In Vitro Cross-Linking MS Reveals SMG1-UPF2-SMG7 Assembly as Molecular Partners within the NMD Surveillance.

mRNAs containing premature stop codons are responsible for various genetic diseases as well as cancers. The truncated proteins synthesized from these aberrant mRNAs are seldom detected due to the nonsense-mediated mRNA decay (NMD) pathway. Such a surveillance mechanism detects most of these aberrant mRNAs and rapidly destroys them from the pool of mRNAs. Here, we implemented chemical cross-linking mass spectrometry (CLMS) techniques to trace novel biology consisting of protein-protein interactions (PPIs) within the NMD machinery. A set of novel complex networks between UPF2 (Regulator of nonsense transcripts 2), SMG1 (Serine/threonine-protein kinase SMG1), and SMG7 from the NMD pathway were identified, among which UPF2 was found as a connection bridge between SMG1 and SMG7. The UPF2 N-terminal formed most interactions with SMG7, and a set of residues emerged from the MIF4G-I, II, and III domains docked with SMG1 or SMG7. SMG1 mediated interactions with initial residues of UPF2, whereas SMG7 formed very few interactions in this region. Modelled structures highlighted that PPIs for UPF2 and SMG1 emerged from the well-defined secondary structures, whereas SMG7 appeared from the connecting loops. Comparing the influence of cancer-derived mutations over different CLMS sites revealed that variants in the PPIs for UPF2 or SMG1 have significant structural stability effects. Our data highlights the protein-protein interface of the SMG1, UPF2, and SMG7 genes that can be used for potential therapeutic approaches. Blocking the NMD pathway could enhance the production of neoantigens or internal cancer vaccines, which could provide a platform to design potential peptide-based vaccines.

Modified Peptide Molecules As Potential Modulators of Shelterin Protein Functions; TRF1.

In this work, we present studies on relatively new and still not well-explored potential anticancer targets which are shelterin proteins, in particular the TRF1 protein can be blocked by in silico designed "peptidomimetic" molecules. TRF1 interacts directly with the TIN2 protein, and this protein-protein interaction is crucial for the proper functioning of telomere, which could be blocked by our novel modified peptide molecules. Our chemotherapeutic approach is based on assumption that modulation of TRF1-TIN2 interaction may be more harmful for cancer cells as cancer telomeres are more fragile than in normal cells. We have shown in vitro within SPR experiments that our modified peptide PEP1 molecule interacts with TRF1, presumably at the site originally occupied by the TIN2 protein. Disturbance of the shelterin complex by studied molecule may not in short term lead to cytotoxic effects, however blocking TRF1-TIN2 resulted in cellular senescence in cellular breast cancer lines used as a cancer model. Thus, our compounds appeared useful as starting model compounds for precise blockage of TRF proteins.

Identification of novel interferon responsive protein partners of human leukocyte antigen A (HLA-A) using cross-linking mass spectrometry (CLMS) approach.

The interferon signalling system elicits a robust cytokine response against a wide range of environmental pathogenic and internal pathological signals, leading to induction of a subset of interferon-induced proteins. We applied DSS (disuccinimidyl suberate) mediated cross-linking mass spectrometry (CLMS) to capture novel protein-protein interactions within the realm of interferon induced proteins. In addition to the expected interferon-induced proteins, we identified novel inter- and intra-molecular cross-linked adducts for the canonical interferon induced proteins, such as MX1, USP18, OAS3, and STAT1. We focused on orthogonal validation of a cohort of novel interferon-induced protein networks formed by the HLA-A protein (H2BFS-HLA-A-HMGA1) using co-immunoprecipitation assay, and further investigated them by molecular dynamics simulation. Conformational dynamics of the simulated protein complexes revealed several interaction sites that mirrored the interactions identified in the CLMS findings. Together, we showcase a proof-of-principle CLMS study to identify novel interferon-induced signaling complexes and anticipate broader use of CLMS to identify novel protein interaction dynamics within the tumour microenvironment.

Organic solvents aggregating and shaping structural folding of protein, a case study of the protease enzyme.

