Twist1 and Twist2 are highly conserved members of the Twist subfamily of bHLH proteins responsible for the transcriptional regulation of the developmental programs in mesenchymal cell lineages. The regulation of such processes requires that Twist1 and Twist2 function as molecular switches to activate and repress target genes by employing several direct and indirect mechanisms. Modes of action by these proteins include direct DNA binding to conserved E-box sequences and recruitment of coactivators or repressors, sequestration of E-protein modulators, and interruption of proper activator/repressor function through protein–protein interactions. Regulatory outcomes of Twist1 and Twist2 are themselves controlled by spatial-temporal expression, phosphoregulation, dimer choice and cellular localization. Although these two proteins are highly conserved and exhibit similar functions in vitro, emerging literature have demonstrated different roles in vivo. The involvement of Twist1 and Twist2 in a broad spectrum of regulatory pathways highlights the importance of understanding their roles in normal development, homeostasis and disease. Here we focus on the mechanistic models of transcriptional regulation and summarize the similarities and differences between Twist1 and Twist2 in the context of myogenesis, osteogenesis, immune system development and cancer.
Hemoglobin I (HbI) from the clam Lucina pectinata is an intriguing hemeprotein that binds and transports H 2 S to sulfide-oxidizing chemoautotrophic bacteria to maintain a symbiotic relationship and to protect the mollusk from H 2 S toxicity. Single point mutations at E7, B10 and E11 positions were introduced in the HbI heme pocket to define the reactivity of sulfide with hemeproteins. The functional and structural properties of mutant and wild type recombinant proteins were first evaluated using the well-known ferrous CO and O 2 derivatives. The effects of these mutations on the ferric environment were then studied in the metaquo and hydrogen sulfide derivatives. The results obtained with the ferrous HbI mutants show that all the E7 substitutions and the PheB10Tyr mutation influence directly CO and O 2 binding and stability while the B10 and E11 substitutions induce distal structural rearrangements that affect ligand entry and escape indirectly. For the metaquo-GlnE7His, PheB10Val, PheB10Leu and the E11 variants, two individual distal structures are suggested, one of which is associated with H-bonding interactions between the E7 residues and the bound water. Similar H-bonding interactions are invoked for these HbI-H 2 S mutant derivatives and the rHbI, altering in turn sulfide reactivity within these protein samples. This is evident in the resonance Raman spectra of these HbI-H 2 S complexes, which show reduction of heme iron as judged by the appearance of the ν 4 oxidation state marker at 1356 cm −1 , indicative of heme-Fe II species. This reduction process depends strongly on distal mutations showing faster reduction for those HbI mutants exhibiting strongest H-bonding interactions. Overall, the results presented here show that: a. H 2 S association is regulated by steric constraints; b. H 2 S release is controlled by two competing reactions involving simple sulfide dissociation and heme reduction; c. at high H 2 S concentrations, reduction of the ferric center dominates; d. reduction of the heme is also enhanced in those HbI mutants having polar distal environments. † This work was supported in part by funds from the National Science Foundation, Cellular Biology (Grant 0544250) and NIH-NIGMS/ MBRS-SCORE S06GM08103-34, NCRR G12RR03051, P20RR016439 and NCMHD T37MD001477. ⊥To whom correspondence should be addressed: Telephone (787) Fax (787)265-5476; E-mail E-mail: sonw@caribe.net. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 June 9. Published in final edited form as:Biochemistry. Hydrogen sulfide (H 2 S) is a well known poisonous gas whose cytotoxic effects have been studied for more than 300 years (1). Interestingly, it has been found recently that H 2 S is produced in mammalian tissues and that, similar to nitric oxide (NO) and carbon monoxide (CO), it may function as a neuromodulator, neuroprotector and/or smooth muscle relaxant/ constrictor (2-5). Endogenous H 2 S is produced at high concentrations in the brain (100 µM) by cystathionine beta-synthase en...
The focal facial dermal dysplasias (FFDDs) are a group of inherited developmental disorders in which the characteristic diagnostic feature is bitemporal scar-like lesions that resemble forceps marks. To date, the genetic defects underlying these ectodermal dysplasias have not been determined. To identify the gene defect causing autosomal-recessive Setleis syndrome (type III FFDD), homozygosity mapping was performed with genomic DNAs from five affected individuals and 26 members of the consanguineous Puerto Rican (PR) family originally described by Setleis and colleagues. Microsatellites D2S1397 and D2S2968 were homozygous in all affected individuals, mapping the disease locus to 2q37.3. Haplotype analyses of additional markers in the PR family and a consanguineous Arab family further limited the disease locus to approximately 3 Mb between D2S2949 and D2S2253. Of the 29 candidate genes in this region, the bHLH transcription factor, TWIST2, was initially sequenced on the basis of its known involvement in murine facial development. Homozygous TWIST2 nonsense mutations, c.324C>T and c.486C>T, were identified in the affected members of the Arab and PR families, respectively. Characterization of the expressed mutant proteins, p.Q65X and p.Q119X, by electrophoretic mobility shift assays and immunoblot analyses indicated that they were truncated and unstable. Notably, Setleis syndrome patients and Twist2 knockout mice have similar facial features, indicating the gene's conserved role in mammalian development. Although human TWIST2 and TWIST1 encode highly homologous bHLH transcription factors, the finding that TWIST2 recessive mutations cause an FFDD and dominant TWIST1 mutations cause Saethre-Chotzen craniocynostosis suggests that they function independently in skin and bone development.
Chronic HIV infection leads to the development of cognitive impairments, designated as HIV-associated neurocognitive disorders (HAND). The secretion of soluble neurotoxic factors by HIV-infected macrophages plays a central role in the neuronal dysfunction and cell death associated with HAND. One potentially neurotoxic protein secreted by HIV-1 infected macrophages is cathepsin B. To explore the potential role of cathepsin B in neuronal cell death after HIV infection, we cultured HIV-1ADA infected human monocyte-derived macrophages (MDM) and assayed them for expression and activity of cathepsin B and its inhibitors, cystatins B and C. The neurotoxic activity of the secreted cathepsin B was determined by incubating cells from the neuronal cell line SK-N-SH with MDM conditioned media (MCM) from HIV-1 infected cultures. We found that HIV-1 infected MDM secreted significantly higher levels of cathepsin B than did uninfected cells. Moreover, the activity of secreted cathepsin B was significantly increased in HIV-infected MDM at the peak of viral production. Incubation of neuronal cells with supernatants from HIV-infected MDM resulted in a significant increase in the numbers of apoptotic neurons, and this increase was reversed by the addition of either the cathepsin B inhibitor CA-074 or a monoclonal antibody to cathepsin B. In situ proximity ligation assays indicated that the increased neurotoxic activity of the cathepsin B secreted by HIV-infected MDM resulted from decreased interactions between the enzyme and its inhibitors, cystatins B and C. Furthermore, preliminary in vivo studies of human post-mortem brain tissue suggested an upregulation of cathepsin B immunoreactivity in the hippocampus and basal ganglia in individuals with HAND. Our results demonstrate that HIV-1 infection upregulates cathepsin B in macrophages, increases cathepsin B activity, and reduces cystatin-cathepsin interactions, contributing to neuronal apoptosis. These findings provide new evidence for the role of cathepsin B in neuronal cell death induced by HIV-infected macrophages.
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