http://192.168.1.40:8080/xmlui/handle/123456789/1064 2026-05-31T12:08:38Z http://192.168.1.40:8080/xmlui/handle/123456789/2440 Protein-4.2 association with band 3 (AE1, SLCA4) in Xenopus oocytes: Effects of three natural protein-4.2 mutations associated with hemolytic anemia Toye, A. M.; Ghosh, S.; Young, M. T.; Jones, G. K.; Sessions, R.B.; Ramaugé, M.; Leclerc, P.; Basu, Joyoti; Delaunay, J.; Tanner, M. J. A. We have investigated the effects of coexpression of protein 4.2 and three protein-4.2 variants with band 3 in the Xenopus oocyte expression system. Normal protein 4.2 increased band-3-specific chloride transport in the oocytes. Protein 4.2 also coimmunoprecipitated with band 3 and colocalized with band 3 at the oocyte plasma membrane. The increase in band-3-mediated chloride transport and coimmunoprecipitation of protein 4.2 required the presence of the N-terminal cytoplasmic domain of band 3. Protein 4.2 also localized to the oocyte plasma membrane in the absence of band 3. The protein-4.2 variants 4.2 Tozeur (R310Q) and 4.2 Komatsu (D175Y) had impaired ability to bind to band 3 and these variants did not localize to the oocyte plasma membrane when expressed on their own or when coexpressed with band 3. Unexpectedly, 4.2 Nippon (A142T) behaved similarly to normal protein 4.2. In the absence of a crystal structure of protein 4.2, we propose a homology model of protein 4.2 based on the structure of the sequence-related protein transglutaminase. Using our results in oocytes and this homology model we speculate how these mutations affect protein 4.2 and result in hereditary spherocytosis. DOI: 10.1182/blood-2004-05-1895 2005-05-15T00:00:00Z http://192.168.1.40:8080/xmlui/handle/123456789/2243 TLR4-dependent NF-κB activation and mitogen- and stress-activated protein kinase 1-triggered phosphorylation events are central to Helicobacter pylori peptidyl prolyl cis-, trans-isomerase (HP0175)-mediated induction of IL-6 release from macrophages Pathak, Sushil Kumar; Basu, Sanchita; Bhattacharyya, Asima; Pathak, Shresh; Banerjee, Anirban; Basu, Joyoti; Kundu, Manikuntala Helicobacter pylori infection is associated with the local production of chemokines and cytokines, of which IL-6 is overexpressed at the margin of gastric ulcer in H. pylori-positive gastritis. Cells of the monocytic lineage are the major sources of IL-6, and mononuclear cell infiltration in the lamina propria is characteristic of H. pylori-induced chronic infection. Our study shows for the first time that a secreted peptidyl prolyl cis-, trans-isomerase, HP0175 elicits IL-6 gene expression and IL-6 release from macrophages. An isogenic strain inactivated in the HSP0175 gene (knockout) was attenuated in its IL-6-inducing ability, which was restored after complementation with the HP017.5 gene. The specificity of the HP0175-induced effect was confirmed by the fact that rHP0175 purified from HEK293 cells could also induce IL-6 release, ruling out the possibility that the observed effect was due to bacterial contaminants. HP0175 was capable of interacting directly with the extracellular domain of TLR4. HP0175-induced IL-6 gene expression was critically dependent on TLR4-dependent NF-kappa B and MAPK activation. TLR4/PI3K-dependent ERK1/2 and p38 MAPK signaling converged upon activation of mitogen- and stress-activated protein kinase 1 (MSK1). The central role of MSK1 was borne out by the fact that silencing of MSK1 expression abrogated HP0175-mediated NF-kappa B-dependent IL-6 gene transcription. MSK1 regulated the recruitment of p65 and phopbo-Ser(10)-histone H3 to the IL-6 promoter. HP0175 therefore regulated IL-6 gene transcription through chromatin modification at the IL-6 promoter. 