The design of β-peptide foldamers targeting the transmembrane (TM) domains of complex natural membrane proteins has been a formidable challenge. A series of β-peptides was designed to stably insert ...in TM orientations in phospholipid bilayers. Their secondary structures and orientation in the phospholipid bilayer was characterized using biophysical methods. Computational methods were then devised to design a β-peptide that targeted a TM helix of the integrin αIIbβ3. The designed peptide (β-CHAMP) interacts with the isolated target TM domain of the protein and activates the intact integrin in vitro.
FOP is a disabling disorder in which skeletal muscle is progressively replaced with bone. Lymphocytes, our model system for examining BMP signaling, cannot signal through the canonical Smad pathway ...unless exogenous Smad1 is supplied, providing a unique cell type in which the BMP–p38 MAPK pathway can be examined. FOP lymphocytes exhibit defects in the BMP–p38 MAPK pathway, suggesting that altered BMP signaling underlies ectopic bone formation in this disease.
Introduction: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by progressive heterotopic ossification of connective tissues. Whereas the primary genetic defect in this condition is unknown, BMP4 mRNA and protein and BMP receptor type IA (BMPRIA) protein are overexpressed in cultured lymphocytes from FOP patients, supporting that altered BMP signaling is involved in this disease. In this study, we examined downstream signaling targets to study the BMP–Smad and BMP–p38 mitogen‐activated protein kinase (MAPK) pathways in FOP.
Materials and Methods: Protein phosphorylation was assayed by immunoblots, and p38 MAPK activity was measured by kinase assays. To examine BMP target genes, the mRNA expression of ID1, ID3, and MSX2 was determined by quantitative real‐time PCR. Statistical analysis was performed using Student's t‐test or ANOVA.
Results: FOP lymphocytes exhibited increased levels of p38 phosphorylation and p38 MAPK activity in response to BMP4 stimulation. Furthermore, in response to BMP4, FOP cells overexpressed the downstream signaling targets ID1 by 5‐fold and ID3 by 3‐fold compared with controls. ID1 and ID3 mRNA induction was specifically blocked with a p38 MAPK inhibitor, but not extracellular signal‐related kinase (ERK) or c‐Jun N‐terminal kinase (JNK) inhibitors. MSX2, a known Smad pathway target gene, is not upregulated in control or FOP cells in response to BMP, suggesting that lymphocytes do not use this limb of the BMP pathway. However, introduction of Smad1 into lymphocytes made the cells competent to regulate MSX2 mRNA after BMP4 treatment.
Conclusions: Lymphocytes are a cell system that signals primarily through the BMP–p38 MAPK pathway rather than the BMP–Smad pathway in response to BMP4. The p38 MAPK pathway is dysregulated in FOP lymphocytes, which may play a role in the pathogenesis of FOP.
Coagulation factor XI (FXI) is a covalent homodimer consisting of two identical subunits of 80 kDa linked by a disulfide bond formed by Cys-321 within the Apple 4 domain of each subunit. Because ...FXIC321S is a noncovalent dimer, residues within the interface between the two subunits must mediate its homodimeric structure. The crystal structure of FXI demonstrates formation of salt bridges between Lys-331 of one subunit and Glu-287 of the other subunit and hydrophobic interactions at the interface of the Apple 4 domains involving Ile-290, Leu-284, and Tyr-329. FXIC321S, FXIC321S,K331A, FXIC321S,E287A, FXIC321S,I290A, FXIC321S,Y329A, FXIC321S,L284A, FXIC321S,K331R, and FXIC321S,H343A were expressed in HEK293 cells and characterized using size exclusion chromatography, analytical ultracentrifugation, electron microscopy, and functional assays. Whereas FXIC321S and FXIC321S,H343A existed in monomer/dimer equilibrium (Kd ∼ 40 nm), all other mutants were predominantly monomers with impaired dimer formation by analytical ultracentrifugation (Kd = 3–38 μm). When converted to the active enzyme, FXIa, all the monomeric mutants activated FIX similarly to wild-type dimeric FXIa. In contrast, these monomeric mutants could not be activated efficiently by FXIIa, thrombin, or autoactivation in the presence of dextran sulfate. We conclude that salt bridges formed between Lys-331 of one subunit and Glu-287 of the other together with hydrophobic interactions at the interface, involving residues Ile-290, Leu-284, and Tyr-329, are essential for homodimer formation. The dimeric structure of FXI is essential for normal proteolytic activation of FXI by FXIIa, thrombin, or FXIa either in solution or on an anionic surface but not for FIX activation by FXIa in solution.
: Identification of gene mutations in Mendelian disorders is often determined by linkage analysis and positional cloning, an approach that is difficult for fibrodysplasia ossificans progressiva ...(FOP) due to a low reproductive fitness that results in a small number of multigenerational families showing inheritance of the disease. Altered signaling pathways can be investigated as a complementary method to identify the consequences of the mutated gene responsible for FOP and to identify potential therapeutic targets. Candidate signaling pathways for FOP are those that malfunctioning could account for the malformation of the great toes during embryonic development and could explain the postnatal progressive heterotopic endochondral ossification. Signaling pathways that fit these criteria are the BMP signaling pathway and its interacting pathways. A large body of data suggest that the BMP‐4 signaling pathway is dysregulated in FOP.
