Myeloproliferative neoplasms (MPNs) are frequently driven by mutations within the C-terminal domain (C-domain) of calreticulin (CRT). CRTDel52 and CRTIns5 are recurrent mutations. Oncogenic transformation requires both mutated CRT and the thrombopoietin receptor (Mpl), but the molecular mechanism of CRT-mediated constitutive activation of Mpl is unknown. We show that the acquired C-domain of CRTDel52 mediates both Mpl binding and disulfide-linked CRTDel52 dimerization. Cysteine mutations within the novel C-domain (C400A and C404A) and the conserved N-terminal domain (N-domain; C163A) of CRTDel52 are required to reduce disulfide-mediated dimers and multimers of CRTDel52. Based on these data and published structures of CRT oligomers, we identify an N-domain dimerization interface relevant to both WT CRT and CRTDel52. Elimination of disulfide bonds and ionic interactions at both N-domain and C-domain dimerization interfaces is required to abrogate the ability of CRTDel52 to mediate cell proliferation via Mpl. Thus, MPNs exploit a natural dimerization interface of CRT combined with C-domain gain of function to achieve cell transformation.

Regulation of gene expression is one of the mechanisms of virulence in pathogenic organisms. In this context, we would like to understand the gene regulation of acetamidase enzyme of Mycobacterium smegmatis, which is the first reported inducible enzyme in mycobacteria. The acetamidase is highly inducible and the expression of this enzyme is increased 100-fold when the substrate acetamide is added. The acetamidase structural gene (amiE) is found immediately downstream of three predicted open reading frames (ORFs). Three of these genes along with a divergently expressed ORF are predicted to form an operon and involved in the regulation of acetamidase enzyme. Here we report expression, purification and functional characterization of AmiA which is one of these predicted ORFs. Electrophoretic mobility shift assays showed that AmiA binds to the region between the amiA and amiD near the predicted promoter (P2). Over-expression of AmiA significantly lowered the expression of acetamidase compared to the wild type as demonstrated by qRT-PCR and SDS-PAGE. We conclude that AmiA binds near P2 promoter and acts as a repressor in the regulation of acetamidase operon. The described work is a further step forward toward broadening the knowledge on understanding of the complex gene regulatory mechanism of Mycobacterium sp.

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Every year, an unacceptably large number of infant deaths occur in developing nations, with premature birth and asphyxia being two of the leading causes. A well-regulated thermal environment is critical for neonatal survival. Advanced incubators currently exist, but they are far too expensive to meet the needs of developing nations. We are developing a thermodynamically advanced low-cost incubator suitable for operation in a low-resource environment. Our design features three innovations: (1) a disposable baby chamber to reduce infant mortality due to nosocomial infections, (2) a passive cooling mechanism using low-cost heat pipes and evaporative cooling from locally found clay pots, and (3) insulated panels and a thermal bank consisting of water that effectively preserve and store heat. We developed a prototype incubator and visited and presented our design to our partnership hospital site in Mysore, India. After obtaining feedback, we have determined realistic, nontrivial design requirements and constraints in order to develop a new prototype incubator for clinical trials in hospitals in India.

Mycobacterium tuberculosis adapts to stress conditions by responding to the signals from its external environment. M. tuberculosis genome encodes 11 eukaryotic like serine/threonine protein kinases (STPK) and their importance in regulating the physiology and virulence of the bacteria are being explored. Previous study from our lab identified the M. tuberculosis STPK, PknI interacts with two peroxidase proteins such as Rv2159c and Rv0148. In this study, we have characterized the biological function behind the PknI-Rv2159c interaction in M. tuberculosis. Point mutation of Ala-Gly-Trp motif identified that only Ala49 and Gly50 amino acids of Rv2159c are responsible for interaction and there is no phosphorylation involved in the PknI-Rv2159c interaction. Rv2159c is a member from the carboxymuconolactone decarboxylase family with peroxidase activity. Enzymatic assays with catalytic site point mutants showed that Cys84 of Rv2159c was responsible for its alkylhydroperoxidase activity. Interestingly, interaction with PknI increased its peroxidase activity by several folds. Gene knockdown of Rv2159c in M. tuberculosis showed increased sensitivity to peroxides such as cumene hydroperoxide and hydrogen peroxide. Proteomic analysis of differentially expressing Rv2159c strains by 2D gel electrophoresis and mass spectrometry revealed the differential abundance of 21 proteins. The total absence of oxidoreductase, GuaB1 suggests the essential role of Rv2159c in redox maintenance. Our findings provide new insights on signaling mechanisms of PknI in maintaining the redox homeostasis during oxidative stresses.

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Myeloproliferative neoplasms (MPNs) are frequently driven by insertions and deletions within the gene encoding calreticulin (CRT). CRTDel52 and CRTIns5 are recurrent mutations. Although oncogenic transformation requires both mutated CRT and the myeloproliferative leukemia protein (Mpl), the molecular mechanism of CRT-mediated constitutive activation of Mpl is unknown. Our studies reveal that the novel C-domain of CRTDel52 encodes specificity both for Mpl binding and for disulfide-mediated CRT dimerization. Disulfide-stabilized CRTDel52 dimers and multimers are observed in MPN patient-derived platelet lysates and in transfected mammalian cells. Cysteine mutations within both the novel C-domain (C400A and C404A) and the conserved N-domain (C163A) of CRTDel52 are required to reduce disulfide-mediated dimers and multimers of CRTDel52. Based on these data and published structures of crystalized CRT oligomers, we tested the relevance of ionic interactions between charged residues proximal to C163 at the N-domain dimerization interface. Charge alteration at these residues affected dimerization and multimerization of both wild type and CRTDel52. Elimination of intermolecular disulfides and disruption of ionic interactions at both proposed dimerization interfaces was required to abrogate the ability of CRTDel52 to induce cytokine-independent cell proliferation via Mpl. Based on these findings, we propose a structural model of the Mpl-activating CRTDel52 unit as a covalently-linked dimer that is stabilized by disulfides and ionic interactions at both the C-domain and N-domain. MPNs exploit a natural dimerization interface of CRT combined with C-domain gain-of-functions to achieve cell transformation.