Differential sensing with arrays of de novo designed peptide assemblies.

Differential sensing attempts to mimic the mammalian senses of smell and taste to identify analytes and complex mixtures. In place of hundreds of complex, membrane-bound G-protein coupled receptors, differential sensors employ arrays of small molecules. Here we show that arrays of computationally designed de novo peptides provide alternative synthetic receptors for differential sensing. We use self-assembling α-helical barrels (αHBs) with central channels that can be altered predictably to vary their sizes, shapes and chemistries. The channels accommodate environment-sensitive dyes that fluoresce upon binding. Challenging arrays of dye-loaded barrels with analytes causes differential fluorophore displacement. The resulting fluorimetric fingerprints are used to train machine-learning models that relate the patterns to the analytes. We show that this system discriminates between a range of biomolecules, drink, and diagnostically relevant biological samples. As αHBs are robust and chemically diverse, the system has potential to sense many analytes in various settings.

Molecular architecture of the autoinhibited kinesin-1 lambda particle.

Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the α-helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative-stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1 in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain-light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drives kinesin-1 activation.

De novo designed peptides for cellular delivery and subcellular localisation.

Increasingly, it is possible to design peptide and protein assemblies de novo from first principles or computationally. This approach provides new routes to functional synthetic polypeptides, including designs to target and bind proteins of interest. Much of this work has been developed in vitro. Therefore, a challenge is to deliver de novo polypeptides efficiently to sites of action within cells. Here we describe the design, characterisation, intracellular delivery, and subcellular localisation of a de novo synthetic peptide system. This system comprises a dual-function basic peptide, programmed both for cell penetration and target binding, and a complementary acidic peptide that can be fused to proteins of interest and introduced into cells using synthetic DNA. The designs are characterised in vitro using biophysical methods and X-ray crystallography. The utility of the system for delivery into mammalian cells and subcellular targeting is demonstrated by marking organelles and actively engaging functional protein complexes.

Kinesin-1 captures RNA cargo in its adaptable coils.

The prototypic and ubiquitous microtubule motor, kinesin-1, uses a variety of adaptor proteins to facilitate the selective transport of diverse cargo within the cell. These cargo adaptors bind to the motor complex through interactions with the kinesin light or heavy chains (KLCs or KHCs). In this issue of Genes & Development, Dimitrova-Paternoga et al. (pp. 976-991) present the first structural characterization of a KHC-cargo adaptor interface. They describe an antiparallel heterotrimeric coiled-coil complex between the carboxy tail of KHC and Tm1-I/C (aTm1), the atypical tropomyosin that is important for oskar mRNA transport in Drosophila oocytes. This interaction enhances direct binding between KHC and RNA. Their findings demonstrate the structural plasticity of the KHC tail as a platform for protein-protein interactions and reveal how a cargo adaptor protein can modify a motor-RNA interface to promote transport.

Molecular mechanism for kinesin-1 direct membrane recognition.

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxyl-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical, and cell biology approaches, we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a subset of long kinesin light-chain paralogs and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids, and it contributes to kinesin-1-dependent lysosome positioning, a canonical activity that, until now, has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a protein-lipid coincidence detection framework for kinesin-1-mediated organelle transport.

Fragment-linking peptide design yields a high-affinity ligand for microtubule-based transport.

Synthetic peptides are attractive candidates to manipulate protein-protein interactions inside the cell as they mimic natural interactions to compete for binding. However, protein-peptide interactions are often dynamic and weak. A challenge is to design peptides that make improved interactions with the target. Here, we devise a fragment-linking strategy-"mash-up" design-to deliver a high-affinity ligand, KinTag, for the kinesin-1 motor. Using structural insights from natural micromolar-affinity cargo-adaptor ligands, we have identified and combined key binding features in a single, high-affinity ligand. An X-ray crystal structure demonstrates interactions as designed and reveals only a modest increase in interface area. Moreover, when genetically encoded, KinTag promotes transport of lysosomes with higher efficiency than natural sequences, revealing a direct link between motor-adaptor binding affinity and organelle transport. Together, these data demonstrate a fragment-linking strategy for peptide design and its application in a synthetic motor ligand to direct cellular cargo transport.

