The position of the Gly-xxx-Gly motif in transmembrane segments modulates dimer affinity.

Although the intrinsic low solubility of membrane proteins presents challenges to their high-resolution structure determination, insight into the amino acid sequence features and forces that stabilize their folds has been provided through study of sequence-dependent helix-helix interactions between single transmembrane (TM) helices. While the stability of helix-helix partnerships mediated by the Gly-xxx-Gly (GG4) motif is known to be generally modulated by distal interfacial residues, it has not been established whether the position of this motif, with respect to the ends of a given TM segment, affects dimer affinity. Here we examine the relationship between motif position and affinity in the homodimers of 2 single-spanning membrane protein TM sequences: glycophorin A (GpA) and bacteriophage M13 coat protein (MCP). Using the TOXCAT assay for dimer affinity on a series of GpA and MCP TM segments that have been modified with either 4 Leu residues at each end or with 8 Leu residues at the N-terminal end, we show that in each protein, centrally located GG4 motifs are capable of stronger helix-helix interactions than those proximal to TM helix ends, even when surrounding interfacial residues are maintained. The relative importance of GG4 motifs in stabilizing helix-helix interactions therefore must be considered not only in its specific residue context but also in terms of the location of the interactive surface relative to the N and C termini of alpha-helical TM segments.     

Potent growth suppressive activity of curcumin in human breast cancer cells: Modulation of Wnt/β-catenin signaling

Abnormal activation of the Wnt/β-catenin signaling pathway and subsequent upregulation of β-catenin driven downstream targets—c-Myc and cyclin D1 is associated with development of breast cancer. The objective of our study was to determine if curcumin could modulate the key elements of Wnt pathway in breast cancer cells; an effect that might underscore its usefulness for chemoprevention/treatment of this malignancy. Curcumin showed a cytotoxic effect on MCF-7 cells with 50% inhibitory concentration (IC₅₀) of 35 μM; while IC₅₀ for MDA-MB-231 cells was 30 μM. Treatment with low cytostatic dose of 20 μM curcumin showed G₂/M arrest in both breast cancer cells. The effect of curcumin (20 μM) treatment on expression of Wnt/β-catenin pathway components in breast cancer cells (MCF-7 and MDA-MB-231) was analyzed by immunofluorescence and Western blotting. Curcumin was found to effectively inhibit the expression of several Wnt/β-catenin pathway components—disheveled, β-catenin, cyclin D1 and slug in both MCF-7 and MDA-MB-231. Immunofluorescence analysis showed that curcumin markedly reduced the nuclear expression of disheveled and β-catenin proteins. Further, the protein levels of the positively regulated β-catenin targets—cyclin D1 and slug, were downregulated by curcumin treatment. The expression levels of two integral proteins of Wnt signaling, GSK3β and E-cadherin were also altered by curcumin treatment. In conclusion, our data demonstrated that the efficacy of curcumin in inhibition of cell proliferation and induction of apoptosis might occur through modulation of β-catenin pathway in human breast cancer cells.     

Functional response of the small multidrug resistance protein EmrE to mutations in transmembrane helix 2

Escherichia coli EmrE is a small multidrug resistance protein encompassing four transmembrane (TM) sequences that oligomerizes to confer resistance to antimicrobials. Here we examined the effects on in vivo protein accumulation and ethidium resistance activity of single residue substitutions at conserved and variable positions in EmrE transmembrane segment 2 (TM2). We found that activity was reduced when conserved residues localized to one TM2 surface were replaced. Our findings suggest that conserved TM2 positions tolerate greater residue diversity than conserved sites in other EmrE TM sequences, potentially reflecting a source of substrate polyspecificity.     

