An integrative structural biology analysis of von Willebrand factor binding and processing by ADAMTS-13 in solution

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An Integrative Structural Biology Analysis of Von Willebrand Factor Binding and Processing by ADAMTS-13 in Solution

Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a ‘fuzzy complex’ that follows a ‘dynamic zipper’ mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis.This study was supported in part by grants from Spanish, French, Danish and Catalan public and private bodies (grant/fellowship references PID2019-107725RG-I00, BES-2015-074583, ANR-10-LABX-12-01, 6108-00031B, 8022-00385B, LF18039, NNF18OC0032724, Novo Nordisk Foundation “Bio-MS”, 2017SGR3 and Fundació “La Marató de TV3” 201815). This work was also supported by EPICS-XS, project 823839, funded by the Horizon 2020 programme of the European Union. The CBS is a member of France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology (FRISBI), which are national infrastructures supported by the French National Research Agency (grants ANR-10-INBS-04-01 and ANR-10-INBS-05, respectively). Finally, we acknowledge the Structural Mass Spectrometry Unit of CIISB, an Instruct-CZ Centre, which was supported by MEYS CR (LM2018127)

Molecular and in vivo studies of a glutamate-class prolyl-endopeptidase for coeliac disease therapy

The digestion of gluten generates toxic peptides, among which a highly immunogenic proline-rich 33-mer from wheat α-gliadin, that trigger coeliac disease. Neprosin from the pitcher plant is a reported prolyl endopeptidase. Here, we produce recombinant neprosin and its mutants, and find that full-length neprosin is a zymogen, which is self-activated at gastric pH by the release of an all-β pro-domain via a pH-switch mechanism featuring a lysine plug. The catalytic domain is an atypical 7+8-stranded β-sandwich with an extended active-site cleft containing an unprecedented pair of catalytic glutamates. Neprosin efficiently degrades both gliadin and the 33-mer in vitro under gastric conditions and is reversibly inactivated at pH > 5. Moreover, co-administration of gliadin and the neprosin zymogen at the ratio 500:1 reduces the abundance of the 33-mer in the small intestine of mice by up to 90%. Neprosin therefore founds a family of eukaryotic glutamate endopeptidases that fulfils requisites for a therapeutic glutenase.This study was supported in part by grants from Spanish and Catalan public and private bodies (grant/fellowship references PID2019-107725RG-I00 to F.X.G.-R., A.R.B., U.E. and T.G.; BES-2016-076877 to S.R.M., BES-2015-074583 to L.A.M., Beatriu de Pinós 2018BP00163 to U.E., 2017SGR3 and Fundació La Marató de TV3 201815 to F.X.G.-R., U.E., A.R.B. and T.G.)

Functional interplays of proteins and proteases: AD13-VWF, TGFß2-¿2M, and the proteolysis of gliadin by neprosin

Tesis doctoral.-- Universitat de Barcelona. Departament de Bioquímica i Biologia Molecular (Farmàcia)The present thesis has established a new and state of the art mammalian protein expression system at the laboratory which has then contributed with high-yield expression for the purification and crystallization of three glycoproteins, namely TGFβ2, ADAMTS13 and Neprosin, which adopt important biomedical roles in human physiology, cardiovascular disorders, and celiac disease treatment, respectively. Proteases are major players in the physiology and pathology of all living organisms. Their often highly sophisticated regulation mechanisms are therefore essential for proper function, and to prevent misdirected spatial and/or temporal proteolytic activity, which in turn may trigger disease. This regulation is achieved through a wide variety of mechanisms including protein-protein interaction, substrate allosteric activation, or their biosynthesis as inactive precursor, so called zymogens. In the present thesis a variety of regulatory mechanisms of proteases and inhibitors were studied by using a combination of biochemical, biophysical and structural techniques. In the first project we comprehensively illuminate the biochemistry of the VWF:ADAMTS-13 axis. ADAMTS13 regulates the multimeric form of Von Willebrand Factor (VWF) in blood circulation by specific, shear-dependent proteolysis. Despite circulating in a seemingly latent form with a very long active plasma half-life, ADAMTS13 is resistant to plasma inhibitors and VWF is the only known substrate, and which involves an allosteric activation of ADAMTS13 by substrate binding. This unprecedented specificity has been attributed to extensive exosite interactions between the AD13-MDTCS domains, where the Disintegrin-like domain (D) approaches to Metalloprotease (M), Cysteine-rich (C), and Spacer (S) domain once the VWF is bound achieving a “tight” conformation. Structural analysis in solution revealed that the enzyme adopts a highly flexible unbound, latent structures and VWF peptide-bound, active structures that significantly differes from the AD13-MDTCS crystal structure. We integrate the experimental results with computational approaches. Thus, we hypothesize that the interaction culminates in a ‘fuzzy complex’ that follows a ‘dynamic zipper’ mechanism involving numerous reversible, weak but additive interactions that result in strong binding and ultimately in cleavage. In the second project a novel family of glutamate peptidases was studied and the crystal structure determined of both the active and zymogen form, establishing the active site, catalytic mechanism and protease class annotation, and describing a unique catalytic dyad composed by two glutamates while showing a close structural homology with eqolisins

