JianFeng Chen

Bistable regulation of integrin adhesiveness by a bipolar metal ion cluster

Integrin α4β7 mediates rolling adhesion in Ca2+ and Ca2+ + Mg2+, and firm adhesion in Mg2+ and Mn2+, mimicking the two key steps in leukocyte accumulation in inflamed vasculature. We mutated an interlinked linear array of three divalent cation-binding sites present in integrin β-subunit I-like domains. The middle, metal ion–dependent adhesion site (MIDAS) is required for both rolling and firm adhesion. One polar site, that adjacent to MIDAS (ADMIDAS), is required for rolling because its mutation results in firm adhesion. The other polar site, the ligand-induced metal binding site (LIMBS), is required for firm adhesion because its mutation results in rolling. The LIMBS mediates the positive regulatory effects of low Ca2+ concentrations, whereas the ADMIDAS mediates the negative regulatory effects of higher Ca2+ concentrations, which are competed by Mn2+. The bipolar sites thus stabilize two alternative phases of adhesion.

Discovery of antiandrogen activity of nonsteroidal scaffolds of marketed drugs.

Finding good drug leads de novo from large chemical libraries, real or virtual, is not an easy task. High-throughput screening is often plagued by low hit rates and many leads that are toxic or exhibit poor bioavailability. Exploiting the secondary activity of marketed drugs, on the other hand, may help in generating drug leads that can be optimized for the observed side-effect target, while maintaining acceptable bioavailability and toxicity profiles. Here, we describe an efficient computational methodology to discover leads to a protein target from safe marketed drugs. We applied an in silico “drug repurposing” procedure for identification of nonsteroidal antagonists against the human androgen receptor (AR), using multiple predicted models of an antagonist-bound receptor. The library of marketed oral drugs was then docked into the best-performing models, and the 11 selected compounds with the highest docking score were tested in vitro for AR binding and antagonism of dihydrotestosterone-induced AR transactivation. The phenothiazine derivatives acetophenazine, fluphenazine, and periciazine, used clinically as antipsychotic drugs, were identified as weak AR antagonists. This in vitro biological activity correlated well with endocrine side effects observed in individuals taking these medications. Further computational optimization of phenothiazines, combined with in vitro screening, led to the identification of a nonsteroidal antiandrogen with improved AR antagonism and marked reduction in affinity for dopaminergic and serotonergic receptors that are the primary target of phenothiazine antipsychotics.

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca2+-binding motifs that have essential roles in integrin biogenesis. The function of another Ca2+-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.

Aberrant activation of integrin α4β7 suppresses lymphocyte migration to the gut

Integrin adhesion molecules mediate lymphocyte migration and homing to normal and inflamed tissues. While the ligand-binding activity of integrins is known to be modulated by conformational changes, little is known about how the appropriate balance of integrin adhesiveness is maintained in order to optimize the migratory capacity of lymphocytes in vivo. In this study we examined the regulation of the gut homing receptor alpha4beta7 integrin by manipulating at the germline level an integrin regulatory domain known as adjacent to metal ion-dependent adhesion site (ADMIDAS). ADMIDAS normally serves to raise the activation threshold of alpha4beta7, thereby stabilizing it in the default nonadhesive state. Lymphocytes from knockin beta7 (D146A) mice, which harbor a disrupted ADMIDAS, not only expressed an alpha4beta7 integrin that persistently adhered to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), but also exhibited perturbed cell migration along MAdCAM-1 substrates resulting from improper de-adhesion of the lymphocyte trailing edge. In vivo, aberrantly activated alpha4beta7 enhanced adhesion to Peyers patch venules, but suppressed lymphocyte homing to the gut, diminishing the capacity of T cells to induce colitis. Our results underscore the importance of a proper balance in the adhesion and de-adhesion of the alpha4beta7 integrin, both for lymphocyte trafficking to the gut and for colitis progression.

Regulation of outside-in signaling and affinity by the β2 I domain of integrin αLβ2

The adhesiveness of integrin αLβ2 is modulated by divalent cations. We mutated three metal ion-binding sites in the β2 I domain. The metal ion-dependent adhesion site (MIDAS) and the ligand-induced metal-binding site are required for ligand binding and sufficient for synergism between Ca2+ and Mg2+. Adjacent to MIDAS (ADMIDAS) mutants are constitutively active but remain bent, with poor exposure of a β2 stalk region epitope. Fluorescence resonance energy transfer between fluorescent protein-fused αL and β2 cytoplasmic domains showed that ADMIDAS mutation abrogated ligand binding-induced spatial separation of cytoplasmic domains. Furthermore, ADMIDAS mutation abolished spreading on ligand-bearing substrates. Thus, β2 I domain metal ion-binding sites regulate αL I domain affinity, and the ADMIDAS is required for outside-in signaling.

