Tatyana Sandalova - Academia.edu (original) (raw)

Papers by Tatyana Sandalova

Spr1654 from Streptococcus pneumoniae plays a key role in the production of un... more Spr1654 from Streptococcus pneumoniae plays a key role in the production of unusual sugars, presumably functioning as a pyridoxal-5′-phosphate (PLP)-dependent aminotransferase. Spr1654 was predicted to catalyse the transferring of amino group to form the amino sugar 2-acetamido-4-amino-2, 4, 6-trideoxygalactose moiety (AATGal), representing a crucial step in biosynthesis of teichoic acids in S. pneumoniae. We have determined the crystal structures of the apo-, PLP- and PMP-bound forms of Spr1654. Spr1654 forms a homodimer, in which each monomer contains one active site. Using spectrophotometry and based on absorbance profiles of PLP- and PMP-formed enzymes, our results indicate that l-glutamate is most likely the preferred amino donor. Structural superposition of the crystal structures of Spr1654 on previously determined structures of other sugar aminotransferases in complex with glutamate and/or UDP-activated sugar allowed us to identify key Spr1654 residues for ligand binding and catalysis. The crystal structures of Spr1654 and in complex with PLP and PMP can direct the future rational design of novel therapeutic compounds against S. pneumoniae.

Open Biology, Apr 1, 2018

Spr1654 from Streptococcus pneumoniae plays a key role in the production of unusual sugars, presu... more Spr1654 from Streptococcus pneumoniae plays a key role in the production of unusual sugars, presumably functioning as a pyridoxal-5 0-phosphate (PLP)-dependent aminotransferase. Spr1654 was predicted to catalyse the transferring of amino group to form the amino sugar 2-acetamido-4amino-2, 4, 6-trideoxygalactose moiety (AATGal), representing a crucial step in biosynthesis of teichoic acids in S. pneumoniae. We have determined the crystal structures of the apo-, PLP-and PMP-bound forms of Spr1654. Spr1654 forms a homodimer, in which each monomer contains one active site. Using spectrophotometry and based on absorbance profiles of PLPand PMP-formed enzymes, our results indicate that L-glutamate is most likely the preferred amino donor. Structural superposition of the crystal structures of Spr1654 on previously determined structures of other sugar aminotransferases in complex with glutamate and/or UDP-activated sugar allowed us to identify key Spr1654 residues for ligand binding and catalysis. The crystal structures of Spr1654 and in complex with PLP and PMP can direct the future rational design of novel therapeutic compounds against S. pneumoniae.

Arthritis & rheumatology, Nov 26, 2019

ObjectiveAutoantibodies targeting histidyl–transfer RNA synthetase (HisRS; anti–Jo‐1) are common ... more ObjectiveAutoantibodies targeting histidyl–transfer RNA synthetase (HisRS; anti–Jo‐1) are common in the idiopathic inflammatory myopathies (IIMs) and antisynthetase syndrome. This study was undertaken to investigate immunity against HisRS in the blood and lungs of patients with IIM/antisynthetase syndrome.MethodsBronchoalveolar lavage (BAL) fluid, BAL fluid cells, and peripheral blood mononuclear cells (PBMCs) from patients with IIM/antisynthetase syndrome (n = 24) were stimulated with full‐length HisRS protein or a HisRS‐derived peptide (HisRS11–23). BAL fluid and PBMCs from patients with sarcoidosis (n = 7) and healthy subjects (n = 12) were included as controls. The CD4+ T cell response was determined according to levels of CD40L up‐regulation and cytokine expression using flow cytometry. Anti–Jo‐1 autoantibody responses in the serum and BAL fluid were assessed by enzyme‐linked immunosorbent assay. Lung biopsy samples from patients with IIM/antisynthetase syndrome (n = 14) were investigated by immunohistochemistry.ResultsIn BAL fluid, CD4+ T cells from 3 of 4 patients with IIM/antisynthetase syndrome responded to stimulation with HisRS protein, as measured by the median fold change in CD40L expresssion in stimulated cells compared to unstimulated cells (median fold change 3.6, interquartile range [IQR] 2.7–14.7), and 2 of 3 patients with IIM/antisynthetase syndrome had the highest responses to HisRS11–23 (median fold change 88, IQR 27–149). In PBMCs, CD4+ T cells from 14 of 18 patients with IIM/antisynthetase syndrome responded to HisRS protein (median fold change 7.38, IQR 2.69–31.86; P < 0.001), whereas a HisRS11–23 response was present in 11 of 14 patients with IIM/antisynthetase syndrome (median fold change 3.4, IQR 1.87–10.9; P < 0.001). In the control group, there was a HisRS11–23 response in 3 of 7 patients with sarcoidosis (median fold change 2.09, IQR 1.45–3.29) and in 5 of 12 healthy controls (median fold change 2, IQR 1.89–2.42). CD4+ T cells from patients with IIM/antisynthetase syndrome displayed a pronounced Th1 phenotype in the BAL fluid when compared to the PBMCs (P < 0.001), producing high amounts of interferon‐γ and interleukin‐2 following stimulation. Anti–Jo‐1 autoantibodies were detected in BAL fluid and germinal center (GC)–like structures were seen in the lung biopsy samples from patients with IIM/antisynthetase syndrome.ConclusionThe results of this study demonstrate a pronounced presence of HisRS‐reactive CD4+ T cells in PBMCs and BAL fluid cells from patients with IIM/antisynthetase syndrome as compared to patients with sarcoidosis and healthy controls. These findings, combined with the presence of anti–Jo‐1 autoantibodies in BAL fluid and GC‐like structures in the lungs, suggest that immune activation against HisRS might take place within the lungs of patients with IIM/antisynthetase syndrome.

