Arginine methylation of caspase-8 controls life/death decisions in extrinsic apoptotic networks.

Procaspase-8 is a key mediator of death receptor (DR)-mediated pathways. Recently, the role of post-translational modifications (PTMs) of procaspase-8 in controlling cell death has received increasing attention. Here, using mass spectrometry screening, pharmacological inhibition and biochemical assays, we show that procaspase-8 can be targeted by the PRMT5/RIOK1/WD45 methylosome complex. Furthermore, two potential methylation sites of PRMT5 on procaspase-8, R233 and R435, were identified in silico. R233 and R435 are highly conserved in mammals and their point mutations are among the most common mutations of caspase-8 in cancer. The introduction of mutations at these positions resulted in inhibitory effects on CD95L-induced caspase-8 activity, effector caspase activation and apoptosis. In addition, we show that procaspase-8 can undergo symmetric di-methylation. Finally, the pharmacological inhibition of PRMT5 resulted in the inhibitory effects on caspase activity and apoptotic cell death. Taken together, we have unraveled the additional control checkpoint in procaspase-8 activation and the arginine methylation network in the extrinsic apoptosis pathway.

SEMA 2.0: web-platform for B-cell conformational epitopes prediction using artificial intelligence.

Prediction of conformational B-cell epitopes is a crucial task in vaccine design and development. In this work, we have developed SEMA 2.0, a user-friendly web platform that enables the research community to tackle the B-cell epitopes prediction problem using state-of-the-art protein language models. SEMA 2.0 offers comprehensive research tools for sequence- and structure-based conformational B-cell epitopes prediction, accurate identification of N-glycosylation sites, and a distinctive module for comparing the structures of antigen B-cell epitopes enhancing our ability to analyze and understand its immunogenic properties. SEMA 2.0 website https://sema.airi.net is free and open to all users and there is no login requirement. Source code is available at https://github.com/AIRI-Institute/SEMAi.

Modulation of extrinsic apoptotic pathway by intracellular glycosylation.

The importance of post-translational modifications (PTMs), particularly O-GlcNAcylation, of cytoplasmic proteins in apoptosis has been neglected for quite a while. Modification of cytoplasmic proteins by a single N-acetylglucosamine sugar is a dynamic and reversible PTM exhibiting properties more like phosphorylation than classical O- and N-linked glycosylation. Due to the sparse information existing, we have only limited understanding of how GlcNAcylation affects cell death. Deciphering the role of GlcNAcylation in cell fate may provide further understanding of cell fate decisions. This review focus on the modulation of extrinsic apoptotic pathway via GlcNAcylation carried out by O-GlcNAc transferase (OGT) or by other bacterial effector proteins.

PROSTATA: a framework for protein stability assessment using transformers.

MOTIVATION: Accurate prediction of change in protein stability due to point mutations is an attractive goal that remains unachieved. Despite the high interest in this area, little consideration has been given to the transformer architecture, which is dominant in many fields of machine learning. RESULTS: In this work, we introduce PROSTATA, a predictive model built in a knowledge-transfer fashion on a new curated dataset. PROSTATA demonstrates advantage over existing solutions based on neural networks. We show that the large improvement margin is due to both the architecture of the model and the quality of the new training dataset. This work opens up opportunities to develop new lightweight and accurate models for protein stability assessment. AVAILABILITY AND IMPLEMENTATION: PROSTATA is available at https://github.com/AIRI-Institute/PROSTATA and https://prostata.airi.net.

Reconstruction of the regulatory hypermethylation network controlling hepatocellular carcinoma development during hepatitis C viral infection.

