Structural Basis for Regulated Proteolysis by the α-Secretase ADAM10.

Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation.

Crystal Structure of a Full-Length Human Tetraspanin Reveals a Cholesterol-Binding Pocket.

Tetraspanins comprise a diverse family of four-pass transmembrane proteins that play critical roles in the immune, reproductive, genitourinary, and auditory systems. Despite their pervasive roles in human physiology, little is known about the structure of tetraspanins or the molecular mechanisms underlying their various functions. Here, we report the crystal structure of human CD81, a full-length tetraspanin. The transmembrane segments of CD81 pack as two largely separated pairs of helices, capped by the large extracellular loop (EC2) at the outer membrane leaflet. The two pairs of helices converge at the inner leaflet to create an intramembrane pocket with additional electron density corresponding to a bound cholesterol molecule within the cavity. Molecular dynamics simulations identify an additional conformation in which EC2 separates substantially from the transmembrane domain. Cholesterol binding appears to modulate CD81 activity in cells, suggesting a potential mechanism for regulation of tetraspanin function.

Inaxaplin for Proteinuric Kidney Disease in Persons with Two APOL1 Variants.

BACKGROUND: Persons with toxic gain-of-function variants in the gene encoding apolipoprotein L1 (APOL1) are at greater risk for the development of rapidly progressive, proteinuric nephropathy. Despite the known genetic cause, therapies targeting proteinuric kidney disease in persons with two APOL1 variants (G1 or G2) are lacking. METHODS: We used tetracycline-inducible APOL1 human embryonic kidney (HEK293) cells to assess the ability of a small-molecule compound, inaxaplin, to inhibit APOL1 channel function. An APOL1 G2-homologous transgenic mouse model of proteinuric kidney disease was used to assess inaxaplin treatment for proteinuria. We then conducted a single-group, open-label, phase 2a clinical study in which inaxaplin was administered to participants who had two APOL1 variants, biopsy-proven focal segmental glomerulosclerosis, and proteinuria (urinary protein-to-creatinine ratio of ≥0.7 to <10 [with protein and creatinine both measured in grams] and an estimated glomerular filtration rate of ≥27 ml per minute per 1.73 m2 of body-surface area). Participants received inaxaplin daily for 13 weeks (15 mg for 2 weeks and 45 mg for 11 weeks) along with standard care. The primary outcome was the percent change from the baseline urinary protein-to-creatinine ratio at week 13 in participants who had at least 80% adherence to inaxaplin therapy. Safety was also assessed. RESULTS: In preclinical studies, inaxaplin selectively inhibited APOL1 channel function in vitro and reduced proteinuria in the mouse model. Sixteen participants were enrolled in the phase 2a study. Among the 13 participants who were treated with inaxaplin and met the adherence threshold, the mean change from the baseline urinary protein-to-creatinine ratio at week 13 was -47.6% (95% confidence interval, -60.0 to -31.3). In an analysis that included all the participants regardless of adherence to inaxaplin therapy, reductions similar to those in the primary analysis were observed in all but 1 participant. Adverse events were mild or moderate in severity; none led to study discontinuation. CONCLUSIONS: Targeted inhibition of APOL1 channel function with inaxaplin reduced proteinuria in participants with two APOL1 variants and focal segmental glomerulosclerosis. (Funded by Vertex Pharmaceuticals; VX19-147-101 ClinicalTrials.gov number, NCT04340362.).

A tail of two sites: a bipartite mechanism for recognition of notch ligands by mind bomb E3 ligases.

Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular, and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C terminus. Together, these studies provide insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential target for therapeutic modulation of Notch signal transduction in disease.

Damage Mechanics of Biological Tissues in Relation to Viscoelasticity.

