Publications by authors named "Luiza Deszcz"

12 Publications

  • Page 1 of 1

HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain.

Nat Chem Biol 2021 Jul 22. Epub 2021 Jul 22.

Research Institute of Molecular Pathology, Vienna BioCenter, Vienna, Austria.

HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates. The protein adopts a remarkable snake-like structure, where the C-terminal HECT domain heads an extended alpha-solenoid body that coils in on itself and houses various protein-protein interaction modules. Our integrative structural analysis shows that this ring structure is highly dynamic, enabling the flexible HECT domain to reach protein targets presented by the various acceptor sites. Together, our data demonstrate how HUWE1 is regulated by its unique structure, adapting a promiscuous E3 ligase to selectively target unassembled orphan proteins.
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http://dx.doi.org/10.1038/s41589-021-00831-5DOI Listing
July 2021

The linear ubiquitin chain assembly complex (LUBAC) generates heterotypic ubiquitin chains.

Elife 2021 06 18;10. Epub 2021 Jun 18.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), Vienna, Austria.

The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L), was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RING-in-between-RING (RBR) domains, a coordinated ubiquitin relay mechanism between the HOIL-1-interacting protein (HOIP) and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L.
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http://dx.doi.org/10.7554/eLife.60660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8245127PMC
June 2021

Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling.

EMBO J 2020 12 20;39(24):e103303. Epub 2020 Nov 20.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria.

HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site-specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)-induced inflammatory signaling. A HOIP K784R mutant is catalytically active but shows reduced induction of an NF-κB reporter relative to wild-type HOIP. HOIP K784 is evolutionarily conserved, equivalent to HOIP K778 in mice. We generated Hoip knock-in mice, which show no overt developmental phenotypes; however, in response to TNF, Hoip mouse embryonic fibroblasts display mildly suppressed NF-κB activation and increased apoptotic markers. On the other hand, HOIP K778R enhances the TNF-induced formation of TNFR complex II and an interaction between TNFR complex II and LUBAC. Loss of the LUBAC component SHARPIN leads to embryonic lethality in Hoip mice, which is rescued by knockout of TNFR1. We propose that site-specific ubiquitination of HOIP regulates a LUBAC-dependent switch between survival and apoptosis in TNF signaling.
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http://dx.doi.org/10.15252/embj.2019103303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737615PMC
December 2020

Molecular features of the UNC-45 chaperone critical for binding and folding muscle myosin.

Nat Commun 2019 10 21;10(1):4781. Epub 2019 Oct 21.

Research Institute of Molecular Pathology, Vienna BioCenter, Vienna, Austria.

Myosin is a motor protein that is essential for a variety of processes ranging from intracellular transport to muscle contraction. Folding and assembly of myosin relies on a specific chaperone, UNC-45. To address its substrate-targeting mechanism, we reconstitute the interplay between Caenorhabditis elegans UNC-45 and muscle myosin MHC-B in insect cells. In addition to providing a cellular chaperone assay, the established system enabled us to produce large amounts of functional muscle myosin, as evidenced by a biochemical and structural characterization, and to directly monitor substrate binding to UNC-45. Data from in vitro and cellular chaperone assays, together with crystal structures of binding-deficient UNC-45 mutants, highlight the importance of utilizing a flexible myosin-binding domain. This so-called UCS domain can adopt discrete conformations to efficiently bind and fold substrate. Moreover, our data uncover the molecular basis of temperature-sensitive UNC-45 mutations underlying one of the most prominent motility defects in C. elegans.
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http://dx.doi.org/10.1038/s41467-019-12667-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803673PMC
October 2019

The IAP family member BRUCE regulates autophagosome-lysosome fusion.

Nat Commun 2018 02 9;9(1):599. Epub 2018 Feb 9.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna BioCenter (VBC), 1030, Vienna, Austria.

Autophagy has an important role in cellular homeostasis by degrading and recycling cytotoxic components. Ubiquitination is known to target cargoes for autophagy; however, key components of this pathway remain elusive. Here we performed an RNAi screen to uncover ubiquitin modifiers that are required for starvation-induced macroautophagy in mammalian cells. Our screen uncovered BRUCE/Apollon/Birc6, an IAP protein, as a new autophagy regulator. Depletion of BRUCE leads to defective fusion of autophagosomes and lysosomes. Mechanistically, BRUCE selectively interacts with two ATG8 members GABARAP and GABARAPL1, as well as with Syntaxin 17, which are all critical regulators of autophagosome-lysosome fusion. In addition, BRUCE colocalizes with LAMP2. Interestingly, a non-catalytic N-terminal BRUCE fragment that is sufficient to bind GABARAP/GABARAPL1 and Syntaxin 17, and to colocalize with LAMP2, rescues autolysosome formation in Bruce cells. Thus, BRUCE promotes autolysosome formation independently of its ubiquitin-conjugating activity and is a regulator of both macroautophagy and apoptosis.
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http://dx.doi.org/10.1038/s41467-018-02823-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5807552PMC
February 2018

Arginine phosphorylation marks proteins for degradation by a Clp protease.