Journal of Clinical Investigation, Mar 12, 2018

the SNPs rs1004443-G, rs3760860-A, and rs3760861-A. Given the ability of UL18 to inhibit NK cell ... more the SNPs rs1004443-G, rs3760860-A, and rs3760861-A. Given the ability of UL18 to inhibit NK cell responses and the apparent importance of NK cells in controlling HCMV replication, we investigated LILRB1 polymorphisms in transplant patients to test the hypothesis that individuals with greater LILRB1 expression on NK cells would exhibit poorer control of HCMV replication. In con-cells, and 20%-70% of NK cells (6, 19, 20). We previously defined several haplotypes of the LILRB1 gene and their relationship to LILRB1 expression on NK cells in healthy individuals (7, 17, 21). More specifically, individuals with the SNPs rs1004443-A, rs3760860-G, and rs3760861-G have higher levels of LILRB1 transcript and surface expression on NK cells (7) than those with

Experimental and Molecular Therapeutics, Jul 1, 2021

Development of biologics or cell therapeutics which target MHC class I:peptide (pMHC) complexes f... more Development of biologics or cell therapeutics which target MHC class I:peptide (pMHC) complexes for recognition and elimination of tumor cells is hindered by low affinity, cross-reactivity or challenging biochemical properties of antibody- and T-cell receptor-based binders. We hypothesized that DARPin® proteins may be particularly effective in solving this problem due to structural characteristics of their antigen binding surface and excellent biophysical properties. Panels of binders highly specific for a given pMHC complex were isolated from DARPin® libraries through several rounds of selection and counter selection on the relevant or irrelevant but structurally similar pMHC complexes using ribosome display. DARPin® binders were successfully isolated against pMHC complexes composed of different MHC class I alleles with various peptides derived from either tumor associated antigens or non-self viral proteins. A selected panel of DARPin® binders specific to HLA-A2 molecule in association with SLLMWITQC (SLL peptide), a peptide derived from NY-ESO-1, was used to create bi-specific T-cell engagers containing another moiety binding to the epsilon component of the CD3 complex, thus allowing highly sensitive analysis of pMHC specificity and potential cross-reactivity. Using a number of cellular assays, including peptide pulsing of TAP-deficient T2 cells, we confirmed high specificity of selected DARPin® proteins to the HLA-A2:SLL complex. Architectural fine tuning and sequence engineering allowed us to further increase the potency of the selected candidates without compromising the specificity. This was manifested as effective T cell mediated activation by relevant DARPin® constructs in the presence of HLA-A2+/NY-ESO-1+ tumor cells. Furthermore, HLA-A2+/NY-ESO-1+ cells but not HLA-A2+/NY-ESO-1- cells were effectively killed in the presence of engineered HLA-A2:SLL-specific DARPin® T-cell engagers. The versatility of the DARPin® platform and the engineering experience gained with NY-ESO-1 did allow us to identify highly potent and specific DARPins® proteins for additional clinically attractive pMHC targets.Alanine and X-scan mutagenesis demonstrated that, in many cases, interactions with several peptide residues located across the entire peptide sequence are critical for DARPin® protein binding to the pMHC complex. These data suggest that peptide residues exposed outside of the MHC peptide binding grove create the focal point of MHC:peptide:DARPin® proteins interactions. Further molecular and cellular analysis of DARPin® protein specificity will be evaluated in order to de-risk for potential clinically relevant toxicity.In conclusion, we show that the DARPin® technology platform may be highly instrumental in developing a new class of anti-cancer therapeutics based on specific targeting of pMHC complexes presented selectively by cancer cells. Citation Format: Natalia Venetz, Sandra Müller, Tim Schulte, Stefanie Fischer, Maria Paladino, Nicole Pina, Nadir Kadri, Sandra Bruckmaier, Andreas Cornelius, Tanja Hospodarsch, Tatyana Sandalova, Victor Levitsky, Adnane Achour, Marcel Walser. Application of the DARPin® technology for specific targeting of tumor-associated MHC class I:peptide complexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1349.

