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Removal of the mRNA 5' cap primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme Dcp2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5'-3'exoribonuclease Xrn1. Kinetoplastida lack Dcp2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

African trypanosomiasis (AT) is a hemoparasitic disease caused by infection with African trypanosomes and it is prevalent in many sub-Saharan African countries, affecting both humans and domestic animals. The disease is transmitted mostly by haematophagous insects of the genus Glossina while taking blood meal, in the process spreading the parasites from an infected animal to an uninfected animal. The disease is fatal if untreated, and the available drugs are generally ineffective and resulting in toxicities. Therefore, it is still pertinent to explore novel methods and targets for drug discovery. Proteolysis-targeting chimeras (PROTACs) present a new strategy for development of therapeutic molecules that mimic cellular proteasomal-mediated protein degradation to target proteins involved in different disease types. PROTACs have been used to degrade proteins involved in various cancers, neurodegenerative diseases, and immune disorders with remarkable success. Here, we highlight the problems associated with the current treatments for AT, discuss the concept of PROTACs and associated targeted protein degradation (TPD) approaches, and provide some insights on the future potential for the use of these emerging technologies (PROTACs and TPD) for the development of new generation of anti-Trypanosoma drugs and the first "TrypPROTACs"., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

COVID-19 pandemic continues to remain a global health concern owing to the emergence of newer variants. Several multi-Omics studies have produced extensive evidence on host-pathogen interactions and potential therapeutic targets. Nonetheless, an increased understanding of host signaling networks regulated by post-translational modifications and their ensuing effect on the cellular dynamics is critical to expanding the current knowledge on SARS-CoV-2 infections. Through an unbiased transcriptomics, proteomics, acetylomics, phosphoproteomics, and exometabolome analysis of a lung-derived human cell line, we show that SARS-CoV-2 Norway/Trondheim-S15 strain induces time-dependent alterations in the induction of type I IFN response, activation of DNA damage response, dysregulated Hippo signaling, among others. We identified interplay of phosphorylation and acetylation dynamics on host proteins and its effect on the altered release of metabolites, especially organic acids and ketone bodies. Together, our findings serve as a resource of potential targets that can aid in designing novel host-directed therapeutic strategies., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Analysis of the protein coding transcriptome by the RNA sequencing requires either enrichment of the desired fraction of coding transcripts or depletion of the abundant non-coding fraction consisting mainly of rRNA. We propose an alternative mRNA enrichment strategy based on the RNA-binding properties of the human IFIT1, an antiviral protein recognizing cap 0 RNA. Here, we compare for Saccharomyces cerevisiae an IFIT1-based mRNA pull-down with yeast targeted rRNA depletion by the RiboMinus method. IFIT1-based RNA capture depletes rRNA more effectively, producing high quality RNA-seq data with an excellent coverage of the protein coding transcriptome, while depleting cap-less transcripts such as mitochondrial or some non-coding RNAs. We propose IFIT1 as a cost effective and versatile tool to prepare mRNA libraries for a variety of organisms with cap 0 mRNA ends, including diverse plants, fungi and eukaryotic microbes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Electrostatic energy has a significant contribution to intermolecular interaction energy, especially in biological systems. Unfortunately, precise quantum mechanics calculations are not feasible for large biological systems; hence, simpler calculation methods are required. We propose a method called UBDB+EPMM (University at Buffalo Pseudoatom DataBank + Exact Potential Multipole Moments), which shortens computational time without losing accuracy. Here, we characterize electrostatic interactions in selected complexes of IFIT proteins with RNA. IFIT proteins are effectors of the innate immune system, and by binding foreign RNA, they prevent the synthesis of viral proteins in human host cells; hence, they block the propagation of viruses. We show that by using the UBDB+EPMM method it is possible to describe protein-RNA interactions not only qualitatively but also quantitatively. Looking at the charge penetration contribution to electrostatic interactions, we find all amino acid residues with strong local interactions. Moreover, we confirm that electrostatic interaction of IFIT5 with pppRNA does not depend on the sequence of the RNA.

