Search

Entamoeba histolytica, a protozoan parasite, is the causative agent of amoebiasis, which is a significant global health concern. The virulence mechanisms underlying its pathogenicity are multifaceted and complex. However, endocytic processes and motility are well accepted virulence determinants. As previously reported, an AGCK family kinase, EhAGCK1 to be involved in trogocytosis exclusively while another one from same family named EhAGCK2 participates in all actin dependent endocytic processes. As the kinase dead mutants of EhAGCK1 showed significant defect in destruction of live host cells and also the localisation pattern of same is distinguishable from EhAGCK2. From observations so far, it appears that former initiates a distinguishable signaling cascade. In this work, we have demonstrated distinct biochemical properties of kinases involved in related yet distinguishable endocytic processes for the first time. Our biochemical characterization highlights distinct ion dependency of EhAGCK1 along with substrate specificity. We also show upstream activator of these kinases, 3-phosphoinositide dependent kinase 1 (PDK1) activity and its role in activating the kinase activity. The kinases exhibit property of autophosphorylation, and which may regulate the kinase activity subsequently. Summarily, these studies show that EhAGCK1 and EhAGCK2 show distinct biochemical properties which further confirm their unique role in related endocytic processes of trogocytosis and phagocytosis. Author summary: The protozoan parasite E. histolytica causes amebiasis in humans. The trophozoites depend on variety of endocytic processes to survive and cause pathogenesis in the gut of host system. Phagocytosis and trogocytosis are two related endocytic processes known to contribute to the pathogenesis by the parasite in vivo. The two processes are phenotypically different, trogocytosis being fast paced resembling nibbling and specific to live host cells. In terms of molecular mechanism of these processes, an AGC family kinase, EhAGCK1 is known to be involved in trogocytosis specifically while another one EhAGCK2 is involved in all types of endocytic processes including fluid uptake. Our biochemical characterization of these two kinases reveal that they have distinct substrate specificity and ion requirement. The overall biochemical assessment indicates the two kinases belonging to same family are involved in the phagocytic and trogocytic pathway distinctly. Hence, although the phagocytosis and trogocytosis at molecular level appear to be related but they also involve steps unique to each pathway. [ABSTRACT FROM AUTHOR]

Intein enzymes catalyze the splicing of their flanking polypeptide chains and have found tremendous biotechnological applications. Their terminal residues form the catalytic core and participate in the splicing reaction. Hence, the neighboring N‐ and C‐terminal extein residues influence the catalytic rate. As these extein residues vary depending on the substrate identity, we tested the influence of 20 amino acids at these sites in the Spl DnaX intein and observed significant variation of spliced product as well as N‐ and C‐terminus cleavage product formation. We investigated the dependence of these reactions on the extein residues by molecular dynamics (MD) simulations on eight extein variants, and found that the conformational sampling of the active‐site residues of the intein enzyme differed among these extein variants. We found that the extein variants that sample higher population of near‐attack conformers (NACs) of the active‐site residues undergo higher product formation in our activity assays. Ground state conformers that closely resemble the transition state are referred to as NACs. Very good correlation was observed between the NAC populations from the MD simulations of eight extein variants and the corresponding product formation from our activity assays. Furthermore, this molecular detail enabled us to elucidate the mechanistic roles of several conserved active‐site residues in the splicing reaction. Overall, this study shows that the catalytic power of Spl DnaX intein enzyme, and most likely other inteins, depends on the efficiency of formation of NACs in the ground state, which is further modulated by the extein residues. [ABSTRACT FROM AUTHOR]

