee; Hao Wu; Yan Zhang; Pier Paolo Pandolfi; Xiao Zhen Zhou; Kun Ping Lu

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency for inhibiting Pin1 function in vivo. By using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)—a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but whose drug target remains elusive—inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the protein encoded by the fusion oncogene PML–RARA and treats APL in APL cell and animal models as well as in human patients. ATRA-induced Pin1 ablation also potently inhibits triple-negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors.


FEBS Letters | 2010

The structure of Get4 reveals an α-solenoid fold adapted for multiple interactions in tail-anchored protein biogenesis

Gunes Bozkurt; Klemens Wild; Stefan Amlacher; Ed Hurt; Bernhard Dobberstein; Irmgard Sinning

Tail‐anchored proteins play important roles in protein translocation, membrane fusion and apoptosis. They are targeted to the endoplasmic reticulum membrane via the guided‐entry of tail‐anchored proteins (Get) pathway. We present the 2 Å crystal structure of Get4 which participates in early steps of the Get pathway. The structure shows an α‐solenoid fold with particular deviations from the regular pairwise arrangement of α‐helices. A conserved hydrophobic groove accommodates the flexible C‐terminal region in trans. The structural organization of the Get4 helical hairpin motifs provides a scaffold for protein–protein interactions in the Get pathway.


Acta Crystallographica Section D-biological Crystallography | 2010

Structural insights into the assembly of the human and archaeal signal recognition particles.

Klemens Wild; Gert Bange; Gunes Bozkurt; Bernd Segnitz; Astrid Hendricks; Irmgard Sinning

The signal recognition particle (SRP) is a conserved ribonucleoprotein (RNP) complex that co-translationally targets membrane and secretory proteins to membranes. The assembly of the particle depends on the proper folding of the SRP RNA, which in mammalia and archaea involves an induced-fit mechanism within helices 6 and 8 in the S domain of SRP. The two helices are juxtaposed and clamped together upon binding of the SRP19 protein to their apices. In the current assembly paradigm, archaeal SRP19 causes the asymmetric loop of helix 8 to bulge out and expose the binding platform for the key player SRP54. Based on a heterologous archaeal SRP19-human SRP RNA structure, mammalian SRP19 was thought not to be able to induce this change, thus explaining the different requirements of SRP19 for SRP54 recruitment. In contrast, the crystal structures of a crenarchaeal and the all-human SRP19-SRP RNA binary complexes presented here show that the asymmetric loop is bulged out in both binary complexes. Differences in SRP assembly between mammalia and archaea are therefore independent of SRP19 and are based on differences in SRP RNA itself. A new SRP-assembly scheme is presented.


Cell Reports | 2012

CD8+ T Cells from Mice Transnuclear for a TCR that Recognizes a Single H-2Kb-Restricted MHV68 Epitope Derived from gB-ORF8 Help Control Infection

Sharvan Sehrawat; Oktay Kirak; Paul-Albert Koenig; Marisa K. Isaacson; Sofia Marques; Gunes Bozkurt; J. Pedro Simas; Rudolph Jaenisch; Hidde L. Ploegh

To study the CD8(+) T cell response against a mouse γ-herpes virus, we generated K(b)-MHV-68-ORF8(604-612)RAG(-/-) CD8(+) T cell receptor transnuclear (TN) mice as a source of virus-specific CD8(+) T cells. K(b)-ORF8-Tet(+) CD8(+) T cells, expanded in the course of a resolving MHV-68 infection, served as a source of nucleus donors. Various in vivo and ex vivo assay criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively in response to viral infection, helped control viral burden, and exhibited a phenotype similar to that of endogenous K(b)-ORF8-Tet(+) cells. When compared to OT-1 cells, TN cells displayed distinct properties in response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the TCR expressed by the TN cells. The availability of MHV-68-specific CD8(+) TCR TN mice provides a new tool for investigating aspects of host-pathogen interactions unique to γ-herpes viruses.


Immunity | 2017

Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling

Anni Kleino; Nancy Ramia; Gunes Bozkurt; Yanfang Shen; Himani Nailwal; Jing Huang; Johanna Napetschnig; Monique Gangloff; Francis Ka-Ming Chan; Hao Wu; Jixi Li; Neal S. Silverman

