PIEZO1 is downregulated in glenohumeral chondrocytes in early cuff tear arthropathy following a massive rotator cuff tear in a mouse model

Introduction: A massive rotator cuff tear (RCT) leads to glenohumeral joint destabilization and characteristic degenerative changes, termed cuff tear arthropathy (CTA). Understanding the response of articular cartilage to a massive RCT will elucidate opportunities to promote homeostasis following restoration of joint biomechanics with rotator cuff repair. Mechanically activated calcium-permeating channels, in part, modulate the response of distal femoral chondrocytes in the knee against injurious loading and inflammation. The objective of this study was to investigate PIEZO1-mediated mechanotransduction of glenohumeral articular chondrocytes in the altered biomechanical environment following RCT to ultimately identify potential therapeutic targets to attenuate cartilage degeneration after rotator cuff repair.Methods: First, we quantified mechanical susceptibility of chondrocytes in mouse humeral head cartilage ex vivo with treatments of specific chemical agonists targeting PIEZO1 and TRPV4 channels. Second, using a massive RCT mouse model, chondrocytes were assessed for mechano-vulnerability, PIEZO1 expression, and calcium signaling activity 14-week post-injury, an early stage of CTA.Results: In native humeral head chondrocytes, chemical activation of PIEZO1 (Yoda1) significantly increased chondrocyte mechanical susceptibility against impact loads, while TRPV4 activation (GSK101) significantly decreased impact-induced chondrocyte death. A massive RCT caused morphologic and histologic changes to the glenohumeral joint with decreased sphericity and characteristic bone bruising of the posterior superior quadrant of the humeral head. At early CTA, chondrocytes in RCT limbs exhibit a significantly decreased functional expression of PIEZO1 compared with uninjured or sham controls.Discussion: In contrast to the hypothesis, PIEZO1 expression and activity is not increased, but rather downregulated, after massive RCT at the early stage of cuff tear arthropathy. These results may be secondary to the decreased axial loading after glenohumeral joint decoupling in RCT limbs

Small molecule dual-inhibitors of TRPV4 and TRPA1 for attenuation of inflammation and pain

TRPV4 ion channels represent osmo-mechano-TRP channels with pleiotropic function and wide-spread expression. One of the critical functions of TRPV4 in this spectrum is its involvement in pain and inflammation. However, few small-molecule inhibitors of TRPV4 are available. Here we developed TRPV4-inhibitory molecules based on modifications of a known TRPV4-selective tool-compound, GSK205. We not only increased TRPV4-inhibitory potency, but surprisingly also generated two compounds that potently co-inhibit TRPA1, known to function as chemical sensor of noxious and irritant signaling. We demonstrate TRPV4 inhibition by these compounds in primary cells with known TRPV4 expression - articular chondrocytes and astrocytes. Importantly, our novel compounds attenuate pain behavior in a trigeminal irritant pain model that is known to rely on TRPV4 and TRPA1. Furthermore, our novel dual-channel blocker inhibited inflammation and pain-associated behavior in a model of acute pancreatitis - known to also rely on TRPV4 and TRPA1. Our results illustrate proof of a novel concept inherent in our prototype compounds of a drug that targets two functionally-related TRP channels, and thus can be used to combat isoforms of pain and inflammation in-vivo that involve more than one TRP channel. This approach could provide a novel paradigm for treating other relevant health conditions

Nanomechanics of Ankyrin Repeat Proteins

<p>Ankyrin repeats (ARs) are polypeptide motifs identified in thousands of proteins. Many AR proteins play a function as scaffolds in protein-protein interactions which may require specific mechanical properties. Also, a number of AR proteins have been proposed to mediate mechanotransduction in a variety of different functional settings. The folding and stability of a number of AR proteins have been studied in detail by chemical and temperature denaturation experiments, yet the mechanic of AR proteins remain largely unknown. In this dissertation, we have researched the mechanical properties of AR proteins by using protein engineering and a combination of atomic force microscopy (AFM)-based single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations. Three kinds of AR proteins were investigated: NI6C (synthetic AR protein), D34 (of ankyrin-R) and gankyrin (oncoprotein). While the main focus of this research was to characterize the response of AR proteins to mechanical forces, our results extended beyond the protein nanomechanics to the understanding of protein folding mechanisms.</p>Dissertatio

Mechanical Unfolding of an Ankyrin Repeat Protein

Ankryin repeat proteins comprise tandem arrays of a 33-residue, predominantly α-helical motif that stacks roughly linearly to produce elongated and superhelical structures. They function as scaffolds mediating a diverse range of protein-protein interactions, and some have been proposed to play a role in mechanical signal transduction processes in the cell. Here we use atomic force microscopy and molecular-dynamics simulations to investigate the natural 7-ankyrin repeat protein gankyrin. We find that gankyrin unfolds under force via multiple distinct pathways. The reactions do not proceed in a cooperative manner, nor do they always involve fully stepwise unfolding of one repeat at a time. The peeling away of half an ankyrin repeat, or one or more ankyrin repeats, occurs at low forces; however, intermediate species are formed that are resistant to high forces, and the simulations indicate that in some instances they are stabilized by nonnative interactions. The unfolding of individual ankyrin repeats generates a refolding force, a feature that may be more easily detected in these proteins than in globular proteins because the refolding of a repeat involves a short contraction distance and incurs a low entropic cost. We discuss the origins of the differences between the force- and chemical-induced unfolding pathways of ankyrin repeat proteins, as well as the differences between the mechanics of natural occurring ankyrin repeat proteins and those of designed consensus ankyin repeat and globular proteins

Minimizing Pulling Geometry Errors in Atomic Force Microscope Single Molecule Force Spectroscopy

In atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS), it is assumed that the pulling angle is negligible and that the force applied to the molecule is equivalent to the force measured by the instrument. Recent studies, however, have indicated that the pulling geometry errors can drastically alter the measured force-extension relationship of molecules. Here we describe a software-based alignment method that repositions the cantilever such that it is located directly above the molecule's substrate attachment site. By aligning the applied force with the measurement axis, the molecule is no longer undergoing combined loading, and the full force can be measured by the cantilever. Simulations and experimental results verify the ability of the alignment program to minimize pulling geometry errors in AFM-SMFS studies

Full Reconstruction of a Vectorial Protein Folding Pathway by Atomic Force Microscopy and Molecular Dynamics Simulations*

During co-translational folding, the nascent polypeptide chain is extruded sequentially from the ribosome exit tunnel and, under severe conformational constraints, is dictated by its one-dimensional geometry. How do such vectorial constraints impact the folding pathway? Here, we combine single-molecule atomic force spectroscopy and steered molecular dynamics simulations to examine protein folding in the presence of one-dimensional constraints that are similar to those imposed on the nascent polypeptide chain. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. We show that fully stretched and then relaxed NI6C starts folding by the formation of local secondary structures, followed by the nucleation of three N-terminal repeats. This rate-limiting step is then followed by the vectorial and sequential folding of the remaining repeats. However, after partial unfolding, when allowed to refold, the C-terminal repeats successively regain structures without any nucleation step by using the intact N-terminal repeats as a template. These results suggest a pathway for the co-translational folding of repeat proteins and have implications for mechanotransduction