INTEGRINS: A BEDSIDE TO BENCH TO BEDSIDE STORY.

I provide a narrative of the path I took to discover the membrane receptors that mediate leukocyte adhesion, now known as ß2 integrins or CD11/CD18. We followed this discovery with the first determination of the 3-D structures of integrins. The latter advance provided the foundation for understanding the unique features of integrins as divalent cation-dependent signaling receptors and as mechanosensitive conduits between the extracellular matrix and the intracellular cytoskeleton. Our structural studies are now opening new paths for taming overactive integrins in disease while minimizing the collateral damage associated with the faulty pharmacodynamics of current integrin inhibitory drugs.

Cryo-EM structures of full-length integrin αIIbß3 in native lipids.

Platelet integrin αIIbß3 is maintained in a bent inactive state (low affinity to physiologic ligand), but can rapidly switch to a ligand-competent (high-affinity) state in response to intracellular signals ("inside-out" activation). Once bound, ligands drive proadhesive "outside-in" signaling. Anti-αIIbß3 drugs like eptifibatide can engage the inactive integrin directly, inhibiting thrombosis but inadvertently impairing αIIbß3 hemostatic functions. Bidirectional αIIbß3 signaling is mediated by reorganization of the associated αIIb and ß3 transmembrane α-helices, but the underlying changes remain poorly defined absent the structure of the full-length receptor. We now report the cryo-EM structures of full-length αIIbß3 in its apo and eptifibatide-bound states in native cell-membrane nanoparticles at near-atomic resolution. The apo form adopts the bent inactive state but with separated transmembrane α-helices, and a fully accessible ligand-binding site that challenges the model that this site is occluded by the plasma membrane. Bound eptifibatide triggers dramatic conformational changes that may account for impaired hemostasis. These results advance our understanding of integrin structure and function and may guide development of safer inhibitors.

Conformational Dynamics in Extended RGD-Containing Peptides.

RGD is a prolific example of a tripeptide used in biomaterials for cell adhesion, but the potency of free or surface-bound RGD tripeptide is orders-of-magnitude less than the RGD domain within natural proteins. We designed a set of peptides with varying lengths, composed of fragments of fibronectin protein whose central three residues are RGD, in order to vary their conformational behavior without changing the binding site's chemical environment. With these peptides, we measure the conformational dynamics and transient structure of the active site. Our studies reveal how flanking residues affect conformational behavior and integrin binding. We find that disorder of the binding site is important to the potency of RGD peptides and that transient hydrogen bonding near the RGD site affects both the energy landscape roughness of the peptides and peptide binding. This phenomenon is independent of longer-range folding interactions and helps explain why short binding sequences, including RGD itself, do not fully replicate the integrin-targeting properties of extracellular matrix proteins. Our studies reinforce that peptide binding is a holistic event and fragments larger than those directly involved in binding should be considered in the design of peptide epitopes for functional biomaterials.

Structure-guided design of pure orthosteric inhibitors of αIIbß3 that prevent thrombosis but preserve hemostasis.

A prevailing dogma is that inhibition of vascular thrombosis by antagonizing platelet integrin αIIbß3 cannot be achieved without compromising hemostasis, thus causing serious bleeding and increased morbidity and mortality. It is speculated that these adverse outcomes result from drug-induced activating conformational changes in αIIbß3 but direct proof is lacking. Here, we report the structure-guided design of peptide Hr10 and a modified form of the partial agonist drug tirofiban that act as "pure" antagonists of αIIbß3, i.e., they no longer induce the conformational changes in αIIbß3. Both agents inhibit human platelet aggregation but preserve clot retraction. Hr10 and modified tirofiban are as effective as partial agonist drugs in inhibiting vascular thrombosis in humanized mice, but neither causes serious bleeding, establishing a causal link between partial agonism and impaired hemostasis. Pure orthosteric inhibitors of αIIbß3 may thus provide safer alternatives for human therapy, and valuable tools to probe structure-activity relationships in integrins.

Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVß3 and α5ß1.

Targeting both integrins αVß3 and α5ß1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins have not been fully elucidated. RGD-containing knottin 2.5F binds selectively to αVß3 and α5ß1, whereas knottin 2.5D is αVß3 specific. To elucidate the structural basis of this selectivity, we determined the structures of 2.5F and 2.5D as apo proteins and in complex with αVß3, and compared their interactions with integrins using molecular dynamics simulations. These studies show that 2.5D engages αVß3 by an induced fit, but conformational selection of a flexible RGD loop accounts for high-affinity selective binding of 2.5F to both integrins. The contrasting binding of the highly flexible low-affinity linear RGD peptides to multiple integrins suggests that a "Goldilocks zone" of conformational flexibility of the RGD loop in 2.5F underlies its selective binding promiscuity to integrins.

uPAR isoform 2 forms a dimer and induces severe kidney disease in mice.

Soluble urokinase plasminogen activator receptor (suPAR) is an immune-derived circulating signaling molecule that has been implicated in chronic kidney disease, such as focal segmental glomerulosclerosis (FSGS). Typically, native uPAR (isoform 1) translates to a 3-domain protein capable of binding and activating integrins, yet the function of additional isoforms generated by alternative splicing is unknown. Here, we characterized mouse uPAR isoform 2 (msuPAR2), encoding domain I and nearly one-half of domain II, as a dimer in solution, as revealed by 3D electron microscopy structural analysis. In vivo, msuPAR2 transgenic mice exhibited signs of severe renal disease characteristic of FSGS with proteinuria, loss of kidney function, and glomerulosclerosis. Sequencing of the glomerular RNAs from msuPAR2-Tg mice revealed a differentially expressed gene signature that includes upregulation of the suPAR receptor Itgb3, encoding ß3 integrin. Crossing msuPAR2-transgenic mice with 3 different integrin ß3 deficiency models rescued msuPAR2-mediated kidney function. Further analyses indicated a central role for ß3 integrin and c-Src in msuPAR2 signaling and in human FSGS kidney biopsies. Administration of Src inhibitors reduced proteinuria in msuPAR2-transgenic mice. In conclusion, msuPAR2 may play an important role in certain forms of scarring kidney disease.

High-Affinity Bent ß2-Integrin Molecules in Arresting Neutrophils Face Each Other through Binding to ICAMs In cis.

Leukocyte adhesion requires ß2-integrin activation. Resting integrins exist in a bent-closed conformation-i.e., not extended (E-) and not high affinity (H-)-unable to bind ligand. Fully activated E+H+ integrin binds intercellular adhesion molecules (ICAMs) expressed on the opposing cell in trans. E-H- transitions to E+H+ through E+H- or through E-H+, which binds to ICAMs on the same cell in cis. Spatial patterning of activated integrins is thought to be required for effective arrest, but no high-resolution cell surface localization maps of activated integrins exist. Here, we developed Super-STORM by combining super-resolution microscopy with molecular modeling to precisely localize activated integrin molecules and identify the molecular patterns of activated integrins on primary human neutrophils. At the time of neutrophil arrest, E-H+ integrins face each other to form oriented (non-random) nanoclusters. To address the mechanism causing this pattern, we blocked integrin binding to ICAMs in cis, which significantly relieved the face-to-face orientation.

Novel Pure αVß3 Integrin Antagonists That Do Not Induce Receptor Extension, Prime the Receptor, or Enhance Angiogenesis at Low Concentrations.

The integrin αVß3 receptor has been implicated in several important diseases, but no antagonists are approved for human therapy. One possible limitation of current small-molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small-molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVß3-mediated cell adhesion to αVß3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVß3 antagonist cilengitide of paradoxically enhancing aortic sprout angiogenesis at concentrations below its IC50, which correlates with cilengitide's enhancement of tumor growth in vivo.

A microfluidic renal proximal tubule with active reabsorptive function.