Human RNA‐binding motif 3 protein (RBM3) is a cold‐shock protein which functions in various aspects of global protein synthesis, cell proliferation and apoptosis by interacting with the components of basal translational machinery. RBM3 plays important roles in tumour progression and cancer metastasis, and also has been shown to be involved in neuroprotection and endoplasmic reticulum stress response. Here, we have solved the solution NMR structure of the N‐terminal 84 residue RNA recognition motif (RRM) of RBM3. The remaining residues are rich in RGG and YGG motifs and are disordered. The RRM domain adopts a βαββαβ topology, which is found in many RNA‐binding proteins. NMR‐monitored titration experiments and molecular dynamic simulations show that the beta‐sheet and two loops form the RNA‐binding interface. Hydrogen bond, pi–pi and pi–cation are the key interactions between the RNA and the RRM domain. NMR, size exclusion chromatography and chemical cross‐linking experiments show that RBM3 forms oligomers in solution, which is favoured by decrease in temperature, thus, potentially linking it to its function as a cold‐shock protein. Temperature‐dependent NMR studies revealed that oligomerization of the RRM domain occurs via nonspecific interactions. Overall, this study provides the detailed structural analysis of RRM domain of RBM3, its interaction with RNA and the molecular basis of its temperature‐dependent oligomerization. [ABSTRACT FROM AUTHOR]

The parasite Entamoeba histolytica is the etiological agent of amoebiasis, a major cause of morbidity and mortality due to parasitic diseases in developing countries. Phagocytosis is an essential mode of obtaining nutrition and has been associated with the virulence behaviour of E. histolytica. Signalling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remains to be elucidated in this parasite. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica and have described some of the molecules that play key roles in the process. Here we showed the involvement of non-Dbl Rho Guanine Nucleotide Exchange Factor, EhGEF in regulation of amoebic phagocytosis by regulating activation of EhRho1. EhGEF was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. Our observation from imaging, pull down experiments and down regulating expression of different molecules suggest that EhGEF interacts with EhRho1 and it is required during initiation of phagocytosis and phagosome formation. Also, biophysical, and computational analysis reveals that EhGEF mediates GTP exchange on EhRho1 via an unconventional pathway. In conclusion, we describe a non-Dbl EhGEF of EhRho1 which is involved in endocytic processes of E. histolytica. Author summary: E. histolytica is causative agent of amoebiasis in humans, which is of major concern in children, as repeated infection leads to stunted physical and mental growth. Parasite display variety of endocytic processes, like phagocytosis, trogocytosis and micropinocytosis to name a few. The molecular mechanism of these processes is still largely unknown. The molecules, which participate in these endocytic processes may prove to be good therapeutic targets, as endocytic processes are necessary for parasite growth, survival, and pathogenesis in host system. Here in this this report, we have focussed on an unconventional EhGEF, which was identified as EhRho1 interacting protein by mass spectrometry. Our microscopy experiments show EhGEF participates in phagocytosis and trogocytosis and is highly dynamic in nature. The molecule also plays a crucial role in activation and recruitment of EhRho1 to the site of phagocytosis, and downregulation of EhGEF mimics phenotype similar to overexpression of activity dead mutant (T34N) of EhRho1. Our finding reveals EhGEF to play important role in actin dependent endocytic processes through EhRho1 and thereby, regulating actin dynamics during the process through EhFormin1 and EhProfilin1. [ABSTRACT FROM AUTHOR]

Intein enzymes catalyze the splicing of their flanking polypeptide chains and have found tremendous biotechnological applications. Their terminal residues form the catalytic core and participate in the splicing reaction. Hence, the neighboring N- and C-terminal extein residues influence the catalytic rate. As these extein residues vary depending on the substrate identity, we tested the influence of 20 amino acids at these sites in the Spl DnaX intein and observed significant variation of spliced product as well as N- and C-terminus cleavage product formation. We investigated the dependence of these reactions on the extein residues by molecular dynamics (MD) simulations on eight extein variants, and found that the conformational sampling of the active-site residues of the intein enzyme differed among these extein variants. We found that the extein variants that sample higher population of near-attack conformers (NACs) of the active-site residues undergo higher product formation in our activity assays. Ground state conformers that closely resemble the transition state are referred to as NACs. Very good correlation was observed between the NAC populations from the MD simulations of eight extein variants and the corresponding product formation from our activity assays. Furthermore, this molecular detail enabled us to elucidate the mechanistic roles of several conserved active-site residues in the splicing reaction. Overall, this study shows that the catalytic power of Spl DnaX intein enzyme, and most likely other inteins, depends on the efficiency of formation of NACs in the ground state, which is further modulated by the extein residues., (© 2023 The Protein Society.)