The Relative Influence of Metal Ion Binding Sites in the I-like Domain and the Interface with the Hybrid Domain on Rolling and Firm Adhesion by Integrin α4β7

We examined the effect of conformational change at the β7 I-like/hybrid domain interface on regulating the transition between rolling and firm adhesion by integrin α4β7. An N-glycosylation site was introduced into the I-like/hybrid domain interface to act as a wedge and to stabilize the open conformation of this interface and hence the open conformation of the α4 β7 headpiece. Wild-type α4β7 mediates rolling adhesion in Ca2+ and Ca2+/Mg2+ but firm adhesion in Mg2+ and Mn2+. Stabilizing the open headpiece resulted in firm adhesion in all divalent cations. The interaction between metal binding sites in the I-like domain and the interface with the hybrid domain was examined in double mutants. Changes at these two sites can either counterbalance one another or be additive, emphasizing mutuality and the importance of multiple interfaces in integrin regulation. A double mutant with counterbalancing deactivating ligand-induced metal ion binding site (LIMBS) and activating wedge mutations could still be activated by Mn2+, confirming the importance of the adjacent to metal ion-dependent adhesion site (ADMIDAS) in integrin activation by Mn2+. Overall, the results demonstrate the importance of headpiece allostery in the conversion of rolling to firm adhesion.

Structural specializations of α4β7, an integrin that mediates rolling adhesion

Electron microscopy and crystallography studies of α4β7 integrin reveal the mechanism by which this atypical integrin enables rolling adhesion prior to integrin activation.

Role of Integrins in Regulating Proteases to Mediate Extracellular Matrix Remodeling

The extracellular matrix (ECM) is an extracellular scaffold composed of complex mixtures of proteins that plays a pivotal role in tumor progression. ECM remodeling is crucial for tumor migration and invasion during the process of metastasis. ECM can be remodeled by several processes including synthesis, contraction and proteolytic degradation. In order to cross through the ECM barriers, malignant cells produce a spectrum of extracellular proteinases including matrix metalloproteinases (MMPs), serine proteases (mainly the urokinase plasminogen activator (uPA) system) and cysteine proteases to degrade ECM components. As major adhesion molecules to support cell attachment to ECM, integrins play critical roles in tumor progression by enhancing tumor cell survival, migration and invasion. Previous studies have shown that integrins can regulate the expression and activity of these proteases through different pathways. This review summarizes the roles of MMPs and uPA system in ECM remodeling and discusses the regulatory functions of integrins on these proteases in invasive tumors.

Leucyl-tRNA Synthetase Consisting of Two Subunits from Hyperthermophilic Bacteria Aquifex aeolicus*

In a hyperthermophilic bacterium, Aquifex aeolicus, leucyl-tRNA synthetase (LeuRS) consists of two non-identical polypeptide subunits (α and β), different from the canonical LeuRS, which has a single polypeptide chain. By PCR, using genome DNA of A. aeolicus as a template, genes encoding the α and β subunits were amplified and cloned in Escherichia coli. The α subunit could not be expressed stably in vivo, whereas the β subunit was overproduced and purified by a simple procedure. The β subunit was inactive in catalysis but was able to bind tRNALeu. Interestingly, the heterodimer αβ-LeuRS could be overproduced in E. coli cells containing both genes and was purified to 95% homogeneity as a hybrid dimer. The kinetics of A. aeolicus LeuRS in pre-steady and steady states and cross-recognition of LeuRS and tRNALeufrom A. aeolicus and E. coli were studied. Magnesium concentration, pH value, and temperature aminoacylation optima were determined to be 12 mm, 7.8, and 70 °C, respectively. Under optimal conditions, A. aeolicus αβ-LeuRS is stable up to 65 °C.

Cation-π interaction regulates ligand-binding affinity and signaling of integrin α4β7

Integrin α4β7 mediates rolling and firm adhesion of leucocytes, two of the critical steps in leukocyte migration and tissue specific homing. Affinity of α4β7 for ligand is dynamically regulated by three interlinked metal ion-binding sites in β7-subunit I domain. In this study, we found that Phe185 (F185), a highly conserved aromatic residue in β7-subunit, links the specificity-determining loop and the synergistic metal ion-binding site (SyMBS) through cation-π interaction. Mutations of F185 that disrupted the SyMBS cation-F185 interaction led to deficient firm cell adhesion mediated by high affinity α4β7, and only slightly affected rolling adhesion mediated by low affinity α4β7. Disruption of SyMBS cation-F185 interaction induced partial extension of integrin ectodomain and separation of cytoplasmic tails, and impaired α4β7-mediated bidirectional signaling. In addition, loss of SyMBS cation-F185 interaction increased paxillin expression and promoted paxillin-integrin binding, leading to deficient cell spreading. Furthermore, integrin α4β7-mediated cell migration was decreased by the abolishment of SyMBS cation-F185 interaction. Thus, these findings reveal a cation-π interaction playing vital roles in the regulation of integrin affinity, signaling, and biological functions.