Journal of Autoimmunity, Aug 1, 2018

ObjectiveACPA-positive RA is associated with distinct HLA-DR alleles and with antibodies targetin... more ObjectiveACPA-positive RA is associated with distinct HLA-DR alleles and with antibodies targeting citrullinated α-enolase. We screened α-enolase for tentative T cell epitopes and focused the present study on aa326–340. Both the frequency and quality of T cell responses were studied in blood and synovial fluid of RA patients.MethodsBinding of native and citrullinated α-eno326–340 HLA-DRB1*04:01 was studied by in vitro competition assays and by DSC, and T cell responses by in vitro culture. HLA-DRB1*04:01 tetramers were used for assessing ex vivo frequency and phenotype of α-enolase-specific T cells. Cross-reactivity, i.e. whether the same T cells could recognize both peptides, was addressed utilizing RA patient samples and HLA-DRB1*04:01-transgenic mice.ResultsFrequencies of T cells recognizing native eno326–340 were similar in blood and synovial fluid, whereas the frequency of cit-eno326–340 T cells was elevated in synovial fluid. The cit-eno-specific, but not native-eno-specific T cells in blood displayed a memory CD45RO phenotype indicating previous exposure to citrullinated enolase. In vitro assays revealed functional responses to both peptides in patient samples and immunized HLA-DRB1*04:01-IE-transgenic mice. Cross-reactivity to the two peptides was observed but was not universal.ConclusionsHLA-DRB1*04:01-restricted CD4 T cells recognizing native and cit-eno326–340 peptides are part of the normal circulating T cell repertoire. We observed more T cells specific for the citrullinated peptide with a memory phenotype in the circulation, which supports the concept that such T cells might be activated outside the joints and subsequently recruited and possibly re-activated in inflamed joints.

FEBS Journal, Aug 28, 2019

The molecular bases of amyloid aggregation propensity are still poorly understood, especially for... more The molecular bases of amyloid aggregation propensity are still poorly understood, especially for proteins that display a stable folded native structure. A prototypic example is human beta-2 microglobulin (β2m), which, when accumulated in patients, gives rise to dialysis-related amyloidosis. Interestingly, although the physiologic concentration of β2m in mice is five times higher than that found in human patients, no amyloid deposits are observed in mice. Moreover, murine β2m not only displays a lower amyloid propensity both in vivo and in vitro, but also inhibits the aggregation of human β2m in vitro. Here we compared human and murine β2m for their aggregation propensity, ability to form soluble oligomers, stability, three-dimensional structure and dynamics. Our results indicate that murine β2m low-aggregation propensity is due to two concomitant aspects: the low aggregation propensity of its primary sequence combined with the absence of high-energy amyloidcompetent conformations under native conditions. The identification of the specific properties Accepted Article This article is protected by copyright. All rights reserved. determining the low aggregation propensity of mouse β2m will help delineate the molecular risk factors which cause a folded protein to aggregate. This article is protected by copyright. All rights reserved. energy obtained for hβ2m resembles that previously determined by replica-averaged metadynamics simulations on a slightly different sequence [18, 63]. The main differences being the lack of a more 'crystal'-like high-energy state and the presence of a slightly more disordered, high-energy, state. This latter was not sampled in a former work [18] because the sampling was not allowed in that region, but was more recently observed by solid state NMR and replica-averaged metadynamics simulations [20]. Circular dichroism Thermal stability experiments were performed in triplicate using three independent batches of protein and were monitored in the far-UV region using a J-810 spectropolarimeter (JASCO Corp., Tokyo, Japan) equipped with a Peltier system for temperature control. The protein concentration was 0.1 mg/mL in 50 mM sodium phosphate pH 7.4. The temperature ramps were carried out from 20 to 95 °C (temperature slope 50 °C/hour) in a 0.1 cm path length cuvette and monitored at 202 nm wavelength. Tm was calculated as the first-derivative minimum of the traces. Spectra before and after unfolding ramp were recorded (260-190 nm). All three β2m variants considered in this work display an irreversible unfolding under the tested conditions. Accession number Atomic coordinates and structure factors for mβ2m have been deposited at the Protein Data Bank, with accession code 6I8C.