Motor-cargo adaptors at the organelle-cytoskeleton interface.

Cytoskeletal motors of the dynein, kinesin and myosin superfamilies maintain and adapt subcellular organelle organization to meet functional demands and support the vesicular transport of material between organelles. These motors require the capacity to specifically recognize the vesicle/organelle to be transported and are capable of selective recognition of multiple cargo. Recent studies have begun to uncover the molecular basis for motor recruitment and have highlighted the role of organelle-associated 'cargo-adaptor' proteins in cellular transport. These adaptors possess sequences and/or structural features that enable both motor recruitment and activation from regulated, inactive, states to enable motility on the cytoskeleton. Motor-cargo adaptor interactions define a key organelle-cytoskeleton interface, acting as crucial regulatory hubs to enable the cell to finely control membrane trafficking and organelle dynamics. Understanding the molecular basis of these interactions may offer new opportunities to control and manipulate cytoskeletal and organelle dynamics for the development of new research tools and potentially therapeutics.

BioCartilage Improves Cartilage Repair Compared With Microfracture Alone in an Equine Model of Full-Thickness Cartilage Loss.

BACKGROUND: Microfracture (MFx) remains a dominant treatment strategy for symptomatic articular cartilage defects. Biologic scaffold adjuncts, such as particulated allograft articular cartilage (BioCartilage) combined with platelet-rich plasma (PRP), offer promise in improving clinical outcomes as an adjunct to MFx. PURPOSE: To evaluate the safety, biocompatibility, and efficacy of BioCartilage and PRP for cartilage repair in a preclinical equine model of full-thickness articular cartilage loss. STUDY DESIGN: Controlled laboratory study. METHODS: Two 10-mm-diameter full-thickness cartilage defects were created in 5 horses in the trochlear ridge of both knees: one proximal (high load) and another distal (low load). Complete blood counts were performed on each peripheral blood and resultant PRP sample. In each horse, one knee received MFx with BioCartilage + PRP, and the other knee received MFx alone. Horses were euthanized at 13 months. Outcomes were assessed with serial arthroscopy, magnetic resonance imaging (MRI), micro-computed tomography (micro-CT), and histology. Statistics were performed using a mixed-effects model with response variable contrasts. RESULTS: No complications occurred. PRP generated in all subjects yielded an increase in platelet fold of 3.8 ± 4.7. Leukocyte concentration decreased in PRP samples by an average fold change of 5 ± 0.1. The overall International Cartilage Repair Society repair score in both the proximal and distal defects was significantly higher (better) in the BioCartilage group compared with MFx (proximal BioCartilage: 7.4 ± 0.51, MFx 4.8 ± 0.1, P = .041; distal BioCartilage: 5.6 ± 0.98, MFx 2.6 ± 1.5, P = .022). BioCartilage-treated proximal defects demonstrated improved histologic scores for repair-host integration (BioCartilage, 96 ± 9; MFx, 68 ± 18; P = .02), base integration (BioCartilage, 100 ± 0; MFx, 70 ± 37; P = .04), and formation of collagen type II (BioCartilage, 82 ± 8; MFx, 58 ± 11; P = .05) compared with the positive control. On MRI, T2 relaxation time was significantly shorter (better) in the superficial region of BioCartilage-treated distal defects compared with MFx (P = .05). There were no significant differences between BioCartilage and MFx on micro-CT analysis. CONCLUSION: BioCartilage with PRP safely improved cartilage repair compared with MFx alone in an equine model of articular cartilage defects up to 13 months after implantation. CLINICAL RELEVANCE: The 1-year results of BioCartilage + PRP suggest that homologous allograft tissue provides a safe and effective augmentation of traditional MFx.

Leukocyte-Reduced Platelet-Rich Plasma Normalizes Matrix Metabolism in Torn Human Rotator Cuff Tendons.

