Biomedical subjects

Renhao Li

Publications and source records attributed to Renhao Li.

8 recordsLinked to original sources

PubMed2005

Recombinant CUB-1 domain polypeptide inhibits the cleavage of ULVWF strings by ADAMTS13 under flow conditions.

The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif) converts the hyperreactive unusually large (UL) forms of von Willebrand factor (VWF) that are newly released from endothelial cells into less active plasma forms by cleaving a peptide bond in the VWF A2 domain. Familial or acquired deficiency of this metalloprotease is associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 belongs to the ADAMTS metalloprotease family, but, unlike other members, it also contains 2 C-terminal CUB domains (complement component Clr/Cls, Uegf, and bone morphogenic protein 1). Mutations in the CUB region have been found in congenital TTP, but deletion of the region did not impair enzyme activity in conventional in vitro assays. We investigated the functions of the CUB domain in ADAMTS13 activity under flow conditions. We found that recombinant CUB-1 and CUB-1+2 polypeptides and synthetic peptides derived from CUB-1 partially blocked the cleavage of ULVWF by ADAMTS13 on the surface of endothelial cells under flow. The polypeptide bound immobilized and soluble forms of ULVWF, and blocked the adhesion of ADAMTS13-coated beads to immobilized ULVWF under flow. These results suggest that the CUB-1 domain may serve as the docking site for ADAMTS13 to bind ULVWF under flow, a critical step to initiate ULVWF proteolysis.

ADAM Proteins↗

PubMed2005

A push-pull mechanism for regulating integrin function.

Homomeric and heteromeric interactions between the alphaIIb and beta3 transmembrane domains are involved in the regulation of integrin alphaIIbbeta3 function. These domains appear to interact in the inactivated state but separate upon integrin activation. Moreover, homomeric interactions may increase the level of alphaIIbbeta3 activity by competing for the heteromeric interaction that specifies the resting state. To test this model, a series of mutants were examined that had been shown previously to either enhance or disrupt the homomeric association of the alphaIIb transmembrane domain. One mutation that enhanced the dimerization of the alphaIIb transmembrane domain indeed induced constitutive alphaIIbbeta3 activation. However, a series of mutations that disrupted homodimerization also led to alphaIIbbeta3 activation. These results suggest that the homo- and heterodimerization motifs overlap in the alphaIIb transmembrane domain, and that mutations that disrupt the alphaIIb/beta3 transmembrane domain heterodimer are sufficient to activate the integrin. The data also imply a mechanism for alphaIIbbeta3 regulation in which the integrin can be shifted from its inactive to its active state by destabilizing an alphaIIb/beta3 transmembrane domain heterodimer and by stabilizing the resulting alphaIIb and beta3 transmembrane domain homodimers.

Animals↗

PubMed2004

Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes.

Homo- and hetero-oligomeric interactions between the transmembrane (TM) helices of integrin alpha and beta subunits may play an important role in integrin activation and clustering. As a first step to understanding these interactions, we used the TOXCAT assay to measure oligomerization of the wild-type alpha(IIb) TM helix and single-site TM domain mutants. TOXCAT measures the oligomerization of a chimeric protein containing a TM helix in the Escherichia coli inner membrane via the transcriptional activation of the gene for chloramphenicol acetyltransferase. We found the amount of chloramphenicol acetyltransferase induced by the wild-type alpha(IIb) TM helix was approximately half that induced by the strongly dimerizing TM helix of glycophorin A, confirming that the alpha(IIb) TM domain oligomerizes in biological membranes. Mutating each of the alpha(IIb) TM domain residues to either Ala, Leu, Ile, or Val revealed that a GXXXG motif mediates oligomerization. Further, we found that the residue preceding each glycine contributed to the oligomerization interface, as did the residue at position i + 4 after the second Gly of GXXXG. Thus, the sequence XXVGXXGGXXXLXX is critical for oligomerization of alpha(IIb) TM helix. These data were used to generate an atomic model of the alpha(IIb) homodimer, revealing a family of structures with right-handed crossing angles of 40 degrees to 60 degrees, consistent with a 4.0-residue periodicity, and with an interface rotated by 50 degrees relative to glycophorin A. Thus, although the alpha(IIb) TM helix makes use of the GXXXG framework, neighboring residues have evolved to engineer its dimerization interface, enabling it to subserve specific and specialized functions.

Amino Acid Motifs↗

PubMed2003

Activation of integrin alphaIIbbeta3 by modulation of transmembrane helix associations.

Transmembrane helices of integrin alpha and beta subunits have been implicated in the regulation of integrin activity. Two mutations, glycine-708 to asparagine-708 (G708N)and methionine-701 to asparagine-701, in the transmembrane helix of the beta3 subunit enabled integrin alphaIIbbeta3 to constitutively bind soluble fibrinogen. Further characterization of the G708N mutant revealed that it induced alphaIIbbeta3 clustering and constitutive phosphorylation of focal adhesion kinase. This mutation also enhanced the tendency of the transmembrane helix to form homotrimers. These results suggest that homomeric associations involving transmembrane domains provide a driving force for integrin activation. They also suggest a structural basis for the coincidence of integrin activation and clustering.

Amino Acid Sequence↗

PubMed2003

Use of phage display to probe the evolution of binding specificity and affinity in integrins.