This study examines the theoretical foundations for the damage mechanics of biological tissues in relation to viscoelasticity. Its primary goal is to provide a mechanistic understanding of well-known experimental observations in biomechanics, which show that the ultimate tensile strength of viscoelastic biological tissues typically increases with increasing strain rate. The basic premise of this framework is that tissue damage occurs when strong bonds, such as covalent bonds in the solid matrix of a biological tissue, break in response to loading. This type of failure is described as elastic damage, under the idealizing assumption that strong bonds behave elastically. Viscoelasticity arises from three types of dissipative mechanisms: (1) Friction between molecules of the same species, which is represented by the tissue viscosity. (2) Friction between fluid and solid constituents of a porous medium, which is represented by the tissue hydraulic permeability. (3) Dissipative reactions arising from weak bonds breaking in response to loading, and reforming in a stress-free state, such as hydrogen bonds and other weak electrostatic bonds. When a viscoelastic tissue is subjected to loading, some of that load may be temporarily supported by those frictional and weak bond forces, reducing the amount of load supported by elastic strong bonds and thus, the extent of elastic damage sustained by those bonds. This protective effect depends on the characteristic time response of viscoelastic mechanisms in relation to the loading history. This study formalizes these concepts by presenting general equations that can model the damage mechanics of viscoelastic tissues.

Continuum Thermodynamics of Constrained Reactive Mixtures.

Mixture theory models continua consisting of multiple constituents with independent motions. In constrained mixtures, all constituents share the same velocity but they may have different reference configurations. The theory of constrained reactive mixtures was formulated to analyze growth and remodeling in living biological tissues. It can also reproduce and extend classical frameworks of damage mechanics and viscoelasticity under isothermal conditions, when modeling bonds that can break and reform. This study focuses on establishing the thermodynamic foundations of constrained reactive mixtures under more general conditions, for arbitrary reactive processes where temperature varies in time and space. By incorporating general expressions for reaction kinetics, it is shown that the residual dissipation statement of the Clausius-Duhem inequality must include a reactive power density, while the axiom of energy balance must include a reactive heat supply density. Both of these functions are proportional to the molar production rate of a reaction, and they depend on the chemical potentials of the mixture constituents. We present novel formulas for the classical thermodynamic concepts of energy of formation and heat of reaction, making it possible to evaluate the heat supply generated by reactive processes from the knowledge of the specific free energy of mixture constituents as well as the reaction rate. We illustrate these novel concepts with mixtures of ideal gases, and isothermal reactive damage mechanics and viscoelasticity, as well as reactive thermoelasticity. This framework facilitates the analysis of reactive tissue biomechanics and physiological and biomedical engineering processes where temperature variations cannot be neglected.

A Finite Element Algorithm for Large Deformation Biphasic Frictional Contact Between Porous-Permeable Hydrated Soft Tissues.

The frictional response of porous and permeable hydrated biological tissues such as articular cartilage is significantly dependent on interstitial fluid pressurization. To model this response, it is common to represent such tissues as biphasic materials, consisting of a binary mixture of a porous solid matrix and an interstitial fluid. However, no computational algorithms currently exist in either commercial or open-source software that can model frictional contact between such materials. Therefore, this study formulates and implements a finite element algorithm for large deformation biphasic frictional contact in the open-source finite element software FEBio. This algorithm relies on a local form of a biphasic friction model that has been previously validated against experiments, and implements the model into our recently-developed surface-to-surface (STS) contact algorithm. Contact constraints, including those specific to pressurized porous media, are enforced with the penalty method regularized with an active-passive augmented Lagrangian scheme. Numerical difficulties specific to challenging finite deformation biphasic contact problems are overcome with novel smoothing schemes for fluid pressures and Lagrange multipliers. Implementation accuracy is verified against semi-analytical solutions for biphasic frictional contact, with extensive validation performed using canonical cartilage friction experiments from prior literature. Essential details of the formulation are provided in this paper, and the source code of this biphasic frictional contact algorithm is made available to the general public.

On the use of constrained reactive mixtures of solids to model finite deformation isothermal elastoplasticity and elastoplastic damage mechanics.