Low solubility of reactants or products in aqueous solutions can result in the enzymatic catalytic reactions that can occur in non-aqueous solutions. In current study we investigated aqueous solutions containing different organic solvents / deep eutectic solvents (DESs) that can influence the protease enzyme's activity, structural, and thermal stabilities. Retroviral aspartic protease enzyme is responsible for the cleavage of the polypeptide precursors into mature viral components, a very crucial step for virus life cycle. In molecular dynamic simulations (MDS), the complex of the protease enzyme with Darunavirwas found highly stable in urea aqueous solution compared to when with the ethylene glycol (EG) or glycerol solvents. Particularly, in different organic solvents the presence of Darunavir induced protein-protein interactions within the protease homodimer. For the systems with EG or glycerol solvents, the flap domains of the enzyme formed an "open" conformation which lead to a weak binding affinity with the drug. Conserved D25 and G27 residues among this family of the aspartic protease enzymes made a stable binding with Darunavir in the urea systems. Unfolding of the protease dimer was initiated due to self-aggregation for the EG or glycerol organic solvents, which formed an "open" conformation for the flap domains. On the contrary lack of such clustering in urea solvent, the protease showed conventional structural folding in the presence or absence of the drug molecule. These novel findings may help to better understand the protease enzymes, which could be controlled by deep eutectic solvents.

The Binding Specificity of PAB1 with Poly(A) mRNA, Regulated by Its Structural Folding.

The poly(A)-binding protein cytoplasmic 1 (PAB1 or PABPC1) protein is associated with the long poly(A) mRNA tails, inducing stability. Herein, we investigated the dynamics of the PABPC1 protein, along with tracing its mRNA binding specificity. During molecular dynamics simulations (MDS), the R176-Y408 amino acids (RRM3-4 domains; RNA recognition motifs) initiated a folded structure that resulted in the formation of different conformations. The RRM4 domain formed high-frequency intramolecular interactions, despite such induced flexibility. Residues D45, Y54, Y56, N58, Q88, and N100 formed long-lasting interactions, and specifically, aromatic residues (Y14, Y54, Y56, W86, and Y140) gained a unique binding pattern with the poly(A) mRNA. In addition, the poly(A) mRNA motif assembled a PABPC1-specific conformation, by inducing movement of the center three nucleotides to face towards RRM1-2 domains. The majority of the high-frequency cancer mutations in PAB1 reside within the RRM4 domain and amino acids engaging in high-frequency interactions with poly(A) mRNA were found to be preserved in different cancer types. Except for the G123C variant, other studied cancer-derived mutants hindered the stability of the protein. Molecular details from this study will provide a detailed understanding of the PABPC1 structure, which can be used to modulate the activity of this gene, resulting in production of mutant peptide or neoantigens in cancer.

The Elephant Evolved p53 Isoforms that Escape MDM2-Mediated Repression and Cancer.

The p53 tumor suppressor is a transcription factor with roles in cell development, apoptosis, oncogenesis, aging, and homeostasis in response to stresses and infections. p53 is tightly regulated by the MDM2 E3 ubiquitin ligase. The p53-MDM2 pathway has coevolved, with MDM2 remaining largely conserved, whereas the TP53 gene morphed into various isoforms. Studies on prevertebrate ancestral homologs revealed the transition from an environmentally induced mechanism activating p53 to a tightly regulated system involving cell signaling. The evolution of this mechanism depends on structural changes in the interacting protein motifs. Elephants such as Loxodonta africana constitute ideal models to investigate this coevolution as they are large and long-living as well as having 20 copies of TP53 isoformic sequences expressing a variety of BOX-I MDM2-binding motifs. Collectively, these isoforms would enhance sensitivity to cellular stresses, such as DNA damage, presumably accounting for strong cancer defenses and other adaptations favoring healthy aging. Here we investigate the molecular evolution of the p53-MDM2 system by combining in silico modeling and in vitro assays to explore structural and functional aspects of p53 isoforms retaining the MDM2 interaction, whereas forming distinct pools of cell signaling. The methodology used demonstrates, for the first time that in silico docking simulations can be used to explore functional aspects of elephant p53 isoforms. Our observations elucidate structural and mechanistic aspects of p53 regulation, facilitate understanding of complex cell signaling, and suggest testable hypotheses of p53 evolution referencing Peto's Paradox.

Self-derived peptides from the SARS-CoV-2 spike glycoprotein disrupting shaping and stability of the homotrimer unit.

