Small molecules that disrupt RAD54-BLM interaction hamper tumor proliferation in colon cancer chemoresistance models.

RAD54 and BLM helicase play pivotal roles during homologous recombination repair (HRR) to ensure genome maintenance. BLM amino acids (aa 181-212) interact with RAD54 and enhance its chromatin remodeling activity. Functionally, this interaction heightens HRR, leading to a decrease in residual DNA damage in colon cancer cells. This contributes to chemoresistance in colon cancer cells against cisplatin, camptothecin, and oxaliplatin, eventually promoting tumorigenesis in preclinical colon cancer mouse models. ChIP-Seq analysis and validation revealed increased BLM and RAD54 corecruitment on the MRP2 promoter in camptothecin-resistant colon cancer cells, leading to BLM-dependent enhancement of RAD54-mediated chromatin remodeling. We screened the Prestwick small-molecule library, with the intent to revert camptothecin- and oxaliplatin-induced chemoresistance by disrupting the RAD54-BLM interaction. Three FDA/European Medicines Agency-approved candidates were identified that could disrupt this interaction. These drugs bound to RAD54, altered its conformation, and abrogated RAD54-BLM-dependent chromatin remodeling on G5E4 and MRP2 arrays. Notably, the small molecules also reduced HRR efficiency in resistant lines, diminished anchorage-independent growth, and hampered the proliferation of tumors generated using camptothecin- and oxaliplatin-resistant colon cancer cells in both xenograft and syngeneic mouse models in BLM-dependent manner. Therefore, the 3 identified small molecules can serve as possible viable candidates for adjunct therapy in colon cancer treatment.

Identification of potential novel inhibitors against the SARS-CoV-2 spike protein: targeting RBD and ACE2 interaction.

The SARS-CoV-2, responsible for the COVID-19 pandemic has wrecked devastation throughout the globe. The SARS-CoV-2 spike (S) glycoprotein plays crucial role in virus attachment, fusion, and entry. This study aims to identify inhibitors targeting the receptor binding domain (RBD) of the S protein using computational and experimental techniques. We carried out virtual screening of four datasets against the S-RBD. Six potential candidate inhibitors were selected for experimental evaluation. Here, we provide experimental evidence that the molecules 9‴-MethyllithosperMate, Epimedin A, Pentagalloylglucose, and Theaflavin-3-gallate have a high binding affinity towards SARS-CoV-2 S-RBD. 9‴-MethyllithosperMate with a KD value of 1.3 nM serves as the best inhibitor, followed by others with KD values in micromolar range. We performed molecular dynamics simulation to assess the binding stability of these inhibitors. Hence, our study reports novel inhibitors against the SARS-CoV-2 S-RBD and their predicted binding mode also suggest the possibility to interfere with the ACE2 binding.Communicated by Ramaswamy H. Sarma.

Unravelling the Biology of EhActo as the First Cofilin From Entamoeba histolytica.

Actin-depolymerising factors (ADF) are a known family of proteins that regulate actin dynamics. Actin regulation is critical for primitive eukaryotes since it drives their key cellular processes. Entamoeba histolytica, a protist human pathogen harbours eleven proteins within this family, however, with no actin depolymerising protein reported to date. We present here the NMR model of EhActo, the first Cofilin from E. histolytica that severs actin filaments and also participates in cellular events like phagocytosis and pseudopod formation. The model typically represents the ADF-homology domain compared to other cofilins. Uniquely, EhActo lacks the critical Serine3 residue present in all known actophorins mediating its phospho-regulation. The second mode of regulation that cofilin's are subjected to is via their interaction with 14-3-3 proteins through the phosphorylated Serine residue and a consensus binding motif. We found a unique interaction between EhActo and 14-3-3 without the presence of the consensus motif or the phosphorylated Serine. These interesting results present unexplored newer mechanisms functional in this pathogen to regulate actophorin. Through our structural and biochemical studies we have deciphered the mechanism of action of EhActo, implicating its role in amoebic biology.

Cdc42/Rac Interactive Binding Containing Effector Proteins in Unicellular Protozoans With Reference to Human Host: Locks of the Rho Signaling.