Functional interplays of proteins and proteases: AD13-VWF, TGFβ2-α2M, and the proteolysis of gliadin by neprosin

[eng] The present thesis has established a new and state of the art mammalian protein expression system at the laboratory which has then contributed with high-yield expression for the purification and crystallization of three glycoproteins, namely TGFβ2, ADAMTS13 and Neprosin, which adopt important biomedical roles in human physiology, cardiovascular disorders, and celiac disease treatment, respectively. Proteases are major players in the physiology and pathology of all living organisms. Their often highly sophisticated regulation mechanisms are therefore essential for proper function, and to prevent misdirected spatial and/or temporal proteolytic activity, which in turn may trigger disease. This regulation is achieved through a wide variety of mechanisms including protein-protein interaction, substrate allosteric activation, or their biosynthesis as inactive precursor, so called zymogens. In the present thesis a variety of regulatory mechanisms of proteases and inhibitors were studied by using a combination of biochemical, biophysical and structural techniques. In the first project we comprehensively illuminate the biochemistry of the VWF:ADAMTS-13 axis. ADAMTS13 regulates the multimeric form of Von Willebrand Factor (VWF) in blood circulation by specific, shear-dependent proteolysis. Despite circulating in a seemingly latent form with a very long active plasma half-life, ADAMTS13 is resistant to plasma inhibitors and VWF is the only known substrate, and which involves an allosteric activation of ADAMTS13 by substrate binding. This unprecedented specificity has been attributed to extensive exosite interactions between the AD13-MDTCS domains, where the Disintegrin-like domain (D) approaches to Metalloprotease (M), Cysteine-rich (C), and Spacer (S) domain once the VWF is bound achieving a “tight” conformation. Structural analysis in solution revealed that the enzyme adopts a highly flexible unbound, latent structures and VWF peptide-bound, active structures that significantly differes from the AD13-MDTCS crystal structure. We integrate the experimental results with computational approaches. Thus, we hypothesize that the interaction culminates in a ‘fuzzy complex’ that follows a ‘dynamic zipper’ mechanism involving numerous reversible, weak but additive interactions that result in strong binding and ultimately in cleavage. In the second project a novel family of glutamate peptidases was studied and the crystal structure determined of both the active and zymogen form, establishing the active site, catalytic mechanism and protease class annotation, and describing a unique catalytic dyad composed by two glutamates while showing a close structural homology with eqolisins. Neprosin was recently discovered in the fluid of the carnivorous pitcher plant Nepenthes ventrata. Given its high activity at low pH, thermic stability and great specificity, neprosin is especially useful in detoxifying gluten proteins including wheat gliadin and the 33-mer immunogenic fragment into non-toxic peptides in a human stomach environment. Additionally, we determined its inhibitory profile against a cohort of peptidase inhibitors. Future approaches will study the effect of neprosin processing of the 33-mer on inflammatory responses in cells and tissue explants to elucidate if neprosin indeed presents an effective and harmless supplementation approach for gluten intolerant patients. In the third project the main protease inhibitor of the blood plasma has been studied; human α2 Macroglobulin (hα2Ms) has a great role on the human physiology by modulating the activity of cytokines such as TGFβ2. We characterised the interaction by a variety of biochemical, biophysical, and binding techniques. Intriguingly, during our crystallographic studies of the complex, we obtained a crystal structure of a TGFβ2 dimer in a novel dimeric assembly of biological unknown function. Overall, the present thesis contributes substantially to the field of structural biochemistry, expanding previous knowledge at the molecular level and adding new regulatory mechanisms, which will hopefully pave the way for the design of specific drugs for therapeutic interventions as well as lead to new approaches for treating coeliac disease through an enzyme supplementation strategy

Recombinant production, purification, crystallization, and structure analysis of human transforming growth factor β2 in a new conformation

© The Author(s) 2019.Transforming growth factor β is a disulfide-linked dimeric cytokine that occurs in three highly related isoforms (TGFβ1–TGFβ3) engaged in signaling functions through binding of cognate TGFβ receptors. To regulate this pathway, the cytokines are biosynthesized as inactive pro-TGFβs with an N-terminal latency-associated protein preceding the mature moieties. Due to their pleiotropic implications in physiology and pathology, TGFβs are privileged objects of in vitro studies. However, such studies have long been limited by the lack of efficient human recombinant expression systems of native, glycosylated, and homogenous proteins. Here, we developed pro-TGFβ2 production systems based on human Expi293F cells, which yielded >2 mg of pure histidine- or Strep-tagged protein per liter of cell culture. We assayed this material biophysically and in crystallization assays and obtained a different crystal form of mature TGFβ2, which adopted a conformation deviating from previous structures, with a distinct dimeric conformation that would require significant rearrangement for binding of TGFβ receptors. This new conformation may be reversibly adopted by a certain fraction of the mature TGβ2 population and represent a hitherto undescribed additional level of activity regulation of the mature growth factor once the latency-associated protein has been separated.This study was supported in part by grants from Spanish and Catalan public and private bodies (grant/fellowship references BFU2015-64487R; MDM-2014-0435; JCI-2012-13573; BES-2015-074583; BES-2013-064651; 2017SGR3; and Fundació “La Marató de TV3” 201815). Te Structural Biology Unit of IBMB is a “María de Maeztu” Unit of Excellence from the Spanish Ministry of Science, Innovation and Universities