Proceedings of the National Academy of Sciences of the United States of America, Feb 26, 2019

Journal of Neuroimmunology, Oct 1, 2014

enzymatically active and inactive forms of PTX. Our results indicate that PTX, through its ADP-ri... more enzymatically active and inactive forms of PTX. Our results indicate that PTX, through its ADP-ribosyltransferase activity, induces two series of events upstream of IL-6: 1) the activation of TLR4 signaling in myeloid cells, leading to pro-IL-1β synthesis; and 2) the formation of a pyrindependent inflammasome that cleaves pro-IL-1β into its active form. In turn, IL-1β stimulates nearby stromal cells to secrete IL-6, which is known to induce vascular changes required for leukocyte adhesion. Without pyrin, PTX does not induce neutrophil adhesion to cerebral capillaries and is less effective at inducing EAE in transgenic mice with encephalitogenic T lymphocytes. This study identifies thefirstmicrobial molecule that activates pyrin, a mechanism by which infections may influence MS and a potential therapeutic target for immune disorders.

Frontiers in Chemistry

Significant advances in mass-spectroscopy (MS) have made it possible to investigate the cellular ... more Significant advances in mass-spectroscopy (MS) have made it possible to investigate the cellular immunopeptidome, a large collection of MHC-associated epitopes presented on the surface of healthy, stressed and infected cells. These approaches have hitherto allowed the unambiguous identification of large cohorts of epitope sequences that are restricted to specific MHC class I and II molecules, enhancing our understanding of the quantities, qualities and origins of these peptide populations. Most importantly these analyses provide essential information about the immunopeptidome in responses to pathogens, autoimmunity and cancer, and will hopefully allow for future tailored individual therapies. Protein post-translational modifications (PTM) play a key role in cellular functions, and are essential for both maintaining cellular homeostasis and increasing the diversity of the proteome. A significant proportion of proteins is post-translationally modified, and thus a deeper understanding ...

PLOS Pathogens, 2019

The 14-3-3 molecular scaffolds promote type I interferon (IFN) responses by stabilizing the inter... more The 14-3-3 molecular scaffolds promote type I interferon (IFN) responses by stabilizing the interaction of RIG-I with the TRIM25 ligase. Viruses have evolved unique strategies to halt this cellular response to support their replication and spread. Here, we report that the ubiquitin deconjugase (DUB) encoded in the N-terminus of the Epstein-Barr virus (EBV) large tegument protein BPLF1 harnesses 14-3-3 molecules to promote TRIM25 autoubiquitination and sequestration of the ligase into inactive protein aggregates. Catalytically inactive BPLF1 induced K48-linked autoubiquitination and degradation of TRIM25 while the ligase was mono-or di-ubiquitinated in the presence of the active viral enzyme and formed cytosolic aggregates decorated by the autophagy receptor p62/SQSTM1. Aggregate formation and the inhibition of IFN response were abolished by mutations of solvent exposed residues in helix-2 of BPLF1 that prevented binding to 14-3-3 while preserving both catalytic activity and binding to TRIM25. 14-3-3 interacted with the Coiled-Coil (CC) domain of TRIM25 in in vitro pulldown, while BPLF1 interacted with both the CC and B-box domains, suggesting that 14-3-3 positions BPLF1 at the ends of the CC dimer, close to known autoubiquitination sites. Our findings provide a molecular understanding of the mechanism by which a viral deubiquitinase inhibits the IFN response and emphasize the role of 14-3-3 proteins in modulating antiviral defenses.