This study presents a framework for plasticity and elastoplastic damage mechanics by treating materials as reactive solids whose internal composition evolves in response to applied loading. Using the framework of constrained reactive mixtures, plastic deformation is accounted for by allowing loaded bonds within the material to break and reform in a stressed state. Bonds which break and reform represent a new generation with a new reference configuration, which is time-invariant and provided by constitutive assumption. The constitutive relation for the reference configuration of each generation may depend on the selection of a suitable yield measure. The choice of this measure and the resulting plastic flow conditions are constrained by the Clausius-Duhem inequality. We show that this framework remains consistent with classical plasticity approaches and principles. Verification of this reactive plasticity framework, which is implemented in the open source FEBio finite element software (febio.org), is performed against standard 2D and 3D benchmark problems. Damage is incorporated into this reactive framework by allowing loaded bonds to break permanently according to a suitable damage measure, where broken bonds can no longer store free energy. Validation is also demonstrated against experimental data for problems involving plasticity and plastic damage. This study demonstrates that it is possible to formulate simple elastoplasticity and elastoplastic damage models within a consistent framework which uses measures of material mass composition as theoretically observable state variables. This theoretical frame can be expanded in scope to account for more complex behaviors.

Direct Osmotic Pressure Measurements in Articular Cartilage Demonstrate Nonideal and Concentration-Dependent Phenomena.

The osmotic pressure in articular cartilage serves an important mechanical function in healthy tissue. Its magnitude is thought to play a role in advancing osteoarthritis. The aims of this study were to: (1) isolate and quantify the magnitude of cartilage swelling pressure in situ; and (2) identify the effect of salt concentration on material parameters. Confined compression stress-relaxation testing was performed on 18 immature bovine and six mature human cartilage samples in solutions of varying osmolarities. Direct measurements of osmotic pressure revealed nonideal and concentration-dependent osmotic behavior, with magnitudes approximately 1/3 those predicted by ideal Donnan law. A modified Donnan constitutive behavior was able to capture the aggregate behavior of all samples with a single adjustable parameter. Results of curve-fitting transient stress-relaxation data with triphasic theory in febio demonstrated concentration-dependent material properties. The aggregate modulus HA increased threefold as the external concentration decreased from hypertonic 2 M to hypotonic 0.001 M NaCl (bovine: HA=0.420±0.109 MPa to 1.266±0.438 MPa; human: HA=0.499±0.208 MPa to 1.597±0.455 MPa), within a triphasic theory inclusive of osmotic effects. This study provides a novel and simple analytical model for cartilage osmotic pressure which may be used in computational simulations, validated with direct in situ measurements. A key finding is the simultaneous existence of Donnan osmotic and Poisson-Boltzmann electrostatic interactions within cartilage.

A Surface-to-Surface Finite Element Algorithm for Large Deformation Frictional Contact in febio.

This study formulates a finite element algorithm for frictional contact of solid materials, accommodating finite deformation and sliding. The algorithm uses a penalty method regularized with an augmented Lagrangian scheme to enforce contact constraints in a nonmortar surface-to-surface approach. Use of a novel kinematical approach to contact detection and enforcement of frictional constraints allows solution of complex problems previously requiring mortar methods or contact smoothing algorithms. Patch tests are satisfied to a high degree of accuracy with a single-pass penalty method, ensuring formulation errors do not affect the solution. The accuracy of the implementation is verified with Hertzian contact, and illustrations demonstrating the ability to handle large deformations and sliding are presented and validated against prior literature. A biomechanically relevant example addressing finger friction during grasping demonstrates the utility of the proposed algorithm. The algorithm is implemented in the open source software febio, and the source code is made available to the general public.

Structure of human POFUT1, its requirement in ligand-independent oncogenic Notch signaling, and functional effects of Dowling-Degos mutations.