Small GTPases are the key to actin cytoskeleton signaling, which opens the lock of effector proteins to forward the signal downstream in several cellular pathways. Actin cytoskeleton assembly is associated with cell polarity, adhesion, movement and other functions in eukaryotic cells. Rho proteins, specifically Cdc42 and Rac, are the primary regulators of actin cytoskeleton dynamics in higher and lower eukaryotes. Effector proteins, present in an inactive state gets activated after binding to the GTP bound Cdc42/Rac to relay a signal downstream. Cdc42/Rac interactive binding (CRIB) motif is an essential conserved sequence found in effector proteins to interact with Cdc42 or Rac. A diverse range of Cdc42/Rac and their effector proteins have evolved from lower to higher eukaryotes. The present study has identified and further classified CRIB containing effector proteins in lower eukaryotes, focusing on parasitic protozoans causing neglected tropical diseases and taking human proteins as a reference point to the highest evolved organism in the evolutionary trait. Lower eukaryotes' CRIB containing proteins fall into conventional effector molecules, PAKs (p21 activated kinase), Wiskoit-Aldrich Syndrome proteins family, and some have unique domain combinations unlike any known proteins. We also highlight the correlation between the effector protein isoforms and their selective specificity for Cdc42 or Rac proteins during evolution. Here, we report CRIB containing effector proteins; ten in Dictyostelium and Entamoeba, fourteen in Acanthamoeba, one in Trypanosoma and Giardia. CRIB containing effector proteins that have been studied so far in humans are potential candidates for drug targets in cancer, neurological disorders, and others. Conventional CRIB containing proteins from protozoan parasites remain largely elusive and our data provides their identification and classification for further in-depth functional validations. The tropical diseases caused by protozoan parasites lack combinatorial drug targets as effective paradigms. Targeting signaling mechanisms operative in these pathogens can provide greater molecules in combatting their infections.

Uncovering the Cyclic AMP Signaling Pathway of the Protozoan Parasite Entamoeba histolytica and Understanding Its Role in Phagocytosis.

Second messenger signaling controls a surprisingly diverse range of processes in several eukaryotic pathogens. Molecular machinery and pathways involving these messengers thus hold tremendous opportunities for therapeutic interventions. Relative to Ca2+ signaling, the knowledge of a crucial second messenger cyclic AMP (cAMP) and its signaling pathway is very scant in the intestinal parasite Entamoeba histolytica. In the current study, mining the available genomic resources, we have for the first time identified the cAMP signal transduction pathway of E. histolytica. Three heptahelical proteins with variable G-protein-coupled receptor domains, heterotrimeric G-proteins (Gα, Gß, and Gγ subunits), soluble adenylyl cyclase, cyclase-associated protein, and enzyme carbonic anhydrase were identified in its genome. We could also identify several putative candidate genes for cAMP downstream effectors such as protein kinase A, A-kinase anchoring proteins, and exchange protein directly activated by the cAMP pathway. Using specific inhibitors against key identified targets, we could observe changes in the intracellular cAMP levels as well as defect in the rate of phagocytosis of red blood cells by the parasite E. histolytica. We thus strongly believe that characterization of some of these unexplored crucial signaling determinants will provide a paradigm shift in understanding the pathogenicity of this organism.

The actin cytoskeleton orchestra in Entamoeba histolytica.

Years of evolution have kept actin conserved throughout various clades of life. It is an essential protein starring in many cellular processes. In a primitive eukaryote named Entamoeba histolytica, actin directs the process of phagocytosis. A finely tuned coordination between various actin-binding proteins (ABPs) choreographs this process and forms one of the virulence factors for this protist pathogen. The ever-expanding world of ABPs always has space to accommodate new and varied types of proteins to the earlier existing repertoire. In this article, we report the identification of 390 ABPs from Entamoeba histolytica. These proteins are part of diverse families that have been known to regulate actin dynamics. Most of the proteins are primarily uncharacterized in this organism; however, this study aims to annotate the ABPs based on their domain arrangements. A unique characteristic about some of the ABPs found is the combination of domains present in them unlike any other reported till date. Calponin domain-containing proteins formed the largest group among all types with 38 proteins, followed by 29 proteins with the infamous BAR domain in them, and 23 proteins belonging to actin-related proteins. The other protein families had a lesser number of members. Presence of exclusive domain arrangements in these proteins could guide us to yet unknown actin regulatory mechanisms prevalent in nature. This article is the first step to unraveling them.