Nature 2016 11 6;539(7627):48-53. Epub 2016 Oct 6.

Research Institute of Molecular Pathology (IMP), Dr-Bohr-Gasse 7, 1030 Vienna, Austria.

Protein turnover is a tightly controlled process that is crucial for the removal of aberrant polypeptides and for cellular signalling. Whereas ubiquitin marks eukaryotic proteins for proteasomal degradation, a general tagging system for the equivalent bacterial Clp proteases is not known. Here we describe the targeting mechanism of the ClpC-ClpP proteolytic complex from Bacillus subtilis. Quantitative affinity proteomics using a ClpP-trapping mutant show that proteins phosphorylated on arginine residues are selectively targeted to ClpC-ClpP. In vitro reconstitution experiments demonstrate that arginine phosphorylation by the McsB kinase is required and sufficient for the degradation of substrate proteins. The docking site for phosphoarginine is located in the amino-terminal domain of the ClpC ATPase, as resolved at high resolution in a co-crystal structure. Together, our data demonstrate that phosphoarginine functions as a bona fide degradation tag for the ClpC-ClpP protease. This system, which is widely distributed across Gram-positive bacteria, is functionally analogous to the eukaryotic ubiquitin-proteasome system.
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http://dx.doi.org/10.1038/nature20122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640040PMC
November 2016

Linear ubiquitination by LUBEL has a role in Drosophila heat stress response.

EMBO Rep 2016 11 4;17(11):1624-1640. Epub 2016 Oct 4.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, Austria

The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL-1L and SHARPIN in mammals. Here, we provide the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila We identify Drosophila CG11321, which we named Linear Ubiquitin E3 ligase (LUBEL), and find that it catalyzes linear ubiquitination in vitro We detect endogenous linear ubiquitin chain-derived peptides by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, we establish linear ubiquitination-defective flies by mutating residues essential for the catalytic activity of LUBEL Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies.
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http://dx.doi.org/10.15252/embr.201642378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090701PMC
November 2016

Induction of autophagy by spermidine promotes longevity.

Nat Cell Biol 2009 Nov 4;11(11):1305-14. Epub 2009 Oct 4.

Institute of Molecular Biosciences, University of Graz, 8010 Graz, Austria.

Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases (HAT), suppressing oxidative stress and necrosis. Conversely, depletion of endogenous polyamines led to hyperacetylation, generation of reactive oxygen species, early necrotic death and decreased lifespan. The altered acetylation status of the chromatin led to significant upregulation of various autophagy-related transcripts, triggering autophagy in yeast, flies, worms and human cells. Finally, we found that enhanced autophagy is crucial for polyamine-induced suppression of necrosis and enhanced longevity.
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http://dx.doi.org/10.1038/ncb1975DOI Listing
November 2009

An antiviral peptide inhibitor that is active against picornavirus 2A proteinases but not cellular caspases.

J Virol 2006 Oct;80(19):9619-27

Max F. Perutz Laboratories, Medical University of Vienna, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria.