The structural spike (S) protein from the SARS-CoV-2 ß-coronavirus is shown to make different pre- and post-fusion conformations within its homotrimer unit. To support the ongoing novel vaccine design and development strategies, we report the structure-based design approach to develop self-derived S peptides. A dataset of crucial regions from the S protein were transformed into linear motifs that could act as the blockers or stabilizers for the S protein homotrimer unit. Among these distinct S peptides, the pep02 (537-QQFGRDIAD-545) and pep07 (821-RDLICAQKFNGLTVLPPLLTDE-842) were found making stable folded binding with the S protein (550-750 and 950-1050 regions). Upon inserting SARS-CoV-2 S variants in the peptide destabilized the complexed S protein structure, resulting an allosteric effect in different functional regions of the protein. Particularly, the molecular dynamics revealed that A544D mutation in the pep02 peptide induced instability for the complexed S protein, whereas the N943K variant from pep09 exhibited an opposite behavior. An increased protein-peptide binding affinity and the stable structural folding were observed in mutated systems, compared to that of the wild type systems. The presence of mutation has induced an "up" active conformation of the spike (RBD) domain, responsible for interacting the host cell receptor. Among the lower affinity peptide datasets (e.g., pep01), the S1 and S2 subunit in the protein formed an "open" conformation, whereas with higher affinity peptides (e.g., pep07) these domains gained a "closed" conformation. These findings propose that our designed self-derived S peptides could replace a single S protein monomer, blocking the homotrimer formation or inducing stability.

Molecular Determinants and Specificity of mRNA with Alternatively-Spliced UPF1 Isoforms, Influenced by an Insertion in the 'Regulatory Loop'.

The nonsense-mediated mRNA decay (NMD) pathway rapidly detects and degrades mRNA containing premature termination codons (PTCs). UP-frameshift 1 (UPF1), the master regulator of the NMD process, has two alternatively-spliced isoforms; one carries 353-GNEDLVIIWLR-363 insertion in the 'regulatory loop (involved in mRNA binding)'. Such insertion can induce catalytic and/or ATPase activity, as determined experimentally; however, the kinetics and molecular level information are not fully understood. Herein, applying all-atom molecular dynamics, we probe the binding specificity of UPF1 with different GC- and AU-rich mRNA motifs and the influence of insertion to the viable control over UPF1 catalytic activity. Our results indicate two distinct conformations between 1B and RecA2 domains of UPF1: 'open (isoform_2; without insertion)' and 'closed (isoform_1; with insertion)'. These structural movements correspond to an important stacking pattern in mRNA motifs, i.e., absence of stack formation in mRNA, with UPF1 isoform_2 results in the 'open conformation'. Particularly, for UPF1 isoform_1, the increased distance between 1B and RecA2 domains has resulted in reducing the mRNA-UPF1 interactions. Lower fluctuating GC-rich mRNA motifs have better binding with UPF1, compared with AU-rich sequences. Except CCUGGGG, all other GC-rich motifs formed a 4-stack pattern with UPF1. High occupancy R363, D364, T627, and G862 residues were common binding GC-rich motifs, as were R363, N535, and T627 for the AU-rich motifs. The GC-rich motifs behave distinctly when bound to either of the isoforms; lower stability was observed with UPF1 isoform_2. The cancer-associated UPF1 variants (P533L/T and A839T) resulted in decreased protein-mRNA binding efficiency. Lack of mRNA stacking poses in the UPF1P533T system significantly decreased UPF1-mRNA binding efficiency and increased distance between 1B-RecA2. These novel findings can serve to further inform NMD-associated mechanistic and kinetic studies.

Structural determinants of peptide-dependent TAP1-TAP2 transit passage targeted by viral proteins and altered by cancer-associated mutations.

The TAP1-TAP2 complex transports antigenic peptide substrates into the endoplasmic reticulum (ER). In ER, the peptides are further processed and loaded on the major histocompatibility class (MHC) I molecules by the peptide loading complex (PLC). The TAP transporters are linked with the PLC; a target for cancers and viral immune evasion. But the mechanisms whereby the cancer-derived mutations in TAP1-TAP2 or viral factors targeting the PLC, interfere peptide transport are only emerging. This study describes that transit of peptides through TAP can take place via two different channels (4 or 8 helices) depending on peptide length and sequence. Molecular dynamics and binding affinity predictions of peptide-transporters demonstrated that smaller peptides (8-10 mers; e.g. AAGIGILTV, SIINFEKL) can transport quickly through the transport tunnel compared to longer peptides (15-mer; e.g. ENPVVHFFKNIVTPR). In line with a regulated and selective peptide transport by TAPs, the immunopeptidome upon IFN-γ treatment in melanoma cells induced the shorter length (9-mer) peptide presentation over MHC-I that exhibit a relatively weak binding affinity with TAP. A conserved distance between N and C terminus residues of the studied peptides in the transport tunnel were reported. Furthermore, by adversely interacting with the TAP transport passage or affecting TAPNBD domains tilt movement, the viral proteins and cancer-derived mutations in TAP1-TAP2 may induce allosteric effects in TAP that block conformation of the tunnel (closed towards ER lumen). Interestingly, some cancer-associated mutations (e.g. TAP1R372Q and TAP2R373H) can specifically interfere with selective transport channels (i.e. for longer-peptides). These results provide a model for how viruses and cancer-associated mutations targeting TAP interfaces can affect MHC-I antigen presentation, and how the IFN-γ pathway alters MHC-I antigen presentation via the kinetics of peptide transport.