Protein O-fucosyltransferase-1 (POFUT1), which transfers fucose residues to acceptor sites on serine and threonine residues of epidermal growth factor-like repeats of recipient proteins, is essential for Notch signal transduction in mammals. Here, we examine the consequences of POFUT1 loss on the oncogenic signaling associated with certain leukemia-associated mutations of human Notch1, report the structures of human POFUT1 in free and GDP-fucose bound states, and assess the effects of Dowling-Degos mutations on human POFUT1 function. CRISPR-mediated knockout of POFUT1 in U2OS cells suppresses both normal Notch1 signaling, and the ligand-independent signaling associated with leukemogenic mutations of Notch1. Normal and oncogenic signaling are rescued by wild-type POFUT1 but rescue is impaired by an active-site R240A mutation. The overall structure of the human enzyme closely resembles that of the Caenorhabditis elegans protein, with an overall backbone RMSD of 0.93 Å, despite primary sequence identity of only 39% in the mature protein. GDP-fucose binding to the human enzyme induces limited backbone conformational movement, though the side chains of R43 and D244 reorient to make direct contact with the fucose moiety in the complex. The reported Dowling-Degos mutations of POFUT1, except for M262T, fail to rescue Notch1 signaling efficiently in the CRISPR-engineered POFUT1-/- background. Together, these studies identify POFUT1 as a potential target for cancers driven by Notch1 mutations and provide a structural roadmap for its inhibition.

Star test image-sampling polarimeter.

We present an analysis and test of an image sampling polarimeter based on the concept of Star Test Polarimetry first introduced by Ramkhalawon. The method makes use of a stress engineered optical element (SEO) placed in the pupil plane of an optical system to induce a polarization dependent point spread function (PSF) at the detector. We describe the calibration requirements of the polarimeter and introduce a new algorithm that can robustly extract the Stokes parameters in a single irradiance measurement. By acquiring statistics on the sampled Stokes parameters of a uniformly illuminated pinhole array, we show that a single frame can provide a root mean square angular error of approximately 10 milliradians on the Poincaré sphere.

Modeling Inelastic Responses Using Constrained Reactive Mixtures.

This study reviews the progression of our research, from modeling growth theories for cartilage tissue engineering, to the formulation of constrained reactive mixture theories to model inelastic responses in any solid material, such as theories for damage mechanics, viscoelasticity, plasticity, and elasto-plastic damage. In this framework, multiple solid generations α can co-exist at any given time in the mixture. The oldest generation is denoted by α=s and is called the master generation, whose reference configuration Xs is observable. The solid generations α are all constrained to share the same velocity vs, but may have distinct reference configurations Xα. An important element of this formulation is that the time-invariant mapping Fαs=∂Xα/∂Xs between these reference configurations is a function of state, whose mathematical formulation is postulated by constitutive assumption. Thus, reference configurations Xα are not observable (α≠s). This formulation employs only observable state variables, such as the deformation gradient Fs of the master generation and the referential mass concentrations ρrα of each generation, in contrast to classical formulations of inelastic responses which rely on internal state variable theory, requiring evolution equations for those hidden variables. In constrained reactive mixtures, the evolution of the mass concentrations is governed by the axiom of mass balance, using constitutive models for the mass supply densities ρˆrα. Classical and constrained reactive mixture approaches share considerable mathematical analogies, as they both introduce a multiplicative decomposition of the deformation gradient, also requiring evolution equations to track some of the state variables. However, they also differ at a fundamental level, since one adopts only observable state variables while the other introduces hidden state variables. In summary, this review presents an alternative foundational approach to the modeling of inelastic responses in solids, grounded in the classical framework of mixture theory.

The Strain Response to Intraocular Pressure Decrease in the Lamina Cribrosa of Patients with Glaucoma.