In the kidney, the renal proximal tubule (PT) reabsorbs solutes into the peritubular capillaries through active transport. Here, we replicate this reabsorptive function in vitro by engineering a microfluidic PT. The microfluidic PT architecture comprises a porous membrane with user-defined submicron surface topography separating two microchannels representing a PT filtrate lumen and a peritubular capillary lumen. Human PT epithelial cells and microvascular endothelial cells in respective microchannels created a PT-like reabsorptive barrier. Co-culturing epithelial and endothelial cells in the microfluidic architecture enhanced viability, metabolic activity, and compactness of the epithelial layer. The resulting tissue expressed tight junctions, kidney-specific morphology, and polarized expression of kidney markers. The microfluidic PT actively performed sodium-coupled glucose transport, which could be modulated by administration of a sodium-transport inhibiting drug. The microfluidic PT reproduces human physiology at the cellular and tissue levels, and measurable tissue function which can quantify kidney pharmaceutical efficacy and toxicity.

Prophylactic orthosteric inhibition of leukocyte integrin CD11b/CD18 prevents long-term fibrotic kidney failure in cynomolgus monkeys.

Ischaemic acute kidney injury (AKI), an inflammatory disease process, often progresses to chronic kidney disease (CKD), with no available effective prophylaxis. This is in part due to lack of clinically relevant CKD models in non-human primates. Here we demonstrate that inhibition of the archetypal innate immune receptor CD11b/CD18 prevents progression of AKI to CKD in cynomolgus monkeys. Severe ischaemia-reperfusion injury of the right kidney, with subsequent periods of the left ureter ligation, causes irreversible right kidney failure 3, 6 or 9 months after AKI. Moreover, prophylactic inactivation of CD11b/CD18, using the orthosteric CD11b/CD18 inhibitor mAb107, improves microvascular perfusion and histopathology, reduces intrarenal pro-inflammatory mediators and salvages kidney function long term. These studies reveal an important early role of CD11b+ leukocytes in post-ischaemic kidney fibrosis and failure, and suggest a potential early therapeutic intervention to mitigate progression of ischaemic AKI to CKD in humans.

Biology and structure of leukocyte ß 2 integrins and their role in inflammation.

Integrins comprise a large family of αß heterodimeric cell adhesion receptors that are expressed on all cells except red blood cells and that play essential roles in the regulation of cell growth and function. The leukocyte integrins, which include members of the ß 1, ß 2, ß 3, and ß 7 integrin family, are critical for innate and adaptive immune responses but also can contribute to many inflammatory and autoimmune diseases when dysregulated. This review focuses on the ß 2 integrins, the principal integrins expressed on leukocytes. We review their discovery and role in host defense, the structural basis for their ligand recognition and activation, and their potential as therapeutic targets.

Talin1 is required for cardiac Z-disk stabilization and endothelial integrity in zebrafish.

Talin (tln) binds and activates integrins to couple extracellular matrix-bound integrins to the cytoskeleton; however, its role in heart development is not well characterized. We identified the defective gene and the resulting cardiovascular phenotypes in zebrafish tln1(fl02k) mutants. The ethylnitrosourea-induced fl02k mutant showed heart failure, brain hemorrhage, and diminished cardiac and vessel lumens at 52 h post fertilization. Positional cloning revealed a nonsense mutation of tln1 in this mutant. tln1, but neither tln2 nor -2a, was dominantly expressed in the heart and vessels. Unlike tln1 and -2 in the mouse heart, the unique tln1 expression in the heart enabled us, for the first time, to determine the critical roles of Tln1 in the maintenance of cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity, partly through regulating F-actin networks in zebrafish. The similar expression profiles of tln1 and integrin ß1b (itgb1b) and synergistic function of the 2 genes revealed that itgb1b is a potential partner for tln1 in the stabilization of cardiac Z-disks and vessel lumens. Taken together, the results of this work suggest that Tln1-mediated Itgß1b plays a crucial role in maintaining cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity in zebrafish and may also help to gain molecular insights into congenital heart diseases.