Human African trypanosomiasis (HAT) is a neglected tropical disease that is caused by flagellated parasites of the genus Trypanosoma . HAT imposes a significant socio-economic burden on many countries in sub-Saharan Africa and its control is hampered by several drawbacks ranging from the ineffectiveness of drugs, complex dosing regimens, drug resistance, and lack of a vaccine. Despite more than a century of research and investigations, the development of a vaccine to tackle HAT is still challenging due to the complex biology of the pathogens. Advancements in computational modeling coupled with the availability of an unprecedented amount of omics data from different organisms have allowed the design of new generation vaccines that offer better antigenicity and safety profile. One of such new generation approaches is a multi-epitope vaccine (MEV) designed from a collection of antigenic peptides. A MEV can stimulate both cellular and humoral immune responses as well as avoiding possible allergenic reactions. Herein, we take advantage of this approach to design a MEV from conserved hypothetical plasma membrane proteins of Trypanosoma brucei gambiense , the trypanosome subspecies that is responsible for the west and central African forms of HAT. The designed MEV is 402 amino acids long (41.5 kDa). It is predicted to be antigenic, non-toxic, to assume a stable 3D conformation, and to interact with a key immune receptor. In addition, immune simulation foresaw adequate immune stimulation by the putative antigen and a lasting memory. Therefore, the designed chimeric vaccine represents a potential candidate that could be used to target HAT., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

A conserved, 26-residue sequence [AA(X 2 )[A/G][G/L](X 2 )GDV[I/L](X 2 )[V/L]NGE(X 1 )V(X 6 )] and corresponding structure repeating module were identified within the HtrA protease family using a non-redundant set (N = 20) of publicly available structures. While the repeats themselves were far from sequence perfect, they had notable conservation to a statistically significant level. Three or more repetitions were identified within each protein despite being statistically expected to randomly occur only once per 1031 residues. This sequence repeat was associated with a six stranded antiparallel β-barrel module, two of which are present in the core of the structures of the PA clan of serine proteases, while a modified version of this module could be identified in the PDZ-like domains. Automated structural alignment methods had difficulties in superimposing these β-barrels, but the use of a target human HtrA2 structure showed that these modules had an average RMSD across the set of structures of less than 2 Å (mean and median). Our findings support Dayhoff's hypothesis that complex proteins arose through duplication of simpler peptide motifs and domains.

The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder the development of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions. In our workflow, we included methods for expression and purification of ClpX, ClpP, SspB and eGFP-degrons, assays of ClpX ATPase activity, of eGFP-degron binding to SspB and for measuring eGFP-degron degradation in vitro and in vivo. Using uniform, precise and sensitive methods under the same conditions on a range of eGFP-degrons allowed us to determine subtle differences in their properties that can affect their potential applications. Our findings can serve as a reference and a resource for developing targeted protein degradation approaches.

Protein repeats are short, highly similar peptide motifs that occur several times within a single protein, for example the TPR and Ankyrin repeats. Understanding the role of mutation in these proteins is complicated by the competing facts that 1) the repeats are much more restricted to a set sequence than non-repeat proteins, so mutations should be harmful much more often because there are more residues that are heavily restricted due to the need of the sequence to repeat and 2) the symmetry of the repeats in allows the distribution of functional contributions over a number of residues so that sometimes no specific site is singularly responsible for function (unlike enzymatic active site catalytic residues). To address this issue, we review the effects of mutations in a number of natural repeat proteins from the tetratricopeptide and Ankyrin repeat families. We find that mutations are context dependent. Some mutations are indeed highly disruptive to the function of the protein repeats while mutations in identical positions in other repeats in the same protein have little to no effect on structure or function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Izert, Szybowska, Górna and Merski.)

A repertoire of proteolysis-targeting signals known as degrons is a necessary component of protein homeostasis in every living cell. In bacteria, degrons can be used in place of chemical genetics approaches to interrogate and control protein function. Here, we provide a comprehensive review of synthetic applications of degrons in targeted proteolysis in bacteria. We describe recent advances ranging from large screens employing tunable degradation systems and orthogonal degrons, to sophisticated tools and sensors for imaging. Based on the success of proteolysis-targeting chimeras as an emerging paradigm in cancer drug discovery, we discuss perspectives on using bacterial degraders for studying protein function and as novel antimicrobials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Izert, Klimecka and Górna.)