ACS Omega, 2022

Presentation of pathogen-derived epitopes by major histocompatibility complex I (MHC-I) can lead ... more Presentation of pathogen-derived epitopes by major histocompatibility complex I (MHC-I) can lead to the activation and expansion of specific CD8 + T cell clones, eventually resulting in the destruction of infected target cells. Altered peptide ligands (APLs), designed to elicit immunogenicity toward a wildtype peptide, may affect the overall stability of MHC-I/peptide (pMHC) complexes and modulate the recognition by T cell receptors (TCR). Previous works have demonstrated that proline substitution at position 3 (p3P) of different MHC-restricted epitopes, including the immunodominant LCMV-derived epitope gp33 and escape variants, may be an effective design strategy to increase epitope immunogenicity. These studies hypothesized that the p3P substitution increases peptide rigidity, facilitating TCR binding. Here, molecular dynamics simulations indicate that the p3P modification rigidifies the APLs in solution predisposing them for the MHC-I loading as well as once bound to H-2D b , predisposing them for TCR binding. Our results also indicate that peptide position 6, key for interaction of H-2D b /gp33 with the TCR P14, takes a suboptimal conformation before as well as after binding to the TCR. Analyses of H-2D b in complex with APLs, in which position 6 was subjected to an L-to D-amino acid modification, revealed small conformational changes and comparable pMHC thermal stability. However, the L-to D-modification reduced significantly the binding to P14 even in the presence of the p3P modification. Our combined data highlight the sensitivity of the TCR for the conformational dynamics of pMHC and provide further tools to dissect and modulate TCR binding and immunogenicity via APLs.

Acta Crystallographica Section A, Aug 29, 2010

Page s34 s34 forms a polyproline-II-like helix that seems to be a common feature of many Gram-pos... more Page s34 s34 forms a polyproline-II-like helix that seems to be a common feature of many Gram-positive cell-wall anchored virulence factors, and particularly of basal pilins. Together, we identified structural characteristics of pilins that direct their incorporation into the pilus polymer.

FEBS Letters, Aug 21, 1998

Errata FEBS 20430 Erratum to: Oct-I promoter region contains octamer sites and TAAT motifs recogn... more Errata FEBS 20430 Erratum to: Oct-I promoter region contains octamer sites and TAAT motifs recognized by Oct proteins (FEBS 20083)

Chemcatchem, Aug 6, 2015

The residues responsible for binding the catalytic water molecule were interchanged between the c... more The residues responsible for binding the catalytic water molecule were interchanged between the closely related enzymes fructose 6‐phosphate aldolase A (FSAA) and transaldolase B (TalB) from Escherichia coli. In FSAA, this water molecule is bound by hydrogen bonds to the side chains of three residues (Gln59, Thr109 and Tyr131), whereas in TalB only two residues (Glu96 and Thr156) participate. Single and double variants were characterised with respect to fructose 6‐phosphate aldolase and transaldolase activity, stability, pH dependence of activity, pKa value of the essential lysine residue and their three dimensional structure. The double variant TalBE96Q F178Y showed improved aldolase activity with an apparent kcat of 4.3 s−1. The experimentally determined pKa values of the catalytic lysine residue revealed considerable differences: In FSAA, this lysine residue is deprotonated at assay conditions (pKa 5.5) whereas it is protonated in TalB (pKa 9.3). Hence, a deprotonation of the catalytic lysine residue, which is a prerequisite for an efficient nucleophilic attack in TalB, is not necessary in FSAA. Based upon these results, we propose a new mechanism for FSAA with Tyr131 as general acid.

Oxidative stress and disease, Dec 11, 2003

Mucosa-associated lymphoid tissue protein 1 (MALT1) plays a key role in adaptive immune responses... more Mucosa-associated lymphoid tissue protein 1 (MALT1) plays a key role in adaptive immune responses by modulating specific intracellular signalling pathways that control the development and proliferation of both T and B cells. Dysfunction of these pathways is coupled to the progress of highly aggressive lymphoma as well as to potential development of an array of different immune disorders. In contrast to other signalling mediators, MALT1 is not only activated through the formation of the CBM complex together with the proteins CARMA1 and Bcl10, but also by acting as a protease that cleaves multiple substrates to promote lymphocyte proliferation and survival via the NF-κB signalling pathway. Herein, we present the partial 1H, 13C Ile/Val/Leu-Methyl resonance assignment of the monomeric apo form of the paracaspase-IgL3 domain of human MALT1. Our results provide a solid ground for future elucidation of both the three-dimensional structure and the dynamics of MALT1, key for adequate develo...