Hepatocellular carcinoma (HCC) has been associated with hepatitis C viral (HCV) infection as a potential risk factor. Nonetheless, the precise genetic regulatory mechanisms triggered by the virus, leading to virus-induced hepatocarcinogenesis, remain unclear. We hypothesized that HCV proteins might modulate the activity of aberrantly methylated HCC genes through regulatory pathways. Virus-host regulatory pathways, interactions between proteins, gene expression, transport, and stability regulation, were reconstructed using the ANDSystem. Gene expression regulation was statistically significant. Gene network analysis identified four out of 70 HCC marker genes whose expression regulation by viral proteins may be associated with HCC: DNA-binding protein inhibitor ID - 1 (ID1), flap endonuclease 1 (FEN1), cyclin-dependent kinase inhibitor 2A (CDKN2A), and telomerase reverse transcriptase (TERT). It suggested the following viral protein effects in HCV/human protein heterocomplexes: HCV NS3(p70) protein activates human STAT3 and NOTC1; NS2-3(p23), NS5B(p68), NS1(E2), and core(p21) activate SETD2; NS5A inhibits SMYD3; and NS3 inhibits CCN2. Interestingly, NS3 and E1(gp32) activate c-Jun when it positively regulates CDKN2A and inhibit it when it represses TERT. The discovered regulatory mechanisms might be key areas of focus for creating medications and preventative therapies to decrease the likelihood of HCC development during HCV infection.

SEMA: Antigen B-cell conformational epitope prediction using deep transfer learning.

One of the primary tasks in vaccine design and development of immunotherapeutic drugs is to predict conformational B-cell epitopes corresponding to primary antibody binding sites within the antigen tertiary structure. To date, multiple approaches have been developed to address this issue. However, for a wide range of antigens their accuracy is limited. In this paper, we applied the transfer learning approach using pretrained deep learning models to develop a model that predicts conformational B-cell epitopes based on the primary antigen sequence and tertiary structure. A pretrained protein language model, ESM-1v, and an inverse folding model, ESM-IF1, were fine-tuned to quantitatively predict antibody-antigen interaction features and distinguish between epitope and non-epitope residues. The resulting model called SEMA demonstrated the best performance on an independent test set with ROC AUC of 0.76 compared to peer-reviewed tools. We show that SEMA can quantitatively rank the immunodominant regions within the SARS-CoV-2 RBD domain. SEMA is available at https://github.com/AIRI-Institute/SEMAi and the web-interface http://sema.airi.net.

Regulation of extrinsic apoptotic signaling by c-FLIP: towards targeting cancer networks.

The extrinsic pathway is mediated by death receptors (DRs), including CD95 (APO-1/Fas) or TRAILR-1/2. Defects in apoptosis regulation lead to cancer and other malignancies. The master regulator of the DR networks is the cellular FLICE inhibitory protein (c-FLIP). In addition to its key role in apoptosis, c-FLIP may exert other cellular functions, including control of necroptosis, pyroptosis, nuclear factor κB (NF-κB) activation, and tumorigenesis. To gain further insight into the molecular mechanisms of c-FLIP action in cancer networks, we focus on the structure, isoforms, interactions, and post-translational modifications of c-FLIP. We also discuss various avenues to target c-FLIP in cancer cells for therapeutic benefit.

Impact of human CD95 mutations on cell death and autoimmunity: a model.

CD95/Fas/APO-1 can trigger apoptotic as well as nonapoptotic pathways in immune cells. CD95 signaling in humans can be inhibited by several mechanisms, including mutations in the gene encoding CD95. CD95 mutations lead to autoimmune disorders, such as autoimmune lymphoproliferative syndrome (ALPS). Gaining further insight into the reported mutations of CD95 and resulting alterations of its signaling networks may provide further understanding of their presumed role in certain autoimmune diseases. For illustrative purposes and to better understand the potential outcomes of CD95 mutations, here we assign their positions to the recently determined 3D structures of human CD95. Based on this, we make certain predictions and speculate on the putative role of CD95 mutation defects in CD95-mediated signaling for certain autoimmune diseases.

Computer analysis of the relation between hydrogen bond stability in SOD1 mutants and the survival time of amyotrophic lateral sclerosis patients.