This study aimed to identify an appropriate simple mathematical model to fit the number of coronavirus disease 2019 (COVID-19) cases at the national level for the early portion of the pandemic, before significant public health interventions could be enacted. The total number of cases for the COVID-19 epidemic over time in 28 countries was analysed and fit to several simple rate models. The resulting model parameters were used to extrapolate projections for more recent data. While the Gompertz growth model (mean R2 = 0.998) best fit the current data, uncertainties in the eventual case limit introduced significant model errors. However, the quadratic rate model (mean R2 = 0.992) fit the current data best for 25 (89%) countries as determined by R2 values of the remaining models. Projection to the future using the simple quadratic model accurately forecast the number of future total number of cases 50% of the time up to 10 days in advance. Extrapolation to the future with the simple exponential model significantly overpredicted the total number of future cases. These results demonstrate that accurate future predictions of the case load in a given country can be made using this very simple model.

Adipose tissue macrophages (ATMs) display tremendous heterogeneity depending on signals in their local microenvironment and contribute to the pathogenesis of obesity. The phosphoinositide 3-kinase (PI3K) signalling pathway, antagonized by the phosphatase and tensin homologue (PTEN), is important for metabolic responses to obesity. We hypothesized that fluctuations in macrophage-intrinsic PI3K activity via PTEN could alter the trajectory of metabolic disease by driving distinct ATM populations. Using mice harbouring macrophage-specific PTEN deletion or bone marrow chimeras carrying additional PTEN copies, we demonstrate that sustained PI3K activity in macrophages preserves metabolic health in obesity by preventing lipotoxicity. Myeloid PI3K signalling promotes a beneficial ATM population characterized by lipid uptake, catabolism and high expression of the scavenger macrophage receptor with collagenous structure (MARCO). Dual MARCO and myeloid PTEN deficiencies prevent the generation of lipid-buffering ATMs, reversing the beneficial actions of elevated myeloid PI3K activity in metabolic disease. Thus, macrophage-intrinsic PI3K signalling boosts metabolic health by driving ATM programmes associated with MARCO-dependent lipid uptake.

We present a method to rapidly identify hydrogen-mediated interactions in proteins (e.g., hydrogen bonds, hydrogen bonds, water-mediated hydrogen bonds, salt bridges, and aromatic π-hydrogen interactions) through heavy atom geometry alone, that is, without needing to explicitly determine hydrogen atom positions using either experimental or theoretical methods. By including specific real (or virtual) partner atoms as defined by the atom type of both the donor and acceptor heavy atoms, a set of unique angles can be rapidly calculated. By comparing the distance between the donor and the acceptor and these unique angles to the statistical preferences observed in the Protein Data Bank (PDB), we were able to identify a set of conserved geometries (15 for donor atoms and 7 for acceptor atoms) for hydrogen-mediated interactions in proteins. This set of identified interactions includes every polar atom type present in the Protein Data Bank except OE1 (glutamate/glutamine sidechain) and a clear geometric preference for the methionine sulfur atom (SD) to act as a hydrogen bond acceptor. This method could be readily applied to protein design efforts.

Background: Protein repeats can confound sequence analyses because the repetitiveness of their amino acid sequences lead to difficulties in identifying whether similar repeats are due to convergent or divergent evolution. We noted that the patterns derived from traditional "dot plot" protein sequence self-similarity analysis tended to be conserved in sets of related repeat proteins and this conservation could be quantitated using a Jaccard metric., Results: Comparison of these dot plots obviated the issues due to sequence similarity for analysis of repeat proteins. A high Jaccard similarity score was suggestive of a conserved relationship between closely related repeat proteins. The dot plot patterns decayed quickly in the absence of selective pressure with an expected loss of 50% of Jaccard similarity due to a loss of 8.2% sequence identity. To perform method testing, we assembled a standard set of 79 repeat proteins representing all the subgroups in RepeatsDB. Comparison of known repeat and non-repeat proteins from the PDB suggested that the information content in dot plots could be used to identify repeat proteins from pure sequence with no requirement for structural information. Analysis of the UniRef90 database suggested that 16.9% of all known proteins could be classified as repeat proteins. These 13.3 million putative repeat protein chains were clustered and a significant amount (82.9%) of clusters containing between 5 and 200 members were of a single functional type., Conclusions: Dot plot analysis of repeat proteins attempts to obviate issues that arise due to the sequence degeneracy of repeat proteins. These results show that this kind of analysis can efficiently be applied to analyze repeat proteins on a large scale.