OBJECTIVE: To measure biomechanical strains in the lamina cribrosa (LC) of living human eyes with intraocular pressure (IOP) lowering. DESIGN: Cohort study. PARTICIPANTS: Patients with glaucoma underwent imaging before and after laser suturelysis after trabeculectomy surgery (29 image pairs; 26 persons). INTERVENTION: Noninvasive imaging of the eye. MAIN OUTCOME MEASURES: Strains in optic nerve head tissue and changes in depths of the anterior border of the LC. RESULTS: Intraocular pressure decreases caused the LC to expand in thickness in the anterior-posterior strain (Ezz = 0.94 ± 1.2%; P = 0.00020) and contract in radius in the radial strain (Err = - 0.19 ± 0.33%; P = 0.0043). The mean LC depth did not significantly change with IOP lowering (1.33 ± 6.26 µm; P = 0.26). A larger IOP decrease produced a larger, more tensile Ezz (P < 0.0001), greater maximum principal strain (Emax; P < 0.0001), and greater maximum shear strain (Γmax; P < 0.0001). The average LC depth change was associated with the Γmax and radial-circumferential shear strain (Erθ; P < 0.02) but was not significantly related to tensile or compressive strains. An analysis by clock hour showed that in temporal clock hours 3 to 6, a more anterior LC movement was associated with a more positive Emax, and in clock hours 3, 5, and 6, it was associated with a more positive Γmax. At 10 o'clock, a more posterior LC movement was related to a more positive Emax (P < 0.004). Greater compliance (strain/ΔIOP) of Emax (P = 0.044), Γmax (P = 0.052), and Erθ (P = 0.018) was associated with a thinner retinal nerve fiber layer. Greater compliance of Emax (P = 0.041), Γmax (P = 0.021), Erθ (P = 0.024), and in-plane shear strain (Erz; P = 0.0069) was associated with more negative mean deviations. Greater compliance of Γmax (P = 0.055), Erθ (P = 0.040), and Erz (P = 0.015) was associated with lower visual field indices. CONCLUSIONS: With IOP lowering, the LC moves either into or out of the eye but, on average, expands in thickness and contracts in radius. Shear strains are nearly as substantial as in-plane strains. Biomechanical strains are more compliant in eyes with greater glaucoma damage. This work was registered at ClinicalTrials.gov as NCT03267849.

Superficial zone chondrocytes can get compacted under physiological loading: A multiscale finite element analysis.

Recent in vivo and in vitro studies have demonstrated that superficial zone (SZ) chondrocytes within articular layers of diarthrodial joints die under normal physiologic loading conditions. In order to further explore the implications of this observation in future investigations, we first needed to understand the mechanical environment of SZ chondrocytes that might cause them to die under physiological sliding contact conditions. In this study we performed a multiscale finite element analysis of articular contact to track the temporal evolution of a SZ chondrocyte's interstitial fluid pressure, hydraulic permeability, and volume under physiologic loading conditions. The effect of the pericellular matrix modulus and permeability was parametrically investigated. Results showed that SZ chondrocytes can lose ninety percent of their intracellular fluid after several hours of intermittent or continuous contact loading, resulting in a reduction of intracellular hydraulic permeability by more than three orders of magnitude. These findings are consistent with loss of cell viability due to the impediment of cellular metabolic pathways induced by the loss of fluid. They suggest that there is a simple mechanical explanation for the vulnerability of SZ chondrocytes to sustained physiological loading conditions. Future studies will focus on validating these specific findings experimentally. STATEMENT OF SIGNIFICANCE: As with any mechanical system, normal 'wear and tear' of cartilage tissue lining joints is expected. Yet incidences of osteoarthritis are uncommon in individuals younger than 45. This counter-intuitive observation suggests there must be an intrinsic repair mechanism compensating for this wear and tear over many decades of life. Recent experimental studies have shown superficial zone chondrocytes die under physiologic loading conditions, suggesting that this repair mechanism may involve cell replenishment. To better understand the mechanical environment of these cells, we performed a multiscale computational analysis of articular contact under loading. Results indicated that normal activities like walking or standing can induce significant loss of intracellular fluid volume, potentially hindering metabolic activity and fluid transport properties, and causing cell death.

Essential role of endocytosis of the type II transmembrane serine protease TMPRSS6 in regulating its functionality.

The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin- and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.

Immature bovine cartilage wear by fatigue failure and delamination.