The replication of many viruses is absolutely dependent on proteolytic cleavage. Infected cells also use this biological mechanism to induce programmed cell death in response to viral infection. Specific inhibitors for both viral and cellular proteases are therefore of vital importance. We have recently shown that the general caspase inhibitor zVAD.fmk inhibits not only caspases, but also the 2Apro of human rhinoviruses (HRVs) (L. Deszcz, J. Seipelt, E. Vassilieva, A. Roetzer, and E. Kuechler, FEBS Lett. 560:51-55, 2004). Here, we describe a derivative of zVAD.fmk that inhibits HRV2 2Apro but that has no effect on caspase 9. This gain in specificity was achieved by replacing the aspartic acid of zVAD.fmk with methionine to generate zVAM.fmk. Methionine was chosen because an oligopeptide with methionine at the P1 position was a much better substrate than an oligopeptide with an alanine residue, which is found at the P1 position of the wild-type HRV2 2Apro cleavage site. zVAM.fmk inhibits the replication of HRV type 2 (HRV2), HRV14, and HRV16. In contrast to zVAD.fmk, however, zVAM.fmk did not inhibit apoptosis induced by puromycin in HeLa cells. zVAM.fmk inhibited in vitro the intermolecular cleavage of eukaryotic initiation factor 4GI (eIF4GI) by HRV2 2Apro at nanomolar concentrations. However, much higher concentrations of zVAM.fmk were required to inhibit HRV14 2Apro cleavage of eIF4GI. In contrast, intramolecular self-processing of HRV14 2Apro was much more susceptible to inhibition by zVAM.fmk than that of HRV2 2Apro, suggesting that zVAM.fmk inhibits HRV2 and HRV14 replication by targeting different reactions of the same proteinase.
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http://dx.doi.org/10.1128/JVI.00612-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1617246PMC
October 2006

Human rhinoviruses inhibit the accessory function of dendritic cells by inducing sialoadhesin and B7-H1 expression.

J Immunol 2005 Jul;175(2):1145-52

Institute of Immunology and Institute of Biochemistry, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria.

Dendritic cells (DC) are professional APCs with an unmatched ability to interact with and activate T cells. There is accumulating evidence that DC not only efficiently stimulate T cell activation but also regulate T cell responses. However, little is known about cell surface structures on DC involved in the regulation of T cell responses. We demonstrate that human rhinoviruses (HRV) can efficiently inhibit the accessory function of DC through induction of inhibitory cell surface receptors. We observed that treatment of DC with HRV14 (R-DC), a member of the major group HRV family, diminished their T cell stimulatory capacity and induced a promiscuous and deep anergic state in cocultured T cells despite high levels of MHC molecules as well as costimulatory molecules, e.g., B7-1 (CD80) and B7-2 (CD86), and independent of inhibitory soluble factors such as IL-10. In contrast, expression of inhibitory B7-H1 molecules was up-regulated and R-DC de novo expressed sialoadhesin (Sn). Most importantly, blocking of B7-H1 and Sn on R-DC with specific mAbs against both receptors reverted the inhibitory phenotype. Thus, inhibitory signals delivered from R-DC to T cells via B7-H1 and Sn were critical for the induction of anergy. These observations suggest that an altered accessory molecule repertoire on DC upon interaction with HRV down-modulates adaptive immune responses during the viral infection.
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http://dx.doi.org/10.4049/jimmunol.175.2.1145DOI Listing
July 2005

Apoptotic events induced by human rhinovirus infection.

J Gen Virol 2005 May;86(Pt 5):1379-1389

Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Medical Biochemistry, Medical University of Vienna, Dr Bohr Gasse 9/3, A-1030 Vienna, Austria.

HeLa and 16HBE14o(-) bronchial epithelium cells infected with human rhinovirus serotype 14 (HRV14) were found to exhibit typical apoptotic morphological alterations, such as cell contraction and nuclear condensation. These events coincided with high-molecular-weight DNA fragmentation, activation of caspase-9 and caspase-3 and poly(ADP-ribose) polymerase cleavage. Caspase activation was preceded by cytochrome c translocation from the mitochondria to the cytoplasm, indicating that apoptosis caused by HRV14 infection was triggered predominantly via the mitochondrial pathway. Apoptosis did not affect HRV14 replication per se, but it facilitated the release of newly formed virus from cells. As apoptosis was fully induced at the time of maximal accumulation of progeny HRV14, it is postulated that apoptosis contributed to the destabilization of the cell and facilitated viral progeny release.
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http://dx.doi.org/10.1099/vir.0.80754-0DOI Listing
May 2005

Antiviral activity of caspase inhibitors: effect on picornaviral 2A proteinase.

FEBS Lett 2004 Feb;560(1-3):51-5

Department of Medical Biochemistry, Division of Biochemistry, University of Vienna, Dr. Bohr Gasse 9/3, A-1030 Vienna, Austria.

Peptide-based fluoromethyl ketones have been considered for many years to be highly specific caspase inhibitors distinctly blocking the progress of apoptosis in a variety of systems. Here we demonstrate that these compounds can significantly reduce rhinovirus multiplication in cell culture. In their methylated forms they block eIF4GI cleavage in vivo and in vitro and inhibit the activity of picornaviral 2A proteinases.
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http://dx.doi.org/10.1016/S0014-5793(04)00069-9DOI Listing
February 2004
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