Regulation of the c-Abl (ABL1) tyrosine kinase is important because of its role in cellular signaling, and its relevance in the leukemiogenic counterpart (BCR-ABL). Both auto-inhibition and full activation of c-Abl are regulated by the interaction of the catalytic domain with the Src Homology 2 (SH2) domain. The mechanism by which this interaction enhances catalysis is not known. We combined computational simulations with mutagenesis and functional analysis to find that the SH2 domain conveys both local and global effects on the dynamics of the catalytic domain. Locally, it regulates the flexibility of the αC helix in a fashion reminiscent of cyclins in cyclin-dependent kinases, reorienting catalytically important motifs. At a more global level, SH2 binding redirects the hinge motion of the N and C lobes and changes the conformational equilibrium of the activation loop. The complex network of subtle structural shifts that link the SH2 domain with the activation loop and the active site may be partially conserved with other SH2-domain containing kinases and therefore offer additional parameters for the design of conformation-specific inhibitors.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of proteins, which are strongly induced downstream of type I interferon signaling. The molecular mechanism of IFIT anti-viral activity has been studied in some detail, including the recently discovered direct binding of viral nucleic acid, the binding to viral and host proteins, and the possible involvement in anti-viral immune signal propagation. The unique structures of some members of the IFIT family have been solved to reveal an internal pocket for non-sequence-specific, but conformation- and modification-specific, nucleic acid binding. This review will focus on recent discoveries, which link IFITs to the anti-viral response, intrinsic to the innate immune system.

Kenny-Caffey syndrome (KCS) and the similar but more severe osteocraniostenosis (OCS) are genetic conditions characterized by impaired skeletal development with small and dense bones, short stature, and primary hypoparathyroidism with hypocalcemia. We studied five individuals with KCS and five with OCS and found that all of them had heterozygous mutations in FAM111A. One mutation was identified in four unrelated individuals with KCS, and another one was identified in two unrelated individuals with OCS; all occurred de novo. Thus, OCS and KCS are allelic disorders of different severity. FAM111A codes for a 611 amino acid protein with homology to trypsin-like peptidases. Although FAM111A has been found to bind to the large T-antigen of SV40 and restrict viral replication, its native function is unknown. Molecular modeling of FAM111A shows that residues affected by KCS and OCS mutations do not map close to the active site but are clustered on a segment of the protein and are at, or close to, its outer surface, suggesting that the pathogenesis involves the interaction with as yet unidentified partner proteins rather than impaired catalysis. FAM111A appears to be crucial to a pathway that governs parathyroid hormone production, calcium homeostasis, and skeletal development and growth., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are innate immune effector molecules that are thought to confer antiviral defence through disruption of protein-protein interactions in the host translation-initiation machinery. However, it was recently discovered that IFITs can directly recognize viral RNA bearing a 5'-triphosphate group (PPP-RNA), which is a molecular signature that distinguishes it from host RNA. Here we report crystal structures of human IFIT5, its complex with PPP-RNAs, and an amino-terminal fragment of IFIT1. The structures reveal a new helical domain that houses a positively charged cavity designed to specifically engage only single-stranded PPP-RNA, thus distinguishing it from the canonical cytosolic sensor of double-stranded viral PPP-RNA, retinoic acid-inducible gene I (RIG-I, also known as DDX58). Mutational analysis, proteolysis and gel-shift assays reveal that PPP-RNA is bound in a non-sequence-specific manner and requires a 5'-overhang of approximately three nucleotides. Abrogation of PPP-RNA binding in IFIT1 and IFIT5 was found to cause a defect in the antiviral response by human embryonic kidney cells. These results demonstrate the mechanism by which IFIT proteins selectively recognize viral RNA, and lend insight into their downstream effector function.