2006-12-01T00:00:00Z http://192.168.1.40:8080/xmlui/handle/123456789/2241 Interaction between FtsW and penicillin-binding protein 3 (PBP3) directs PBP3 to mid-cell, controls cell septation and mediates the formation of a trimeric complex involving FtsZ, FtsW and PBP3 in mycobacteria Datta, Pratik; Dasgupta, Arunava; Singh, Anil Kumar; Mukherjee, Partha; Kundu, Manikuntala; Basu, Joyoti In bacteria, biogenesis of cell wall at the division site requires penicillin-binding protein 3 (PBP3) (or FtsI). Using pull-down, bacterial two-hybrid, and peptide-based interaction assays, we provide evidence that FtsW of Mycobacterium tuberculosis (FtsW(MTB)) interacts with PBP3 through two extracytoplasmic loops. Pro(306) in the larger loop and Pro(386) in the smaller loop of FtsW are crucial for these interactions. Fluorescence microscopy shows that conditional silencing of ftsW in Mycobacterium smegmatis prevents cell septation and positioning of PBP3 at mid-cell. Pull-down assays and conditional depletion of FtsW in M. smegmatis provide evidence that FtsZ, FtsW and PBP3 of mycobacteria are capable of forming a ternary complex, with FtsW acting as a bridging molecule. Bacterial three-hybrid analysis suggests that in M. tuberculosis, the interaction (unique to mycobacteria) of FtsZ with the cytosolic C-tail of FtsW strengthens the interaction of FtsW with PBP3. ftsW of M. smegmatis could be replaced by ftsW of M. tuberculosis. FtsW(MTB) could support formation of the FtsZ-FtsW-PBP3 ternary complex in M. smegmatis. Our findings raise the possibility that in the genus Mycobacterium binding of FtsZ to the C-tail of FtsW may modulate its interactions with PBP3, thereby potentially regulating septal peptidoglycan biogenesis. DOI: 10.1111/j.1365-2958.2006.05491.x 2006-12-01T00:00:00Z http://192.168.1.40:8080/xmlui/handle/123456789/2065 Positive feedback and noise activate the stringent response regulator rel in mycobacteria Sureka, Kamakshi; Ghosh, B.; Dasgupta, A.; Basu, J.; Kundu, M.; Bose, Indrani Phenotypic heterogeneity in an isogenic, microbial population enables a subset of the population to persist under stress. In mycobacteria, stresses like nutrient and oxygen deprivation activate the stress response pathway involving the two-component system MprAB and the sigma factor, SigE. SigE in turn activates the expression of the stringent response regulator, rel. The enzyme polyphosphate kinase 1 (PPK1) regulates this pathway by synthesizing polyphosphate required for the activation of MprB. The precise manner in which only a subpopulation of bacterial cells develops persistence, remains unknown. Rel is required for mycobacterial persistence. Here we show that the distribution of rel expression levels in a growing population of mycobacteria is bimodal with two distinct peaks corresponding to low (L) and high (H) expression states, and further establish that a positive feedback loop involving the mprAB operon along with stochastic gene expression are responsible for the phenotypic heterogeneity. Combining single cell analysis by flow cytometry with theoretical modeling, we observe that during growth, noise-driven transitions take a subpopulation of cells from the L to the H state within a “window of opportunity” in time preceding the stationary phase. It is these cells which adapt to nutrient depletion in the stationary phase via the stringent response. We find evidence of hysteresis in the expression of rel in response to changing concentrations of PPK1. Hysteresis promotes robustness in the maintenance of the induced state. Our results provide, for the first time, evidence that bistability and stochastic gene expression could be important for the development of “heterogeneity with an advantage” in mycobacteria and suggest strategies for tackling tuberculosis like targeting transitions from the low to the high rel expression state. DOI: 10.1371/journal.pone.0001771 2008-03-12T00:00:00Z