Biochemical and biophysical characterization of nucleotide binding domain of Trehalose transporter from Mycobacterium tuberculosis.

The SugABC is an ABC transporter in Mycobacterium tuberculosis which is proposed to be involved in the process of Trehalose import, but till date the proteins of this transporter complex have not been functionally characterized. This transport process is driven by the nucleotide binding domain SugC of SugABC transporter. To understand the functional role of SugC, we expressed and purified the protein in E.coli. Our purification result shows, Mtb SugC exists as a monomer in solution but forms dimers upon binding to ATP. It is stable at pH 7.5 as analyzed by CD spectroscopy and showed maximum activity at this pH as estimated by Michaelis-Menten's kinetics for Mg-ATP at a KM of 0.15 mM. The SugCH193A mutant was observed to have a reduced catalytic activity implying that H193 is one of the residues involved in the hydrolysis of ATP. The molecular modeling further revealed that, like E.coli MalK, MtbSugC also has an ATPase domain and a regulatory domain. Despite having low sequence homology with other nucleotide binding domains of ABC transporters, the structure and functional motifs of MtbSugC are conserved. Thus, we show that SugC is a functional ATPase domain of SugABC transporter in Mycobacterium tuberculosis.

Surface Plasmon Resonance Analysis of the Protein-protein Binding Specificity Using Autolab ESPIRIT.

Direct protein-protein interactions are known to regulate a wide range of cellular activities. To understand these contacts one can employ various experimental methods like Dynamic Light Scattering (DLS), Fluorescence Resonance Energy Transfer (FRET), Isothermal titration calorimetry (ITC), Chemical crosslinking, Co-immunoprecipitation (Co-IP), Surface Plasmon Resonance (SPR) and many more. Among these, SPR stands out as a quick, label-free, reliable, and accurate quantitation technique. We have used SPR to elucidate the linkage between 14-3-3 Protein 3 (EhP3) and the actin cytoskeleton in the protist pathogen Entamoeba histolytica. It allowed us to screen EhP3 binding with several actin-binding/actin regulatory proteins (Coactosin, Actophorin, Twinfilin, Profilin, and Filamin). Our screening results suggested Coactosin as an important interacting partner of EhP3. A complete kinetic analysis indeed confirmed that EhCoactosin binds EhP3 with an affinity constant of 3 µM.

Biochemical and biophysical characterization of the smallest pyruvate kinase from Entamoeba histolytica.

Entamoeba histolytica infection is highly prevalent in developing countries across the globe. The ATP synthesis in this pathogen is solely dependent on the glycolysis pathway where pyruvate kinase (Pyk) catalyzes the final reaction. Here, we have cloned, overexpressed and purified the pyruvate kinase (EhPyk) from E. histolytica. EhPyk is the shortest currently known Pyk till date as it contains only two of the three characterized domains when compared to the other homologues and our phylogenetic analysis places it on a distinct branch from the known type I/II Pyks. Our purification results suggested that it exists as a homodimer in solution. The kinetic characterization showed that EhPyk has maximum activity at pH 7.5 where it exhibited Michaelis-Menten's kinetics for phosphoenolpyruvate with a Km of 0.23 mM, and it lost its activity at both the acidic pH 4.0 and basic pH 10.0. We also determined the key secondary structural elements of EhPyk at different pH values. MD simulation of EhPyk structure at different pH values suggested that it is most stable at pH 7.0, while least stable at pH 10.0 followed by pH 4.0. Together, our computational simulations correlate well with the experimental studies. In summary, this study expands the current understanding of the EhPyk identified earlier in the amoebic genome and provides the first characterization of this bacterially expressed protein.