Numerous small non-coding RNAs (sRNAs) in bacteria modulate rates of translation initiation and degradation of target mRNAs, which they recognize through base-pairing facilitated by the RNA chaperone Hfq. Recent evidence indicates that the ternary complex of Hfq, sRNA and mRNA guides endoribonuclease RNase E to initiate turnover of both the RNAs. We show that a sRNA not only guides RNase E to a defined site in a target RNA, but also allosterically activates the enzyme by presenting a monophosphate group at the 5'-end of the cognate-pairing "seed." Moreover, in the absence of the target the 5'-monophosphate makes the sRNA seed region vulnerable to an attack by RNase E against which Hfq confers no protection. These results suggest that the chemical signature and pairing status of the sRNA seed region may help to both 'proofread' recognition and activate mRNA cleavage, as part of a dynamic process involving cooperation of RNA, Hfq and RNase E., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The RNA degradosome is a massive multi-enzyme assembly that occupies a nexus in RNA metabolism and post-transcriptional control of gene expression in Escherichia coli and many other bacteria. Powering RNA turnover and quality control, the degradosome serves also as a machine for processing structured RNA precursors during their maturation. The capacity to switch between destructive and processing modes involves cooperation between degradosome components and is analogous to the process of RNA surveillance in other domains of life. Recruitment of components and cellular compartmentalisation of the degradosome are mediated through small recognition domains that punctuate a natively unstructured segment within a scaffolding core. Dynamic in conformation, variable in composition and non-essential under certain laboratory conditions, the degradosome has nonetheless been maintained throughout the evolution of many bacterial species, due most likely to its diverse contributions in global cellular regulation. We describe the role of the degradosome and its components in RNA decay pathways in E. coli, and we broadly compare these pathways in other bacteria as well as archaea and eukaryotes. We discuss the modular architecture and molecular evolution of the degradosome, its roles in RNA degradation, processing and quality control surveillance, and how its activity is regulated by non-coding RNA. Parallels are drawn with analogous machinery in organisms from all life domains. Finally, we conjecture on roles of the degradosome as a regulatory hub for complex cellular processes.

Antiviral innate immunity relies on the recognition of microbial structures. One such structure is viral RNA that carries a triphosphate group on its 5' terminus (PPP-RNA). By an affinity proteomics approach with PPP-RNA as the 'bait', we found that the antiviral protein IFIT1 (interferon-induced protein with tetratricopeptide repeats 1) mediated binding of a larger protein complex containing other IFIT family members. IFIT1 bound PPP-RNA with nanomolar affinity and required the arginine at position 187 in a highly charged carboxy-terminal groove of the protein. In the absence of IFIT1, the growth and pathogenicity of viruses containing PPP-RNA was much greater. In contrast, IFIT proteins were dispensable for the clearance of pathogens that did not generate PPP-RNA. On the basis of this specificity and the great abundance of IFIT proteins after infection, we propose that the IFIT complex antagonizes viruses by sequestering specific viral nucleic acids.

TANK-binding kinase 1 (TBK1) and inducible IκB-kinase (IKK-i) are central regulators of type-I interferon induction. They are associated with three adaptor proteins called TANK, Sintbad (or TBKBP1) and NAP1 (or TBKBP2, AZI2) whose functional relationship to TBK1 and IKK-i is poorly understood. We performed a systematic affinity purification-mass spectrometry approach to derive a comprehensive TBK1/IKK-i molecular network. The most salient feature of the network is the mutual exclusive interaction of the adaptors with the kinases, suggesting distinct alternative complexes. Immunofluorescence data indicated that the individual adaptors reside in different subcellular locations. TANK, Sintbad and NAP1 competed for binding of TBK1. The binding site for all three adaptors was mapped to the C-terminal coiled-coil 2 region of TBK1. Point mutants that affect binding of individual adaptors were used to reconstitute TBK1/IKK-i-deficient cells and dissect the functional relevance of the individual kinase-adaptor edges within the network. Using a microarray-derived gene expression signature of TBK1 in response virus infection or poly(I∶C) stimulation, we found that TBK1 activation was strictly dependent on the integrity of the TBK1/TANK interaction.

The Escherichia coli endoribonuclease RNase E is an essential enzyme having key roles in mRNA turnover and the processing of several structured RNA precursors, and it provides the scaffold to assemble the multienzyme RNA degradosome. The activity of RNase E is inhibited by the protein RraA, which can interact with the ribonuclease's degradosome-scaffolding domain. Here, we report that RraA can bind to the RNA helicase component of the degradosome (RhlB) and the two RNA-binding sites in the degradosome-scaffolding domain of RNase E. In the presence of ATP, the helicase can facilitate the exchange of RraA for RNA stably bound to the degradosome. Our data suggest that RraA can affect multiple components of the RNA degradosome in a dynamic, energy-dependent equilibrium. The multidentate interactions of RraA impede the RNA-binding and ribonuclease activities of the degradosome and may result in complex modulation and rerouting of degradosome activity.