&NA; In the Drosophila immune response, bacterial derived diaminopimelic acid‐type peptidoglycan binds the receptors PGRP‐LC and PGRP‐LE, which through interaction with the adaptor protein Imd leads to activation of the NF‐&kgr;B homolog Relish and robust antimicrobial peptide gene expression. PGRP‐LC, PGRP‐LE, and Imd each contain a motif with some resemblance to the RIP Homotypic Interaction Motif (RHIM), a domain found in mammalian RIPK proteins forming functional amyloids during necroptosis. Here we found that despite sequence divergence, these Drosophila cryptic RHIMs formed amyloid fibrils in vitro and in cells. Amyloid formation was required for signaling downstream of Imd, and in contrast to the mammalian RHIMs, was not associated with cell death. Furthermore, amyloid formation constituted a regulatable step and could be inhibited by Pirk, an endogenous feedback regulator of this pathway. Thus, diverse sequence motifs are capable of forming amyloidal signaling platforms, and the formation of these platforms may present a regulatory point in multiple biological processes. Graphical Abstract Figure. No caption available. HighlightsFormation of functional Imd amyloid fibril is required for Drosophila NF‐&kgr;B signalingCryptic RHIM motifs in peptidoglycan receptors PGRP‐LC and PGRP‐LE form amyloidsProtofibrils of PGRP‐LC and PGRP‐LE seed Imd amyloid polymerization via Imd cRHIMsPirk, an NF‐&kgr;B target, acts as a feedback inhibitor of Imd amyloid formation &NA; Kleino et al. show that amyloid formation is required for activation of the Drosophila Imd pathway upon recognition of bacterial peptidoglycans. Amyloid formation involves a motif resembling one found in necroptosis‐associated mammalian proteins and can be negatively regulated, suggesting that amyloidal signaling platforms may present a regulatory point in multiple biological processes.


Biophysical Journal | 2017

Mapping the Broad Structural and Mechanical Properties of Amyloid Fibrils

Guillaume Lamour; Roy Nassar; Patrick H. W. Chan; Gunes Bozkurt; Jixi Li; Jennifer M. Bui; Calvin K. Yip; Thibault Mayor; Hongbin Li; Hao Wu; Jörg Gsponer

Amyloids are fibrillar nanostructures of proteins that are assembled in several physiological processes in human cells (e.g., hormone storage) but also during the course of infectious (prion) and noninfectious (nonprion) diseases such as Creutzfeldt-Jakob and Alzheimers diseases, respectively. How the amyloid state, a state accessible to all proteins and peptides, can be exploited for functional purposes but also have detrimental effects remains to be determined. Here, we measure the nanomechanical properties of different amyloids and link them to features found in their structure models. Specifically, we use shape fluctuation analysis and sonication-induced scission in combination with full-atom molecular dynamics simulations to reveal that the amyloid fibrils of the mammalian prion protein PrP are mechanically unstable, most likely due to a very low hydrogen bond density in the fibril structure. Interestingly, amyloid fibrils formed by HET-s, a fungal protein that can confer functional prion behavior, have a much higher Youngs modulus and tensile strength than those of PrP, i.e., they are much stiffer and stronger due to a tighter packing in the fibril structure. By contrast, amyloids of the proteins RIP1/RIP3 that have been shown to be of functional use in human cells are significantly stiffer than PrP fibrils but have comparable tensile strength. Our study demonstrates that amyloids are biomaterials with a broad range of nanomechanical properties, and we provide further support for the strong link between nanomechanics and β-sheet characteristics in the amyloid core.


Scientific Reports | 2017

Disulfide Bond Formation and N-Glycosylation Modulate Protein-Protein Interactions in GPI-Transamidase (GPIT)

Lina Yi; Gunes Bozkurt; Qiubai Li; Stanley M. Lo; Anant K. Menon; Hao Wu

Glycosylphosphatidylinositol (GPI) transamidase (GPIT), the enzyme that attaches GPI anchors to proteins as they enter the lumen of the endoplasmic reticulum, is a membrane-bound hetero-pentameric complex consisting of Gpi8, Gpi16, Gaa1, Gpi17 and Gab1. Here, we expressed and purified the luminal domain of Saccharomyces cerevisiae (S. cerevisiae) Gpi8 using different expression systems, and examined its interaction with insect cell expressed luminal domain of S. cerevisiae Gpi16. We found that the N-terminal caspase-like domain of Gpi8 forms a disulfide-linked dimer, which is strengthened by N-glycosylation. The non-core domain of Gpi8 following the caspase-like domain inhibits this dimerization. In contrast to the previously reported disulfide linkage between Gpi8 and Gpi16 in human and trypanosome GPIT, our data show that the luminal domains of S. cerevisiae Gpi8 and S. cerevisiae Gpi16 do not interact directly, nor do they form a disulfide bond in the intact S. cerevisiae GPIT. Our data suggest that subunit interactions within the GPIT complex from different species may vary, a feature that should be taken into account in future structural and functional studies.


Cell | 2018

The Structure of the Necrosome RIPK1-RIPK3 Core, a Human Hetero-Amyloid Signaling Complex

Miguel Mompeán; Wenbo Li; Jixi Li; Ségolène Laage; Ansgar B. Siemer; Gunes Bozkurt; Hao Wu; Ann E. McDermott

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Hao Wu

Boston Children's Hospital

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Hidde L. Ploegh

Massachusetts Institute of Technology

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Ed Hurt

Heidelberg University

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