Negative Regulation of TGFß Signaling by Stem Cell Antigen-1 Protects against Ischemic Acute Kidney Injury.

Acute kidney injury, often caused by an ischemic insult, is associated with significant short-term morbidity and mortality, and increased risk of chronic kidney disease. The factors affecting the renal response to injury following ischemia and reperfusion remain to be clarified. We found that the Stem cell antigen-1 (Sca-1), commonly used as a stem cell marker, is heavily expressed in renal tubules of the adult mouse kidney. We evaluated its potential role in the kidney using Sca-1 knockout mice submitted to acute ischemia reperfusion injury (IRI), as well as cultured renal proximal tubular cells in which Sca-1 was stably silenced with shRNA. IRI induced more severe injury in Sca-1 null kidneys, as assessed by increased expression of Kim-1 and Ngal, rise in serum creatinine, abnormal pathology, and increased apoptosis of tubular epithelium, and persistent significant renal injury at day 7 post IRI, when recovery of renal function in control animals was nearly complete. Serum creatinine, Kim-1 and Ngal were slightly but significantly elevated even in uninjured Sca-1-/- kidneys. Sca-1 constitutively bound both TGFß receptors I and II in cultured normal proximal tubular epithelial cells. Its genetic loss or silencing lead to constitutive TGFß receptor-mediated activation of canonical Smad signaling even in the absence of ligand and to KIM-1 expression in the silenced cells. These studies demonstrate that by normally repressing TGFß-mediated canonical Smad signaling, Sca-1 plays an important in renal epithelial cell homeostasis and in recovery of renal function following ischemic acute kidney injury.

The α-subunit regulates stability of the metal ion at the ligand-associated metal ion-binding site in ß3 integrins.

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin ßA domain of the ß-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two ß3 integrins αIIbß3 and αVß3, but a potential role of this interaction on stability of the metal ion at LIMBS in ß3 integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated ß3 integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded αIIbß3 versus αVß3, the result of stronger interactions of LIMBS with αV, which reduce stability of the LIMBS metal ion in αVß3. Replacing the αIIb-LIMBS interface residue Phe(191) in αIIb (equivalent to Trp(179) in αV) with Trp strengthened this interface and destabilized the metal ion at LIMBS in αIIbß3; a Trp(179) to Phe mutation in αV produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular αIIbß3 and a W179/F substitution in αVß3 reduced and increased, respectively, the apparent affinity of Mn(2+) to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in αVß3 structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.

Collective epithelial migration drives kidney repair after acute injury.

Acute kidney injury (AKI) is a common and significant medical problem. Despite the kidney's remarkable regenerative capacity, the mortality rate for the AKI patients is high. Thus, there remains a need to better understand the cellular mechanisms of nephron repair in order to develop new strategies that would enhance the intrinsic ability of kidney tissue to regenerate. Here, using a novel, laser ablation-based, zebrafish model of AKI, we show that collective migration of kidney epithelial cells is a primary early response to acute injury. We also show that cell proliferation is a late response of regenerating kidney epithelia that follows cell migration during kidney repair. We propose a computational model that predicts this temporal relationship and suggests that cell stretch is a mechanical link between migration and proliferation, and present experimental evidence in support of this hypothesis. Overall, this study advances our understanding of kidney repair mechanisms by highlighting a primary role for collective cell migration, laying a foundation for new approaches to treatment of AKI.

Atomic basis for the species-specific inhibition of αV integrins by monoclonal antibody 17E6 is revealed by the crystal structure of αVß3 ectodomain-17E6 Fab complex.

The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVß3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn(2+). The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVß3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins.

Structural basis for pure antagonism of integrin αVß3 by a high-affinity form of fibronectin.