BACKGROUND: The optimal platelet-rich plasma (PRP) for treatment of supraspinatus tendinopathy has not been determined. PURPOSE: To evaluate the effect of low- versus high-leukocyte concentrated PRP products on catabolic and anabolic mediators of matrix metabolism in diseased rotator cuff tendons. STUDY DESIGN: Controlled laboratory study. METHODS: Diseased supraspinatus tendons were treated with PRP made by use of 2 commercial systems: Arthrex Autologous Conditioned Plasma Double Syringe System (L(lo) PRP) and Biomet GPS III Mini Platelet Concentrate System (L(hi) PRP). Tendon explants were placed in 6-well plates and cultured in L(lo) PRP, L(hi) PRP, or control media (Dulbecco's Modified Eagle Medium + 10% fetal bovine serum) for 96 hours. Tendons were processed for hematoxylin-eosin histologic results and were scored with the modified Bonar scale. Group 1 tendons were defined as moderate tendinopathy (Bonar score <3); group 2 tendons were assessed as severely affected (Bonar score = 3). Transforming growth factor ß-1 (TGFß-1), interleukin-1ß (IL-1ß), interleukin-1 receptor antagonist (IL-1Ra), interleukin-6 (IL-6), interleukin-8 (IL-8), and matrix metalloproteinase-9 (MMP-9) concentrations in PRP media were measured by use of enzyme-linked immunosorbent assay after 96 hours of culture with diseased tendon. Tendon messenger RNA expression of collagen type I (COL1A1), collagen type III (COL3A1), cartilage oligomeric matrix protein (COMP), MMP-9, MMP-13, and IL-1ß was measured with real-time quantitative polymerase chain reaction. RESULTS: Leukocytes and platelets were significantly more concentrated in L(hi) PRP compared with L(lo) PRP. Increased IL-1ß was present in L(hi) PRP after culture with group 1 tendons. IL-6 was increased in L(hi) PRP after culture with group 2 tendons. Both TGFß-1 and MMP-9 were increased in L(hi) PRP after culture with either tendon group. In L(lo) PRP cultures, IL-1Ra:IL-1ß in PRP used as media and COL1A1:COL3A1 gene expression were increased for group 1 tendon cultures. Gene expression of MMP-9 and IL-1ß was increased in group 2 tendons cultured in L(lo) PRP. There was no significant difference in the expression of MMP-13 or COMP in either group of tendons cultured in L(lo) PRP or L(hi) PRP. CONCLUSION: L(lo) PRP promotes normal collagen matrix synthesis and decreases cytokines associated with matrix degradation and inflammation to a greater extent than does L(hi) PRP in moderately degenerative tendons. In severely degenerative tendons, neither PRP preparation enhanced matrix synthesis. CLINICAL RELEVANCE: L(lo) PRP may promote healing in moderately degenerative rotator cuff tendons.

Induced pluripotent stem cells have similar immunogenic and more potent immunomodulatory properties compared with bone marrow-derived stromal cells in vitro.

AIM: To evaluate the in vitro immunogenic and immunomodulatory properties of induced pluripotent stem cells (iPSCs) compared with bone marrow-derived mesenchymal stromal cells (MSCs). MATERIALS & METHODS: Mouse embryonic fibroblasts (MEFs) were isolated from C3HeB/FeJ and C57BL/6J mice, and reprogrammed to generate iPSCs. Mixed leukocyte reactions were performed using MHC-matched and -mismatched responder leukocytes and stimulator leukocytes, iPSCs or MSCs. To assess immunogenic potential, iPSCs and MSCs were used as stimulator cells for responder leukocytes. To assess immunomodulatory properties, iPSCs and MSCs were cultured in the presence of stimulator and responder leukocytes. MEFs were used as a control. RESULTS: iPSCs had similar immunogenic properties but more potent immunomodulatory effects than MSCs. Co-culture of MHC-mismatched leukocytes with MHC-matched iPSCs resulted in significantly less responder T-cell proliferation than observed for MHC-mismatched leukocytes alone and at more responder leukocyte concentrations than with MSCs. In addition, MHC-mismatched iPSCs significantly reduced responder T-cell proliferation when co-cultured with MHC-mismatched leukocytes, while MHC-mismatched MSCs did not. CONCLUSION: These results provide important information when considering the use of iPSCs in place of MSCs in both regenerative and transplantation medicine.