Functional Interfaces, Biological Pathways, and Regulations of Interferon-Related DNA Damage Resistance Signature (IRDS) Genes.

Interferon (IFN)-related DNA damage resistant signature (IRDS) genes are a subgroup of interferon-stimulated genes (ISGs) found upregulated in different cancer types, which promotes resistance to DNA damaging chemotherapy and radiotherapy. Along with briefly discussing IFNs and signalling in this review, we highlighted how different IRDS genes are affected by viruses. On the contrary, different strategies adopted to suppress a set of IRDS genes (STAT1, IRF7, OAS family, and BST2) to induce (chemo- and radiotherapy) sensitivity were deliberated. Significant biological pathways that comprise these genes were classified, along with their frequently associated genes (IFIT1/3, IFITM1, IRF7, ISG15, MX1/2 and OAS1/3/L). Major upstream regulators from the IRDS genes were identified, and different IFN types regulating these genes were outlined. Functional interfaces of IRDS proteins with DNA/RNA/ATP/GTP/NADP biomolecules featured a well-defined pharmacophore model for STAT1/IRF7-dsDNA and OAS1/OAS3/IFIH1-dsRNA complexes, as well as for the genes binding to GDP or NADP+. The Lys amino acid was found commonly interacting with the ATP phosphate group from OAS1/EIF2AK2/IFIH1 genes. Considering the premise that targeting IRDS genes mediated resistance offers an efficient strategy to resensitize tumour cells and enhances the outcome of anti-cancer treatment, this review can add some novel insights to the field.

Viruses, cancer and non-self recognition.

Virus-host interactions form an essential part of every aspect of life, and this review is aimed at looking at the balance between the host and persistent viruses with a focus on the immune system. The virus-host interaction is like a cat-and-mouse game and viruses have developed ingenious mechanisms to manipulate cellular pathways, most notably the major histocompatibility (MHC) class I pathway, to reside within infected cell while evading detection and destruction by the immune system. However, some of the signals sensing and responding to viral infection are derived from viruses and the fact that certain viruses can prevent the infection of others, highlights a more complex coexistence between the host and the viral microbiota. Viral immune evasion strategies also illustrate that processes whereby cells detect and present non-self genetic material to the immune system are interlinked with other cellular pathways. Immune evasion is a target also for cancer cells and a more detailed look at the interfaces between viral factors and components of the MHC class I peptide-loading complex indicates that these interfaces are also targets for cancer mutations. In terms of the immune checkpoint, however, viral and cancer strategies appear different.

Recognition Dynamics of Cancer Mutations on the ERp57-Tapasin Interface.

Down regulation of the major histocompatibility class (MHC) I pathway plays an important role in tumour development, and can be achieved by suppression of HLA expression or mutations in the MHC peptide-binding pocket. The peptide-loading complex (PLC) loads peptides on the MHC-I molecule in a dynamic multi-step assembly process. The effects of cancer variants on ERp57 and tapasin components from the MHC-I pathway is less known, and they could have an impact on antigen presentation. Applying computational approaches, we analysed whether the ERp57-tapasin binding might be altered by missense mutations. The variants H408R(ERp57) and P96L, D100A, G183R(tapasin) at the protein-protein interface improved protein stability (ΔΔG) during the initial screen of 14 different variants. The H408R(ERp57) and P96L(tapasin) variants, located close to disulphide bonds, were further studied by molecular dynamics (MD). Identifying intramolecular a-a' domain interactions, MD revealed open and closed conformations of ERp57 in the presence and absence of tapasin. In wild-type and mutant ERp57-tapasin complexes, residues Val97, Ser98, Tyr100, Trp405, Gly407(ERp57) and Asn94, Cys95, Arg97, Asp100(tapasin) formed common H-bond interactions. Moreover, comparing the H-bond networks for P96L and H408R with each other, suggests that P96L(tapasin) improved ERp57-tapasin binding more than the H408R(ERp57) mutant. During MD, the C-terminus domain (that binds MHC-I) in tapasin from the ERp57(H408R)-tapasin complex moved away from the PLC, whereas in the ERp57-tapasin(P96L) system was oppositely displaced. These findings can have implications for the function of PLC and, ultimately, for the presentation of MHC-I peptide complex on the tumour cell surface.