Transmembrane (TM) helices engage in homomeric and heteromeric interactions that play essential roles in the folding and assembly of TM proteins. However, features that explain their propensity to ...interact homomerically or heteromerically and determine the strength of these interactions are poorly understood. Integrins provide an ideal model system for addressing these questions because the TM helices of full-length integrins interact heteromerically when integrins are inactive, but isolated TM helices are also able to form homodimers or homooligomers in micelles and bacterial membranes. We sought to determine the features defining specificity for homointeractions versus heterointeractions by conducting a comprehensive comparison of the homomeric and heteromeric interactions of integrin αIIbβ3 TM helices in biological membranes. Using the TOXCAT assay, we found that residues V700, M701, A703, I704, L705, G708, L709, L712, and L713, which are located on the same face of the β3 helix, mediate homodimer formation. We then characterized the β3 heterodimer by measuring the ability of β3 helix mutations to cause ligand binding to αIIbβ3. We found that mutating V696, L697, V700, M701, A703. I704, L705, G708, L712, and L713, but not the small residue–X3–small residue motif S699–X3–A703, caused constitutive αIIbβ3 activation, as well as persistent focal adhesion kinase phosphorylation dependent on αIIbβ3 activation. Because αIIb and β3 use the same face of their respective TM helices for homomeric and heteromeric interactions, the interacting surface on each has an intrinsic “stickiness” predisposing towards helix–helix interactions in membranes. The residues responsible for heterodimer formation comprise a network of interdigitated side chains with considerable geometric complementarity; mutations along this interface invariably destabilize heterodimer formation. By contrast, residues responsible for homomeric interactions are dispersed over a wider surface. While most mutations of these residues are destabilizing, some stabilized homooligomer formation. We conclude that the αIIbβ3 TM heterodimer shows the hallmark of finely tuned heterodimeric interaction, while homomeric interaction is less specific.
The present study was undertaken to determine whether the Bowman-Birk inhibitor (BBI) could prevent colon carcinogenesis in rats treated with dimethylhydrazine (DMH) and whether there were adverse ...side effects associated with treatment with BBI for cancer prevention. BBI was evaluated in the forms of purified BBI (PBBI) or an extract of soybeans enriched in BBI, termed BBI concentrate (BBIC). The results demonstrate that PBBI and BBIC reduced the incidence and frequency of tumors in DMH-treated rats compared with animals treated with DMH alone. Autoclaved BBIC, in which the protease inhibitor activity of BBI was destroyed, had a weak and statistically insignificant, suppressive effect on DMH-induced colon carcinogenesis in rats, suggesting that the protease inhibitor activity of BBI is likely to be responsible for the anticarcinogenic activity of BBIC. Soy molasses, which contains soy isoflavones, did not have an effect on colon cancer carcinogenesis in DMH-treated rats. Similar to results from previous studies (Nauss et al. JNCI 73, 915-924, 1984), the most aggressive, malignant colon adenocarcinomas developed within or in association with gut-associated lymphoid tissue aggregates. No adverse side effects on the pancreas or animal growth were observed in rats treated with PBBI or BBIC. These results demonstrate that PBBI and BBIC may be used to prevent colon cancer without significant adverse side effects.
Mesangial cells and podocytes express integrins α1β1 and α2β1, which are the two major collagen receptors that regulate multiple cellular functions, including extracellular matrix homeostasis. ...Integrin α1β1 protects from glomerular injury by negatively regulating collagen production, but the role of integrin α2β1 in renal injury is unclear. Here, we subjected wild-type and integrin α2-null mice to injury with adriamycin or partial renal ablation. In both of these models, integrin α2-null mice developed significantly less proteinuria and glomerulosclerosis. In addition, selective pharmacological inhibition of integrin α2β1 significantly reduced adriamycin-induced proteinuria, glomerular injury, and collagen deposition in wild-type mice. This inhibitor significantly reduced collagen synthesis in wild-type, but not integrin α2-null, mesangial cells in vitro, demonstrating that its effects are integrin α2β1-dependent. Taken together, these results indicate that integrin α2β1 contributes to glomerular injury by positively regulating collagen synthesis and suggest that its inhibition may be a promising strategy to reduce glomerular injury and proteinuria.
Transforming growth factor β (TGF‐β) has well‐documented roles in chondrocyte maturation and endochondral ossification, but the mechanisms of TGF‐β activation during these processes remain unclear. ...In this study, we analyzed TGF‐β activation in chick embryo resting, proliferating, and hypertrophic chondrocytes in culture. We found that both levels and activation of TGF‐β increased substantially with maturation. The majority of TGF‐β produced by resting cells over culture time remained latent, but a larger portion produced by proliferating and hypertrophic cells was activated with increasing maturation. Zymography of gelatin gels revealed that matrix metalloprotease 2 (MMP‐2) and MMP‐9 were expressed by each population and that MMP‐13 characterized hypertrophic chondrocytes and to a lesser extent proliferating chondrocytes in late cultures. Treatment with pharmacologic agents revealed that both MMPs and serine proteases are involved in activation. However, because inhibition of MMPs almost completely prevented TGF‐β activation, MMPs appear crucial for activation. During culture, inclusion of the tetracycline‐derived, collagenase/gelatinase inhibitor chemically modified nonantimicrobial tetracycline (CMT‐8) at concentrations specific for MMP‐13 inhibition resulted in complete inhibition of TGF‐β activation by proliferating and hypertrophic chondrocytes. These results show that TGF‐β production, release, and activation are regulated developmentally in chondrocytes. Our findings point to a strict mode of regulation of this potent factor to elicit diverse and highly specific effects during chondrocyte maturation and ossification.
PDF