BACKGROUND AND OBJECTIVE: Mutations in the SOD1 protein can lead to the death of motor neurons, which, in turn, causes an incurable disease called amyotrophic lateral sclerosis (ALS). At the same time, the mechanism of the onset and development of this disease is not fully understood and is often contradictory. METHODS: Accelerated Molecular Dynamics as implemented in the OpenMM library, principal component analysis, regression analysis, random forest method. RESULTS: The stability of hydrogen bonds in 72 mutants of the SOD1 protein was calculated. Principal component analysis was carried out. Based on ten principal components acting as predictors, a multiple linear regression model was constructed. An analysis of the correlation of these ten principal components with the initial values of the stability of hydrogen bonds made it possible to characterize the contribution of known structurally and functionally important sites in the SOD1 to the scatter of ALS patients' survival time. CONCLUSION: Such an analysis made it possible to put forward hypotheses about the relationship between the stabilizing and destabilizing effects of mutations in different structurally and functionally important regions of SOD1 with the patients's survival time.

Pharmacological targeting of c-FLIPL and Bcl-2 family members promotes apoptosis in CD95L-resistant cells.

The development of efficient combinatorial treatments is one of the key tasks in modern anti-cancer therapies. An apoptotic signal can either be induced by activation of death receptors (DR) (extrinsic pathway) or via the mitochondria (intrinsic pathway). Cancer cells are characterized by deregulation of both pathways. Procaspase-8 activation in extrinsic apoptosis is controlled by c-FLIP proteins. We have recently reported the small molecules FLIPinB/FLIPinBγ targeting c-FLIPL in the caspase-8/c-FLIPL heterodimer. These small molecules enhanced caspase-8 activity in the death-inducing signaling complex (DISC), CD95L/TRAIL-induced caspase-3/7 activation and subsequent apoptosis. In this study to increase the pro-apoptotic effects of FLIPinB/FLIPinBγ and enhance its therapeutic potential we investigated costimulatory effects of FLIPinB/FLIPinBγ in combination with the pharmacological inhibitors of the anti-apoptotic Bcl-2 family members such as ABT-263 and S63845. The combination of these inhibitors together with FLIPinB/FLIPinBγ increased CD95L-induced cell viability loss, caspase activation and apoptosis. Taken together, our study suggests new approaches for the development of combinatorial anti-cancer therapies specifically targeting both intrinsic and extrinsic apoptosis pathways.

The role of death domain proteins in host response upon SARS-CoV-2 infection: modulation of programmed cell death and translational applications.

The current pandemic of novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) poses a significant global public health threat. While urgent regulatory measures in control of the rapid spread of this virus are essential, scientists around the world have quickly engaged in this battle by studying the molecular mechanisms and searching for effective therapeutic strategies against this deadly disease. At present, the exact mechanisms of programmed cell death upon SARS-CoV-2 infection remain to be elucidated, though there is increasing evidence suggesting that cell death pathways play a key role in SARS-CoV-2 infection. There are several types of programmed cell death, including apoptosis, pyroptosis, and necroptosis. These distinct programs are largely controlled by the proteins of the death domain (DD) superfamily, which play an important role in viral pathogenesis and host antiviral response. Many viruses have acquired the capability to subvert the program of cell death and evade the host immune response, mainly by virally encoded gene products that control cell signaling networks. In this mini-review, we will focus on SARS-CoV-2, and discuss the implication of restraining the DD-mediated signaling network to potentially suppress viral replication and reduce tissue damage.

ANDDigest: a new web-based module of ANDSystem for the search of knowledge in the scientific literature.

