In silico specificity determination of neoantigen-reactive T-lymphocytes

2021 ◽  
Vol 67 (3) ◽  
pp. 251-258
Author(s):  
A.E. Kniga ◽  
I.V. Polyakov ◽  
A.V. Nemukhin
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Molecular Docking ◽  
Protein Interactions ◽  
Structural Features ◽  
Immune Recognition ◽  
Protein Protein Interactions ◽  
Binary Classifiers

Effective personalized immunotherapies of the future will need to capture not only the peculiarities of the patient’s tumor but also of his immune response to it. In this study, using results of in vitro high-throughput specificity assays, and combining comparative models of pMHCs and TCRs using molecular docking, we have constructed all-atom models for the putative complexes of all their possible pairwise TCR-pMHC combinations. For the models obtained we have calculated a dataset of physics-based scores and have trained binary classifiers that perform better compared to their solely sequence-based counterparts. These structure-based classifiers pinpoint the most prominent energetic terms and structural features characterizing the type of protein-protein interactions that underlies the immune recognition of tumors by T cells.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

Protein Interaction Domains and Post-Translational Modifications: Structural Features and Drug Discovery Applications

2020 ◽  
Vol 27 (37) ◽  
pp. 6306-6355 ◽  
Author(s):  
Marian Vincenzi ◽  
Flavia Anna Mercurio ◽  
Marilisa Leone
Keyword(s):  
Drug Discovery ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Structural Information ◽  
Protein Complexes ◽  
Structural Features ◽  
Protein Protein Interactions ◽  
Modular Architecture ◽  
Post Translational Modifications ◽  
Interaction Domains

Background:: Many pathways regarding healthy cells and/or linked to diseases onset and progression depend on large assemblies including multi-protein complexes. Protein-protein interactions may occur through a vast array of modules known as protein interaction domains (PIDs). Objective:: This review concerns with PIDs recognizing post-translationally modified peptide sequences and intends to provide the scientific community with state of art knowledge on their 3D structures, binding topologies and potential applications in the drug discovery field. Method:: Several databases, such as the Pfam (Protein family), the SMART (Simple Modular Architecture Research Tool) and the PDB (Protein Data Bank), were searched to look for different domain families and gain structural information on protein complexes in which particular PIDs are involved. Recent literature on PIDs and related drug discovery campaigns was retrieved through Pubmed and analyzed. Results and Conclusion:: PIDs are rather versatile as concerning their binding preferences. Many of them recognize specifically only determined amino acid stretches with post-translational modifications, a few others are able to interact with several post-translationally modified sequences or with unmodified ones. Many PIDs can be linked to different diseases including cancer. The tremendous amount of available structural data led to the structure-based design of several molecules targeting protein-protein interactions mediated by PIDs, including peptides, peptidomimetics and small compounds. More studies are needed to fully role out, among different families, PIDs that can be considered reliable therapeutic targets, however, attacking PIDs rather than catalytic domains of a particular protein may represent a route to obtain selective inhibitors.

scholarly journals Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 510
Author(s):  
Maho Yamamoto ◽  
Rina Kondo ◽  
Haruka Hozumi ◽  
Seita Doi ◽  
Miwako Denda ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Biochemical Characterization ◽  
S100 Protein ◽  
High Mobility ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Protein Protein Interactions ◽  
High Mobility Group Protein ◽  
Pulldown Assay

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311–342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311–347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

scholarly journals Ways into Understanding HIF Inhibition

Cancers ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 159
Author(s):  
Tina Schönberger ◽  
Joachim Fandrey ◽  
Katrin Prost-Fingerle
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Protein Protein Interactions ◽  
Low Oxygen ◽  
Drug Candidates ◽  
Open Questions ◽  
Wide Range ◽  
Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

scholarly journals Structural design principles for specific ultra-high affinity interactions between colicins/pyocins and immunity proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Avital Shushan ◽  
Mickey Kosloff
Keyword(s):  
Protein Interactions ◽  
Structural Elements ◽  
Protein Protein Interactions ◽  
Immunity Protein ◽  
Rational Engineering ◽  
High Affinity ◽  
Comparative Structure ◽  
Polar Interactions ◽  
Exquisite Specificity