This study investigated wear damage of immature bovine articular cartilage using reciprocal sliding of tibial cartilage strips against glass or cartilage. Experiments were conducted in physiological buffered saline (PBS) or mature bovine synovial fluid (SF). A total of 63 samples were tested, of which 47 exhibited wear damage due to delamination of the cartilage surface initiated in the middle zone, with no evidence of abrasive wear. There was no difference between the friction coefficient of damaged and undamaged samples, showing that delamination wear occurs even when friction remains low under a migrating contact area configuration. No difference was observed in the onset of damage or in the friction coefficient between samples tested in PBS or SF. The onset of damage occurred earlier when testing cartilage against glass versus cartilage against cartilage, supporting the hypothesis that delamination occurs due to fatigue failure of the collagen in the middle zone, since stiffer glass produces higher strains and tensile stresses under comparable loads. The findings of this study are novel because they establish that delamination of the articular surface, starting in the middle zone, may represent a primary mechanism of failure. Based on preliminary data, it is reasonable to hypothesize that delamination wear via subsurface fatigue failure is similarly the primary mechanism of human cartilage wear under normal loading conditions, albeit requiring far more cycles of loading than in immature bovine cartilage.

Tissue engineered autologous cartilage-bone grafts for temporomandibular joint regeneration.

Joint disorders can be detrimental to quality of life. There is an unmet need for precise functional reconstruction of native-like cartilage and bone tissues in the craniofacial space and particularly for the temporomandibular joint (TMJ). Current surgical methods suffer from lack of precision and comorbidities and frequently involve multiple operations. Studies have sought to improve craniofacial bone grafts without addressing the cartilage, which is essential to TMJ function. For the human-sized TMJ in the Yucatan minipig model, we engineered autologous, biologically, and anatomically matched cartilage-bone grafts for repairing the ramus-condyle unit (RCU), a geometrically intricate structure subjected to complex loading forces. Using image-guided micromilling, anatomically precise scaffolds were created from decellularized bone matrix and infused with autologous adipose-derived chondrogenic and osteogenic progenitor cells. The resulting constructs were cultured in a dual perfusion bioreactor for 5 weeks before implantation. Six months after implantation, the bioengineered RCUs maintained their predefined anatomical structure and regenerated full-thickness, stratified, and mechanically robust cartilage over the underlying bone, to a greater extent than either autologous bone-only engineered grafts or acellular scaffolds. Tracking of implanted cells and parallel bioreactor studies enabled additional insights into the progression of cartilage and bone regeneration. This study demonstrates the feasibility of TMJ regeneration using anatomically precise, autologous, living cartilage-bone grafts for functional, personalized total joint replacement. Inclusion of the adjacent tissues such as soft connective tissues and the TMJ disc could further extend the functional integration of engineered RCUs with the host.

A new inhibitor of the ß-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, ß-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of ß-arrestin recruitment to the receptor and ß-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between ß-arrestin and the ß2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/ß-arrestin complexes. This selective ß-arrestin/ß2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical ß2-adrenergic (ß2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect ß-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and ß2AR, supporting the concept of ß-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

Determine the equilibrium mechanical properties of articular cartilage from the short-term indentation response.

Indentation testing is widely used to evaluate the mechanical properties of articular cartilage. However, most curve-fitting solutions for indentation analysis require the deformation data of cartilage at the equilibrium state, which often takes the tissue hours to reach. The lengthy testing time reduces the efficiency of indentation, increases the chance of tissue deterioration, and prevents in vivo applications. To overcome these limitations, a novel technique based on principal component analysis (PCA) was developed in this study, which can predict the full indentation creep curve based on the first few minutes' deformation history and the principal components. The accuracy of this technique was confirmed using the indentation data from 40 temporomandibular joint condylar cartilage samples and 17 bovine knee joint samples. The mechanical properties determined by biphasic curve-fitting using predicted and experimental data are in good agreement, with the difference between the two less than 5%. For TMJ and knee cartilages, it is found that any number of full tests beyond eight will not lead to any increase larger than 1% in the accuracy, indicating a low sample number required for prediction. In addition, the principal components of indentation creep curves are consistent for the same type of cartilage tested with identical protocols, but significantly different between two distinct cartilages. Therefore PCA may also represent a new method to compare the mechanical behaviors of different cartilages, as it avoids the assumptions associated with mechanical constitutive models and relies purely on the experimental data.