BACKGROUND: The rapid growth of scientific literature has rendered the task of finding relevant information one of the critical problems in almost any research. Search engines, like Google Scholar, Web of Knowledge, PubMed, Scopus, and others, are highly effective in document search; however, they do not allow knowledge extraction. In contrast to the search engines, text-mining systems provide extraction of knowledge with representations in the form of semantic networks. Of particular interest are tools performing a full cycle of knowledge management and engineering, including automated retrieval, integration, and representation of knowledge in the form of semantic networks, their visualization, and analysis. STRING, Pathway Studio, MetaCore, and others are well-known examples of such products. Previously, we developed the Associative Network Discovery System (ANDSystem), which also implements such a cycle. However, the drawback of these systems is dependence on the employed ontologies describing the subject area, which limits their functionality in searching information based on user-specified queries. RESULTS: The ANDDigest system is a new web-based module of the ANDSystem tool, permitting searching within PubMed by using dictionaries from the ANDSystem tool and sets of user-defined keywords. ANDDigest allows performing the search based on complex queries simultaneously, taking into account many types of objects from the ANDSystem's ontology. The system has a user-friendly interface, providing sorting, visualization, and filtering of the found information, including mapping of mentioned objects in text, linking to external databases, sorting of data by publication date, citations number, journal H-indices, etc. The system provides data on trends for identified entities based on dynamics of interest according to the frequency of their mentions in PubMed by years. CONCLUSIONS: The main feature of ANDDigest is its functionality, serving as a specialized search for information about multiple associative relationships of objects from the ANDSystem's ontology vocabularies, taking into account user-specified keywords. The tool can be applied to the interpretation of experimental genetics data, the search for associations between molecular genetics objects, and the preparation of scientific and analytical reviews. It is presently available at https://anddigest.sysbio.ru/ .

Controlling Cell Death through Post-translational Modifications of DED Proteins.

Apoptosis is a form of programmed cell death, deregulation of which occurs in multiple disorders, including neurodegenerative and autoimmune diseases as well as cancer. The formation of a death-inducing signaling complex (DISC) and death effector domain (DED) filaments are critical for initiation of the extrinsic apoptotic pathway. Post-translational modifications (PTMs) of DED-containing DISC components such as FADD, procaspase-8, and c-FLIP comprise an additional level of apoptosis regulation, which is necessary to overcome the threshold for apoptosis induction. In this review we discuss the influence of PTMs of FADD, procaspase-8, and c-FLIP on DED filament assembly and cell death induction, with a focus on the 3D organization of the DED filament.

Learning the changes of barnase mutants thermostability from structural fluctuations obtained using anisotropic network modeling.

In biotechnology applications, rational design of new proteins with improved physico-chemical properties includes a number of important tasks. One of the greatest practical and fundamental challenges is the design of highly thermostable protein enzymes that maintain catalytic activity at high temperatures. This problem may be solved by introducing mutations into the wild-type enzyme protein. In this work, to predict the impact of such mutations in barnase protein we applied the anisotropic network modeling approach, revealing atomic fluctuations in structural regions that are changed in mutants compared to the wild-type protein. A regression model was constructed based on these structural features that can allow one to predict the thermal stability of new barnase mutants. Moreover, the analysis of regression model provides a mechanistic explanation of how the structural features can contribute to the thermal stability of barnase mutants.

Dissecting DISC regulation via pharmacological targeting of caspase-8/c-FLIPL heterodimer.

Pharmacological targeting via small molecule-based chemical probes has recently acquired an emerging importance as a valuable tool to delineate molecular mechanisms. Induction of apoptosis via CD95/Fas and TRAIL-R1/2 is triggered by the formation of the death-inducing signaling complex (DISC). Caspase-8 activation at the DISC is largely controlled by c-FLIP proteins. However molecular mechanisms of this control have just started to be uncovered. In this study we report the first-in-class chemical probe targeting c-FLIPL in the heterodimer caspase-8/c-FLIPL. This rationally designed small molecule was aimed to imitate the closed conformation of the caspase-8 L2' loop and thereby increase caspase-8 activity after initial processing of the heterodimer. In accordance with in silico predictions, this small molecule enhanced caspase-8 activity at the DISC, CD95L/TRAIL-induced caspase activation, and subsequent apoptosis. The generated computational model provided further evidence for the proposed effects of the small molecule on the heterodimer caspase-8/c-FLIPL. In particular, the model has demonstrated that boosting caspase-8 activity by the small molecule at the early time points after DISC assembly is crucial for promoting apoptosis induction. Taken together, our study allowed to target the heterodimer caspase-8/c-FLIPL and get new insights into molecular mechanisms of its activation.