AbstractThe interactions of the antibiotic proteins colicins/pyocins with immunity proteins is a seminal model system for studying protein–protein interactions and specificity. Yet, a precise and quantitative determination of which structural elements and residues determine their binding affinity and specificity is still lacking. Here, we used comparative structure-based energy calculations to map residues that substantially contribute to interactions across native and engineered complexes of colicins/pyocins and immunity proteins. We show that the immunity protein α1–α2 motif is a unique structurally-dissimilar element that restricts interaction specificity towards all colicins/pyocins, in both engineered and native complexes. This motif combines with a diverse and extensive array of electrostatic/polar interactions that enable the exquisite specificity that characterizes these interactions while achieving ultra-high affinity. Surprisingly, the divergence of these contributing colicin residues is reciprocal to residue conservation in immunity proteins. The structurally-dissimilar immunity protein α1–α2 motif is recognized by divergent colicins similarly, while the conserved immunity protein α3 helix interacts with diverse colicin residues. Electrostatics thus plays a key role in setting interaction specificity across all colicins and immunity proteins. Our analysis and resulting residue-level maps illuminate the molecular basis for these protein–protein interactions, with implications for drug development and rational engineering of these interfaces.

scholarly journals EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-β/SMAD signalling

2021 ◽  
Author(s):  
Liqing Jia ◽  
Xiaolu Ge ◽  
Chao Du ◽  
Linna Chen ◽  
Yanhong Zhou ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Protein Interactions ◽  
Elongation Factor ◽  
Protein Translation ◽  
Protein Protein Interactions ◽  
Promising Target ◽  
Tgfβ Receptor ◽  
Mesenchymal Transformation

Abstract Background Eukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD). Methods Immunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein–protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP). Results In this study, we report that EEF1A2 mediates the epithelial–mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFβ Receptor (TβR)-I, and TβRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD. Conclusions These findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals In Silico and In Vitro Studies on the Protein-Protein Interactions between Brugia malayi Immunomodulatory Protein Calreticulin and Human C1q

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e106413 ◽  
Author(s):  
Sunita Yadav ◽  
Smita Gupta ◽  
Chandrabose Selvaraj ◽  
Pawan Kumar Doharey ◽  
Anita Verma ◽  
...  
Keyword(s):  
Protein Interactions ◽  
In Silico ◽  
Brugia Malayi ◽  
In Vitro Studies ◽  
Protein Protein Interactions ◽  
Immunomodulatory Protein

scholarly journals T helper cell polarisation as a measure of the maturation of the immune response

2003 ◽  
Vol 12 (5) ◽  
pp. 285-292 ◽  
Author(s):  
Scott B. Cameron ◽  
Ellen H. Stolte ◽  
Anthony W. Chow ◽  
Huub F. J. Savelkoul
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Intracellular Cytokine Staining ◽  
T Helper Cell ◽  
Helper Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intracellular Cytokine ◽  
T Helper

Background:T helper cell polarisation is important under chronic immune stimulatory conditions and drives the type of the evolving immune response. Mice treated with superantigensin vivodisplay strong effects on Thsubset differentiation. The aim of the study was to detect the intrinsic capacity of T cells to polarise under variousex vivoconditions.Methods:Purified CD4+T cells obtained from superantigen-treated mice were cultured under Thpolarising conditionsin vitro. By combining intracellular cytokine staining and subsequent flow cytometric analysis with quantitative cytokine measurements in culture supernatants by enzyme-linked immunosorbent assay (ELISA), the differential Thpolarising capacity of the treatment can be detected in a qualitative and quantitative manner.Results and conclusions:BALB/c mice were shown to be biased to develop strong Th2 polarised immune responses using Th0 stimulation of purified CD4+T cells from phosphate-buffered saline-treated mice. Nevertheless, our analysis methodology convincingly showed that even in these mice, Toxic Shock Syndrome Toxin-1 treatmentin vivoresulted in a significantly stronger Th1 polarising effect than control treatment. Our results indicate that populations of Thcells can be assessed individually for their differential Th1 or Th2 maturation capacityin vivoby analysing robustin vitropolarisation cultures combined with intracellular cytokine staining and ELISA.

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