The specific binding of RGD-containing proteins to integrin is a function of both the conformation of and the local sequence surrounding the RGD motif. To study the effect of these factors on integrin binding affinity and specificity, we obtained RGD-containing ligands specific for different integrins presented on the same protein scaffold. The beta-turn region between two anti-parallel beta-strands on the loop I of tendamistat, an inhibitor of alpha-amylase, was extended by two residues and randomized in a phagemid library. This library and two subsequently constructed RGD-containing loop I libraries were biopanned with purified integrins alphaIIbbeta3, alphaVbeta3 and alphaVbeta5 individually. The sequence analysis of selected tendamistat variants and characterization by phage ELISA revealed that phage adhesion is mediated exclusively by an RGD motif located at only two out of four possible positions on loop I. Further, sequences flanking the RGD motif were specific for different integrin targets. Interestingly, selected tendamistat variants mimic natural integrin ligands, both in sequence similarity and in integrin binding specificity, indicating that various ligand specificity patterns can be generated by driving towards maximum affinity in the integrin-ligand complexes.

Amino Acid Motifs↗

PubMed2002

Characterization of the monomeric form of the transmembrane and cytoplasmic domains of the integrin beta 3 subunit by NMR spectroscopy.

We have characterized a membrane protein containing residues P688-T762 of the integrin beta3 subunit, encompassing its transmembrane and cytoplasmic domains, by nuclear magnetic resonance spectroscopy. Under conditions in which it is monomeric in dodecylphosphocholine micelles, the protein consists mainly of alpha-helical structures. An amino-terminal helix corresponding to the beta3 transmembrane helix extends into the membrane-proximal region of the cytoplasmic domain. Moreover, following an apparent hinge at residues H722-D723, residues K725-A735 are mostly alpha-helical. In the presence of membrane-mimicking detergents, the cytoplasmic domain connected to the transmembrane helix is substantially ordered at pH 4.8 and 50 degrees C. Its carboxyl-terminal end takes on a turn-helix configuration characteristic of the immunoreceptor tyrosine-based activation motif. These structural features of the beta3 subunit should help to explain its interaction with numerous cytosolic interacting proteins and begin to illuminate the mechanism of integrin activation.

Amino Acid Sequence↗

PubMed2002

A highly destabilizing mutation, G37A, of the bovine pancreatic trypsin inhibitor retains the average native conformation but greatly increases local flexibility.

A point mutation, G37A, on the surface of bovine pancreatic trypsin inhibitor (BPTI) destabilizes the protein by approximately 5 kcal/mol, which is very high for addition of one methyl group. In wild-type (WT) BPTI, Gly 37 HN is in an unusual NH-aromatic-NH network of interactions with the ring of Tyr 35 and the side chain HN of Asn 44. G37A was designed to disrupt this interaction, since the phi and psi backbone angles of G37 are not favorable for an amino acid containing a beta-carbon. Investigations of the structure and dynamics by NMR methods show that G37A retains the average WT structure. The NH-aromatic-NH interactions remain intact, as indicated by NOEs and the large upfield ring current shift (approximately 4 ppm) of A37 HN. The NMR structure, confirmed by molecular modeling calculations, requires phi and psi backbone angles that are highly destabilizing when alanine is in position 37. Although the average structure is essentially unchanged, the dynamics are altered dramatically. Many residues in the region of the mutation have increased flexibility, as probed by aromatic ring flip rates and native state hydrogen exchange. We conclude that a large fraction of the destabilization arises from maintaining A37 in a high-energy conformation. This suggests that disruption of the NH-aromatic-NH network is energetically very costly, and may involve other cooperatively linked interactions. The results illustrate the importance of the Gly-Gly sequence at positions 36 and 37 and the 37 HN-35 aromatic interaction to the stability, folding, and dynamics of the BPTI.

Alanine↗

PubMed2002

Native-like interactions favored in the unfolded bovine pancreatic trypsin inhibitor have different roles in folding.

Folding kinetics of a series of bovine pancreatic trypsin inhibitor (BPTI) variants with similar stabilities and structures have been measured. All are strongly destabilized relative to WT. In Y21A, F22A, Y23A, G37A, and F45A, the three native disulfide bonds are retained. In RM(14-38), Cys14 and Cys38 thiols are methylated while C30-C51 and C5-C55 disulfides remain intact. At pH 2 and 20 degrees C, relaxation rate constants of the major kinetic phase range from approximately 10 ms to 0.71 s in the absence of denaturant. All mutants except G37A exhibit standard two-state behavior. Y21A, F22A, and Y23A fold much more slowly than other mutants. The experiments were designed to test the hypothesis that native-like structure detected in the unfolded BPTI is important in folding. Two native-like contacts are implied by NOEs in reduced and unfolded BPTI, between residues Tyr23 and Ala25, and between Gly37 NH and the Tyr35 ring. The results support an earlier hypothesis that formation of the central beta-hairpin, monitored by a local native interaction between Tyr23 and Ala25, is crucial to initiation of BPTI folding. The second native-like contact is important, not in folding initiation, but in preventing a kinetic trap later in the process. Evidence for this comes from mutant G37A, which behaves very differently from the others in displaying a phenomenon called rollover. G37A is, to our knowledge, the first reported case in which a single-site replacement causes rollover, while the wild type and all other known mutants of the same protein show typical two-state chevron plots. The best explanation is that the G37A mutation introduces a kinetic trap of the type described by Chan and Dill [(1998) Proteins 30, 2-33]. In native BPTI, there is an unusual polar interaction between the ring of Tyr35 and the backbone NH of Gly37. Our results suggest that the NH-aromatic interaction between residues 37 and 35 is important throughout folding in stabilizing native-like loop conformations and in preventing the flexible loops from being trapped in nonfunctional conformations during later stages of folding.

Animals↗