Integrins are important therapeutic targets. However, current RGD-based anti-integrin drugs are also partial agonists, inducing conformational changes that trigger potentially fatal immune reactions and paradoxical cell adhesion. Here we describe the first crystal structure of αVß3 bound to a physiologic ligand, the tenth type III RGD domain of wild-type fibronectin (wtFN10), or to a high-affinity mutant (hFN10) shown here to act as a pure antagonist. Comparison of these structures revealed a central π-π interaction between Trp1496 in the RGD-containing loop of hFN10 and Tyr122 of the ß3 subunit that blocked conformational changes triggered by wtFN10 and trapped hFN10-bound αVß3 in an inactive conformation. Removing the Trp1496 or Tyr122 side chains or reorienting Trp1496 away from Tyr122 converted hFN10 into a partial agonist. These findings offer new insights into the mechanism of integrin activation and a basis for the design of RGD-based pure antagonists.

Structure of the kidney slit diaphragm adapter protein CD2-associated protein as determined with electron microscopy.

CD2-associated protein (CD2AP) is a multidomain scaffolding protein that has a critical role in renal function. CD2AP is expressed in glomerular podocytes at the slit diaphragm, a modified adherens junction that comprises the protein filtration barrier of the kidney, and interacts with a number of protein ligands involved in cytoskeletal remodeling, membrane trafficking, cell motility, and cell survival. The structure of CD2AP is unknown. We used electron microscopy and single particle image analysis to determine the three-dimensional structure of recombinant full-length CD2AP and found that the protein is a tetramer in solution. Image reconstruction of negatively stained protein particles generated a structure at 21 Å resolution. The protein assumed a roughly spherical, very loosely packed structure. Analysis of the electron density map revealed that CD2AP consists of a central coiled-coil domain, which forms the tetramer interface, surrounded by four symmetry-related motifs, each containing three globular domains corresponding to the three SH3 domains. The spatial organization exposes the binding sites of all 12 SH3 domains in the tetramer, allowing simultaneous binding to multiple targets. Determination of the structure of CD2AP provides novel insights into the biology of this slit diaphragm protein and lays the groundwork for characterizing the interactions between key molecules of the slit diaphragm that control glomerular filtration.

Stress hematopoiesis is regulated by the Krüppel-like transcription factor ZBP-89.

Previous studies have shown that ZBP-89 (Zfp148) plays a critical role in erythroid lineage development, with its loss at the embryonic stage causing lethal anemia and thrombocytopenia. Its role in adult hematopoiesis has not been described. We now show that conditional deletion of ZBP-89 in adult mouse hematopoietic stem/progenitor cells (HSPC) causes anemia and thrombocytopenia that are transient in the steady state, but readily uncovered following chemically induced erythro/megakaryopoietic stress. Unexpectedly, stress induced by bone marrow transplantation of ZBP89(-/-) HSPC also resulted in a myeloid-to-B lymphoid lineage switch in bone marrow recipients. The erythroid and myeloid/B lymphoid lineage anomalies in ZBP89(-/-) HSPC are reproduced in vitro in the ZBP-89-silenced multipotent hematopoietic cell line FDCP-Mix A4, and are associated with the upregulation of PU.1 and downregulation of SCL/Tal1 and GATA-1 in ZBP89-deficient cells. Chromatin immunoprecipitation and luciferase reporter assays show that ZBP-89 is a direct repressor of PU.1 and activator of SCL/Tal1 and GATA-1. These data identify an important role for ZBP-89 in regulating stress hematopoiesis in adult mouse bone marrow.

Abatacept in B7-1-positive proteinuric kidney disease.

Abatacept (cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin fusion protein [CTLA-4-Ig]) is a costimulatory inhibitor that targets B7-1 (CD80). The present report describes five patients who had focal segmental glomerulosclerosis (FSGS) (four with recurrent FSGS after transplantation and one with primary FSGS) and proteinuria with B7-1 immunostaining of podocytes in kidney-biopsy specimens. Abatacept induced partial or complete remissions of proteinuria in these patients, suggesting that B7-1 may be a useful biomarker for the treatment of some glomerulopathies. Our data indicate that abatacept may stabilize ß1-integrin activation in podocytes and reduce proteinuria in patients with B7-1-positive glomerular disease.