APOBEC-related mutagenesis and neo-peptide hydrophobicity: implications for response to immunotherapy.

Tumor-associated neo-antigens are mutated peptides that allow the immune system to recognize the affected cell as foreign. Cells carrying excessive mutation load often develop mechanisms of tolerance. PD-L1/PD-1 checkpoint immunotherapy is a highly promising approach to overcome these protective signals and induce tumor shrinkage. Yet, the nature of the neo-antigens driving those beneficial responses remains unclear. Here, we show that APOBEC-related mutagenesis - a mechanism at the crossroads between anti-viral immunity and endogenous nucleic acid editing - increases neo-peptide hydrophobicity (a feature of immunogenicity), as demonstrated by in silico computation and in the TCGA pan-cancer cohort, where APOBEC-related mutagenesis was also strongly associated with immune marker expression. Moreover, APOBEC-related mutagenesis correlated with immunotherapy response in a cohort of 99 patients with diverse cancers, and this correlation was independent of the tumor mutation burden (TMB). Combining APOBEC-related mutagenesis estimate and TMB resulted in greater predictive ability than either parameter alone. Based on these results, further investigation of APOBEC-related mutagenesis as a marker of response to anti-cancer checkpoint blockade is warranted

Sequential Logic Model Deciphers Dynamic Transcriptional Control of Gene Expressions

Cellular signaling involves a sequence of events from ligand binding to membrane receptors through transcription factors activation and the induction of mRNA expression. The transcriptional-regulatory system plays a pivotal role in the control of gene expression. A novel computational approach to the study of gene regulation circuits is presented here.Based on the concept of finite state machine, which provides a discrete view of gene regulation, a novel sequential logic model (SLM) is developed to decipher control mechanisms of dynamic transcriptional regulation of gene expressions. The SLM technique is also used to systematically analyze the dynamic function of transcriptional inputs, the dependency and cooperativity, such as synergy effect, among the binding sites with respect to when, how much and how fast the gene of interest is expressed. expression and additional activities of binding sites are required. Further analyses suggest detailed mechanism of R switch activity where indirect dependency occurs in between UI activity and R switch during specification to differentiation stage. is a promising step for further application of the proposed method

Deciphering the Arginine-Binding Preferences at the Substrate-Binding Groove of Ser/Thr Kinases by Computational Surface Mapping

Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P−2 and P−5, commonly occupied by Arginine (Arg) in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P−2/P−5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P−5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering). The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P−2 and P−5 positions

BCR-ABL residues interacting with ponatinib are critical to preserve the tumorigenic potential of the oncoprotein

Patients with chronic myeloid leukemia in whom tyrosine kinase inhibitors (TKIs) fail often present mutations in the BCR-ABL catalytic domain. We noticed a lack of substitutions involving 4 amino acids (E286, M318, I360, and D381) that form hydrogen bonds with ponatinib. We therefore introduced mutations in each of these residues, either preserving or altering their physicochemical properties. We found that E286, M318, I360, and D381 are dispensable for ABL and BCR-ABL protein stability but are critical for preserving catalytic activity. Indeed, only a "conservative" I360T substitution retained kinase proficiency and transforming potential. Molecular dynamics simulations of BCR-ABLI360T revealed differences in both helix αC dynamics and protein-correlated motions, consistent with a modified ATP-binding pocket. Nevertheless, this mutant remained sensitive to ponatinib, imatinib, and dasatinib. These results suggest that changes in the 4 BCR-ABL residues described here would be selected against by a lack of kinase activity or by maintained responsiveness to TKIs. Notably, amino acids equivalent to those identified in BCR-ABL are conserved in 51% of human tyrosine kinases. Hence, these residues may represent an appealing target for the design of pharmacological compounds that would inhibit additional oncogenic tyrosine kinases while avoiding the emergence of resistance due to point mutations.This work was supported by an investigator grant to P.V. from Associazione Italiana per la Ricerca sul Cancro (AIRC) and by funding from the Biotechnology and Biological Sciences Research Council (BB/I023291/1 and BB/H018409/1 to AP and FF). P.B. is the recipient of an AIRC - Marie Curie fellowship

GPU.proton.DOCK: Genuine Protein Ultrafast proton equilibria consistent DOCKing

GPU.proton.DOCK (Genuine Protein Ultrafast proton equilibria consistent DOCKing) is a state of the art service for in silico prediction of protein–protein interactions via rigorous and ultrafast docking code. It is unique in providing stringent account of electrostatic interactions self-consistency and proton equilibria mutual effects of docking partners. GPU.proton.DOCK is the first server offering such a crucial supplement to protein docking algorithms—a step toward more reliable and high accuracy docking results. The code (especially the Fast Fourier Transform bottleneck and electrostatic fields computation) is parallelized to run on a GPU supercomputer. The high performance will be of use for large-scale structural bioinformatics and systems biology projects, thus bridging physics of the interactions with analysis of molecular networks. We propose workflows for exploring in silico charge mutagenesis effects. Special emphasis is given to the interface-intuitive and user-friendly. The input is comprised of the atomic coordinate files in PDB format. The advanced user is provided with a special input section for addition of non-polypeptide charges, extra ionogenic groups with intrinsic pKa values or fixed ions. The output is comprised of docked complexes in PDB format as well as interactive visualization in a molecular viewer. GPU.proton.DOCK server can be accessed at http://gpudock.orgchm.bas.bg/

A single F153Sβ3 mutation causes constitutive integrin αIIbβ3 activation in a variant form of Glanzmann thrombasthenia

This report identifies a novel variant form of the inherited bleeding disorder Glanzmann thrombasthenia, exhibiting only mild bleeding in a physically active individual. The platelets cannot aggregate ex vivo with physiologic agonists of activation, although microfluidic analysis with whole blood displays moderate ex vivo platelet adhesion and aggregation consistent with mild bleeding. Immunocytometry shows reduced expression of αIIbβ3 on quiescent platelets that spontaneously bind/store fibrinogen, and activation-dependent antibodies (ligand-induced binding site-319.4 and PAC-1) report β3 extension suggesting an intrinsic activation phenotype. Genetic analysis reveals a single F153Sβ3 substitution within the βI-domain from a heterozygous T556C nucleotide substitution of ITGB3 exon 4 in conjunction with a previously reported IVS5(+1)G\u3eA splice site mutation with undetectable platelet messenger RNA accounting for hemizygous expression of S153β3. F153 is completely conserved among β3 of several species and all human β-integrin subunits suggesting that it may play a vital role in integrin structure/function. Mutagenesis of αIIb-F153Sβ3 also displays reduced levels of a constitutively activated αIIb-S153β3 on HEK293T cells. The overall structural analysis suggests that a bulky aromatic, nonpolar amino acid (F,W)153β3 is critical for maintaining the resting conformation of α2- and α1-helices of the βI-domain because small amino acid substitutions (S,A) facilitate an unhindered inward movement of the α2- and α1-helices of the βI-domain toward the constitutively active αIIbβ3 conformation, while a bulky aromatic, polar amino acid (Y) hinders such movements and restrains αIIbβ3 activation. The data collectively demonstrate that disruption of F153β3 can significantly alter normal integrin/platelet function, although reduced expression of αIIb-S153β3 may be compensated by a hyperactive conformation that promotes viable hemostasis