Insights into the Effects of Cancer Associated Mutations at the UPF2 and ATP-Binding Sites of NMD Master Regulator: UPF1.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that recognizes post-transcriptionally abnormal transcripts and mediates their degradation. The master regulator of NMD is UPF1, an enzyme with intrinsic ATPase and helicase activities. The cancer genomic sequencing data has identified frequently mutated residues in the CH-domain and ATP-binding site of UPF1. In silico screening of UPF1 stability change as a function over 41 cancer mutations has identified five variants with significant effects: K164R, R253W, T499M, E637K, and E833K. To explore the effects of these mutations on the associated energy landscape of UPF1, molecular dynamics simulations (MDS) were performed. MDS identified stable H-bonds between residues S152, S203, S205, Q230/R703, and UPF2/AMPPNP, and suggest that phosphorylation of Serine residues may control UPF1-UPF2 binding. Moreover, the alleles K164R and R253W in the CH-domain improved UPF1-UPF2 binding. In addition, E637K and E833K alleles exhibited improved UPF1-AMPPNP binding compared to the T499M variant; the lower binding is predicted from hindrance caused by the side-chain of T499M to the docking of the tri-phosphate moiety (AMPPNP) into the substrate site. The dynamics of wild-type/mutant systems highlights the flexible nature of the ATP-binding region in UPF1. These insights can facilitate the development of drug discovery strategies for manipulating NMD signaling in cell systems using chemical tools.

Molecular basis and quantitative assessment of TRF1 and TRF2 protein interactions with TIN2 and Apollo peptides.

Shelterin is a six-protein complex (TRF1, TRF2, POT1, RAP1, TIN2, and TPP1) that also functions in smaller subsets in regulation and protection of human telomeres. Two closely related proteins, TRF1 and TRF2, make high-affinity contact directly with double-stranded telomeric DNA and serve as a molecular platform. Protein TIN2 binds to TRF1 and TRF2 dimer-forming domains, whereas Apollo makes interaction only with TRF2. To elucidate the molecular basis of these interactions, we employed molecular dynamics (MD) simulations of TRF1TRFH-TIN2TBM and TRF2TRFH-TIN2TBM/ApolloTBM complexes and of the isolated proteins. MD enabled a structural and dynamical comparison of protein-peptide complexes including H-bond interactions and interfacial residues that may regulate TRF protein binding to the given peptides, especially focusing on interactions described in crystallographic data. Residues with a selective function in both TRF1TRFH and TRF2TRFH and forming a stable hydrogen bond network with TIN2TBM or ApolloTBM peptides were traced. Our study revealed that TIN2TBM forms a well-defined binding mode with TRF1TRFH as compared to TRF2TRFH, and that the binding pocket of TIN2TBM is deeper for TRF2TRFH protein than ApolloTBM. The MD data provide a basis for the reinterpretation of mutational data obtained in crystallographic work for the TRF proteins. Together, the previously determined X-ray structure and our MD provide a detailed view of the TRF-peptide binding mode and the structure of TRF1/2 binding pockets. Particular TRF-peptide interactions are very specific for the formation of each protein-peptide complex, identifying TRF proteins as potential targets for the design of inhibitors/drugs modulating telomere machinery for anticancer therapy.

Molecular Modeling and Evaluation of Novel Dibenzopyrrole Derivatives as Telomerase Inhibitors and Potential Drug for Cancer Therapy.

During previous years, many studies on synthesis, as well as on anti-tumor, anti-inflammatory and anti-bacterial activities of the pyrazole derivatives have been described. Certain pyrazole derivatives exhibit important pharmacological activities and have proved to be useful template in drug research. Considering importance of pyrazole template, in current work the series of novel inhibitors were designed by replacing central ring of acridine with pyrazole ring. These heterocyclic compounds were proposed as a new potential base for telomerase inhibitors. Obtained dibenzopyrrole structure was used as a novel scaffold structure and extension of inhibitors was done by different functional groups. Docking of newly designed compounds in the telomerase active site (telomerase catalytic subunit TERT) was carried out. All dibenzopyrrole derivatives were evaluated by three docking programs: CDOCKER, Ligandfit docking (Scoring Functions) and AutoDock. Compound C_9g, C_9k and C_9l performed best in comparison to all designed inhibitors during the docking in all methods and in interaction analysis. Introduction of pyrazole and extension of dibenzopyrrole in compounds confirm that such compound may act as potential telomerase inhibitors.