Recombinant production of human α2-macroglobulin variants and interaction studies with recombinant G-related α2-macroglobulin binding protein and latent transforming growth factor-β2

© The Author(s) 2019.α2-Macroglobulins (α2Ms) regulate peptidases, hormones and cytokines. Mediated by peptidase cleavage, they transit between native, intact forms and activated, induced forms. α2Ms have been studied over decades using authentic material from primary sources, which was limited by sample heterogeneity and contaminants. Here, we developed high-yield expression systems based on transient transfection in Drosophila Schneider 2 and human Expi293F cells, which produced pure human α2M (hα2M) at ~1.0 and ~0.4 mg per liter of cell culture, respectively. In both cases, hα2M was mainly found in the induced form. Shorter hα2M variants encompassing N-/C-terminal parts were also expressed and yielded pure material at ~1.6/~1.3 and ~3.2/~4.6 mg per liter of insect or mammalian cell culture, respectively. We then analyzed the binding of recombinant and authentic hα2M to recombinant latent human transforming growth factor-β2 (pro-TGF-β2) and bacterial G-related α2M binding protein (GRAB) by surface plasmon resonance, multiple-angle laser light scattering, size-exclusion chromatography, fluorogenic labelling, gel electrophoresis and Western-blot analysis. Two GRAB molecules formed stable complexes of high affinity with native and induced authentic hα2M tetramers. The shorter recombinant hα2M variants interacted after preincubation only. In contrast, pro-TGF-β2 did not interact, probably owing to hindrance by the N-terminal latency-associated protein of the cytokine.This study was supported in part by grants from Spanish and Catalan public and private bodies (grant/fellowship references BFU2015-64487R; MDM-2014-0435; JCI-2012-13573; BES-2015-074583; BES2013-064651; 2017SGR3; and Fundació “La Marató de TV3” 201815). Te Structural Biology Unit of IBMB is a “María de Maeztu” Unit of Excellence from the Spanish Ministry of Science, Innovation and Universities

Analysis of the inhibiting activity of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) on matrix metalloproteinases

© The Author(s) 2020.Matrix metalloproteinases (MMPs) occur in 23 human paralogues with key functions in physiology, and their activity is controlled by protein inhibitors. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), which is essential for embryogenesis and tumour suppression, has been reported to inhibit MMPs. Here, we developed eukaryotic and bacterial expression systems for different RECK variants and analysed their inhibitory capacity against representative MMPs in vitro. We could not detect any significant inhibition. Instead, we found that partially purified RECK from the conditioned medium of transfected Expi293F cells but not that of ExpiCHO-S or Drosophila Schneider cells contained a contaminant with proteolytic activity. The contaminant was removed through treatment with a small-molecule serine peptidase inhibitor and additional chromatographic purification. A tantamount contaminant was further detected in an equivalent expression system of the N-terminal fragment of the proteoglycan testican 3, but not in those of two other proteins. These results indicate that previous reports of inhibitory activity of recombinant RECK on MMPs, which were performed with partially purified samples, were probably masked by a coeluting contaminant present in the supernatant of HEK293-derived cells. Thus, RECK is probably not a direct inhibitor of MMP catalytic activity but may still regulate MMPs through other mechanisms.This study was supported in part by grants from Spanish and Catalan public and private bodies (grant/ fellowship references BFU2015–64487R, MDM-2014–0435, BES-2015–074583, BES-2016–076877, 2017SGR3 and Fundació “La Marató de TV3” 201815)

Impact of late presentation of HIV infection on short-, mid- and long-term mortality and causes of death in a multicenter national cohort : 2004-2013

To analyze the impact of late presentation (LP) on overall mortality and causes of death and describe LP trends and risk factors (2004-2013). Cox models and logistic regression were used to analyze data from a nation-wide cohort in Spain. LP is defined as being diagnosed when CD4 < 350 cells/ml or AIDS. Of 7165 new HIV diagnoses, 46.9% (CI:45.7-48.0) were LP, 240 patients died.First-year mortality was the highest (aHR = 10.3[CI:5.5-19.3]); between 1 and 4 years post-diagnosis, aHR = 1.9(1.2-3.0); an