Long and short isoforms of c-FLIP act as control checkpoints of DED filament assembly.

The assembly of the death-inducing signaling complex (DISC) and death effector domain (DED) filaments at CD95/Fas initiates extrinsic apoptosis. Procaspase-8 activation at the DED filaments is controlled by short and long c-FLIP isoforms. Despite apparent progress in understanding the assembly of CD95-activated platforms and DED filaments, the detailed molecular mechanism of c-FLIP action remains elusive. Here, we further addressed the mechanisms of c-FLIP action at the DISC using biochemical assays, quantitative mass spectrometry, and structural modeling. Our data strongly indicate that c-FLIP can bind to both FADD and procaspase-8 at the DED filament. Moreover, the constructed in silico model shows that c-FLIP proteins can lead to the formation of the DISCs comprising short DED filaments as well as serve as bridging motifs for building a cooperative DISC network, in which adjacent CD95 DISCs are connected by DED filaments. This network is based on selective interactions of FADD with both c-FLIP and procaspase-8. Hence, c-FLIP proteins at the DISC control initiation, elongation, and composition of DED filaments, playing the role of control checkpoints. These findings provide new insights into DISC and DED filament regulation and open innovative possibilities for targeting the extrinsic apoptosis pathway.

Delineating the role of c-FLIP/NEMO interaction in the CD95 network via rational design of molecular probes.

BACKGROUND: Structural homology modeling supported by bioinformatics analysis plays a key role in uncovering new molecular interactions within gene regulatory networks. Here, we have applied this powerful approach to analyze the molecular interactions orchestrating death receptor signaling networks. In particular, we focused on the molecular mechanisms of CD95-mediated NF-κB activation and the role of c-FLIP/NEMO interaction in the induction of this pathway. RESULTS: To this end, we have created the homology model of the c-FLIP/NEMO complex using the reported structure of the v-FLIP/NEMO complex, and rationally designed peptides targeting this complex. The designed peptides were based on the NEMO structure. Strikingly, the experimental in vitro validation demonstrated that the best inhibitory effects on CD95-mediated NF-κB activation are exhibited by the NEMO-derived peptides with the substitution D242Y of NEMO. Furthermore, we have assumed that the c-FLIP/NEMO complex is recruited to the DED filaments formed upon CD95 activation and validated this assumption in silico. Further insight into the function of c-FLIP/NEMO complex was provided by the analysis of evolutionary conservation of interacting regions which demonstrated that this interaction is common in distinct mammalian species. CONCLUSIONS: Taken together, using a combination of bioinformatics and experimental approaches we obtained new insights into CD95-mediated NF-κB activation, providing manifold possibilities for targeting the death receptor network.

Organization and evolution of the chalcone synthase gene family in bread wheat and relative species.

BACKGROUND: Flavonoid compounds are secondary plant metabolites, having a functional importance in plant development, protection from pathogens and unfavorable environmental factors. Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoids; it is involved in biosynthesis of all classes of flavonoid compounds. Nevertheless, the Chs gene family in bread wheat (Triticum aestivum L.) has been not characterized yet. The aim of the current study was to investigate structural and functional organization of the Chs genes and evolution of this gene family in bread wheat and relative species. RESULTS: The nucleotide sequences of the eight Chs copies in T. aestivum were identified. Among them, two homoeologous sets of the Chs genes were located on the short (Chs-A1, -B1, -D1) and the long (Chs-A4, -B4, -D4) arms of homoeologous group 2 chromosomes. Two paralogous gene copies in the B-genome (Chs-B2, -B3) were located in the distal regions of 2BS chromosome. To clarify the origin of Chs duplications in the B-genome the phylogenetic analysis with the Chs sequences of Triticum and Aegilops species carrying ancestral genomes was conducted. It was estimated that the first duplication event occurred in the genome of the common ancestor of Triticum and Aegilops genera about 10-12 million years ago (MYA), then another copy was formed in the ancestor of the B-genome about 6-7 MYA. A homology modeling revealed high sequence similarity of bread wheat CHS enzymes. A number of short deletions in coding regions of some Chs sequences are not expected to have any significant functional effects. Estimation of transcriptional activity of the Chs copies along with a comparative analysis of their promoters structure suggested their functional specialization, which likely contributed to the maintaining of the duplicated Chs genes in wheat genome. CONCLUSIONS: From possible ten Chs copies in bread wheat genome, eight members of this family retained their intact structure and activity, while two copies appear to be lost at the level of diploid and tetraploid ancestors. Transcriptional assay along with a comparative analysis of the cis-regulatory elements revealed their functional diversification. The multiple functions supported by the Chs family are assumed to be a driving force for duplications of the Chs gene and their retention in plant genome.

