A unique mRNA decapping complex in trypanosomes.

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.

Targeted protein degradation might present a novel therapeutic approach in the fight against African trypanosomiasis.

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".

ß-Sitosterol could serve as a dual inhibitor of Trypanosoma congolense sialidase and phospholipase A2: in vitro kinetic analyses and molecular dynamic simulations.

The involvement of Trypanosoma congolense sialidase alongside phospholipase A2 has been widely accepted as the major contributing factor to anemia during African animal trypanosomiasis. The enzymes aid the parasite in scavenging sialic acid and fatty acids necessary for survival in the infected host, but there are no specific drug candidates against the two enzymes. This study investigated the inhibitory effects of ß-sitosterol on the partially purified T. congolense sialidase and phospholipase A2. Purification of the enzymes using DEAE cellulose column led to fractions with highest specific activities of 8016.41 and 39.26 µmol/min/mg for sialidase and phospholipase A2, respectively. Inhibition kinetics studies showed that ß-sitosterol is non-competitive and an uncompetitive inhibitor of sialidase and phospholipase A2 with inhibition binding constants of 0.368 and 0.549 µM, respectively. Molecular docking of the compound revealed binding energies of - 8.0 and - 8.6 kcal/mol against the sialidase and phospholipase A2, respectively. Furthermore, 100 ns molecular dynamics simulation using GROMACS revealed stable interaction of ß-sitosterol with both enzymes. Hydrogen bond interactions between the ligand and Glu284 and Leu102 residues of the sialidase and phospholipase A2, respectively, were found to be the major stabilizing forces. In conclusion, ß-sitosterol could serve as a dual inhibitor of T. congolense sialidase and phospholipase A2; hence, the compound could be exploited further in the search for newer trypanocides.

The Role of Electrostatic Interactions in IFIT5-RNA Complexes Predicted by the UBDB+EPMM Method.

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.

Immunoinformatic design of a putative multi-epitope vaccine candidate against Trypanosoma brucei gambiense.

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.

A Uniform Benchmark for Testing SsrA-Derived Degrons in the Escherichia coli ClpXP Degradation Pathway.

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.

Applications of Bacterial Degrons and Degraders - Toward Targeted Protein Degradation in Bacteria.

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.

The Effect of Mutations in the TPR and Ankyrin Families of Alpha Solenoid Repeat Proteins.

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.