Regulation of RANKL by biomechanical loading in fibrochondrocytes of meniscus

OBJECTIVE: We sought to determine whether fibrochondrocytes from menisci express receptor activator of NF-kappaB (RANK), its ligand (RANKL), or osteoprotegerin (OPG) and, if so, whether their expression is modulated by dynamic mechanical loading under inflammatory and normal conditions. METHODS: Fibrochondrocytes from rat menisci were subjected to cyclic tensile strain (CTS) at various magnitudes and frequencies in the presence or absence of interleukin (IL)-1beta for up to 24 h. In order to determine whether a possible regulatory effect of mechanical loading on RANKL and its receptors under inflamed conditions is sustained, cells were stimulated with IL-1beta for 24 h while being subjected to CTS only for the initial 4 and 8h, respectively. Regulation of RANKL, RANK, and OPG expression and synthesis were determined by semiquantitative and real-time PCR, Western blotting, and immunofluorescence. RESULT: Fibrochondrocytes constitutively expressed low levels of RANKL and RANK but marked levels of OPG. IL-1beta upregulated expression and synthesis of RANKL and RANK significantly (p<0.05), whereas expression of OPG was unaffected following 4 and 24 h. When fibrochondrocytes were simultaneously subjected to CTS and IL-1beta, expression of RANKL and RANK was significantly (p<0.05) downregulated as compared to that of IL-1beta-stimulated unstretched cells. The inhibitory effect of CTS on the IL-1beta-induced upregulation of RANKL and RANK was sustained as well as magnitude and frequency dependent. CONCLUSIONS: Our study provides evidence that RANKL and its receptors are expressed in fibrochondrocytes from meniscus. These data also demonstrate that dynamic mechanical loading can modify the expression of RANKL and RANK in inflammatory conditions.     

MHC class I-restricted presentation of maleylated protein binding to scavenger receptors

Pathways for loading exogenous protein-derived peptides on MHC class I are thought to be present mainly in monocyte-lineage cells and to involve phagocytosis- or macropinocytosis-mediated antigenic leakage into either cytosol or extracellular milieu to give peptide access to MHC class I. We show that maleylation of OVA enhanced its presentation to an OVA-specific MHC class I-restricted T cell line by both macrophages and B cells. This enhanced presentation involved uptake through receptors of scavenger receptor (SR)-like ligand specificity, was TAP-1-independent, and was inhibited by low levels (2 mM) of ammonium chloride. No peptide loading of bystander APCs by maleylated (maleyl) OVA-pulsed macrophages was detected. Demaleylated maleyl-OVA showed enhanced MHC class I-restricted presentation through receptor-mediated uptake and remained highly sensitive to 2 mM ammonium chloride. However, if receptor binding of maleyl-OVA was inhibited by maleylated BSA, the residual presentation was relatively resistant to 2 mM ammonium chloride. Maleyl-OVA directly introduced into the cytosol via osmotic lysis of pinosomes was poorly presented, confirming that receptor-mediated presentation of exogenous maleyl-OVA was unlikely to involve a cytosolic pathway. Demaleylated maleyl-OVA was well presented as a cytosolic Ag, consistent with the dependence of cytosolic processing on protein ubiquitination. Thus, receptor-specific delivery of exogenous protein Ags to APCs can result in enhanced MHC class I-restricted presentation, suggesting that the exogenous pathway of peptide loading for MHC class I may be a constitutive property dependent mainly on the quantity of Ag taken up by APCs.     

Genetic diversity of <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> isolates from certain agro-climatic regions of India by using RAPD markers

Genetic diversity analysis of Macrophomina phaseolina isolates obtained from different host range and diverse geographical locations in India was carried out using RAPD fingerprinting. Of the thirteen 10-mer random primers used, primer OPB-08 gave the maximum polymorphism and the UPGMA clustering could separate 50 isolates in to ten groups at more than 65% similarity level. The ten clusters correlated well with the geographical locations with exceptions for isolates obtained from Eastern and Western Ghats. There was a segregation of isolates from these two geographical locations in to two clusters thus, distributing 10 genotypes in to eight geographical locations. All the isolates M. phaseolina irrespective of their host and geographical origin, exhibited two representative monomorphic bands at 250 bp and 1 kb, presence of these bands suggests that isolates might have evolved from a common ancestor but due to geographical isolation fallowed by natural selection and genetic drift might have segregated in to subpopulations. Genetic similarity in the pathogenic population reflects the dispersal of single lineage in all locations in India.