DNase and RNase activities of fresh cow milk lactoferrin.

Lactoferrin (LF) is an Fe3+ -binding glycoprotein first recognized in milk and then in other epithelial secretions and barrier body fluids to which many different functions have been attributed to LF, including protection from iron-induced lipid peroxidation, immunomodulation, cell growth regulation, DNA and RNA binding, as well as transcriptional activation, еtс. The polyfunctional physiological role of LF is still unclear, but it has been suggested to be responsible for primary defense against microbial and viral infections. Here, we present the first evidence that LF preparations isolated from milk of 18 cows of different breeds possess various levels of metal-dependent DNase and metal-independent RNase activities. For univocal assignment of DNase and RNase activities to cow LF, it was subjected to SDS-PAGE using gels with copolymerized calf thymus DNA or polymeric yeast RNA. In situ analysis was revealed DNase and RNase activities only in the gel zones corresponding to homogeneous LF. In contrast to human LF, cow LF possesses a relatively low cytotoxicity towards human tumor cells. The discovery that cow LF has these activities may contribute to understanding the multiple physiological functions of this extremely polyfunctional protein, including its protective role against microbial and viral infections. The computational spatial model of cow LF complex with DNA was obtained: according to the model positively charged residues of LF contact with DNA.

Design, Synthesis and Molecular Modeling Study of Conjugates of ADP and Morpholino Nucleosides as A Novel Class of Inhibitors of PARP-1, PARP-2 and PARP-3.

We report on the design, synthesis and molecular modeling study of conjugates of adenosine diphosphate (ADP) and morpholino nucleosides as potential selective inhibitors of poly(ADP-ribose)polymerases-1, 2 and 3. Sixteen dinucleoside pyrophosphates containing natural heterocyclic bases as well as 5-haloganeted pyrimidines, and mimicking a main substrate of these enzymes, nicotinamide adenine dinucleotide (NAD+)-molecule, have been synthesized in a high yield. Morpholino nucleosides have been tethered to the ß-phosphate of ADP via a phosphoester or phosphoramide bond. Screening of the inhibiting properties of these derivatives on the autopoly(ADP-ribosyl)ation of PARP-1 and PARP-2 has shown that the effect depends upon the type of nucleobase as well as on the linkage between ADP and morpholino nucleoside. The 5-iodination of uracil and the introduction of the P-N bond in NAD+-mimetics have shown to increase inhibition properties. Structural modeling suggested that the P-N bond can stabilize the pyrophosphate group in active conformation due to the formation of an intramolecular hydrogen bond. The most active NAD+ analog against PARP-1 contained 5-iodouracil 2'-aminomethylmorpholino nucleoside with IC50 126 ± 6 µM, while in the case of PARP-2 it was adenine 2'-aminomethylmorpholino nucleoside (IC50 63 ± 10 µM). In silico analysis revealed that thymine and uracil-based NAD+ analogs were recognized as the NAD+-analog that targets the nicotinamide binding site. On the contrary, the adenine 2'-aminomethylmorpholino nucleoside-based NAD+ analogs were predicted to identify as PAR-analogs that target the acceptor binding site of PARP-2, representing a novel molecular mechanism for selective PARP inhibition. This discovery opens a new avenue for the rational design of PARP-1/2 specific inhibitors.