Quality Control of Procollagen in Cells.

Collagen is the most abundant protein in mammals. A unique feature of collagen is its triple-helical structure formed by the Gly-Xaa-Yaa repeats. Three single chains of procollagen make a trimer, and the triple-helical structure is then folded in the endoplasmic reticulum (ER). This unique structure is essential for collagen's functions in vivo, including imparting bone strength, allowing signal transduction, and forming basement membranes. The triple-helical structure of procollagen is stabilized by posttranslational modifications and intermolecular interactions, but collagen is labile even at normal body temperature. Heat shock protein 47 (Hsp47) is a collagen-specific molecular chaperone residing in the ER that plays a pivotal role in collagen biosynthesis and quality control of procollagen in the ER. Mutations that affect the triple-helical structure or result in loss of Hsp47 activity cause the destabilization of procollagen, which is then degraded by autophagy. In this review, we present the current state of the field regarding quality control of procollagen.

Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver.

Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic ß-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-ß1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.

Cartilage-like protein hydrogels engineered via entanglement.

Load-bearing tissues, such as muscle and cartilage, exhibit high elasticity, high toughness and fast recovery, but have different stiffness (with cartilage being significantly stiffer than muscle)1-8. Muscle achieves its toughness through finely controlled forced domain unfolding-refolding in the muscle protein titin, whereas articular cartilage achieves its high stiffness and toughness through an entangled network comprising collagen and proteoglycans. Advancements in protein mechanics and engineering have made it possible to engineer titin-mimetic elastomeric proteins and soft protein biomaterials thereof to mimic the passive elasticity of muscle9-11. However, it is more challenging to engineer highly stiff and tough protein biomaterials to mimic stiff tissues such as cartilage, or develop stiff synthetic matrices for cartilage stem and progenitor cell differentiation12. Here we report the use of chain entanglements to significantly stiffen protein-based hydrogels without compromising their toughness. By introducing chain entanglements13 into the hydrogel network made of folded elastomeric proteins, we are able to engineer highly stiff and tough protein hydrogels, which seamlessly combine mutually incompatible mechanical properties, including high stiffness, high toughness, fast recovery and ultrahigh compressive strength, effectively converting soft protein biomaterials into stiff and tough materials exhibiting mechanical properties close to those of cartilage. Our study provides a general route towards engineering protein-based, stiff and tough biomaterials, which will find applications in biomedical engineering, such as osteochondral defect repair, and material sciences and engineering.

Cotranslational prolyl hydroxylation is essential for flavivirus biogenesis.

Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.

A conserved strategy to attack collagen: The activator domain in bacterial collagenases unwinds triple-helical collagen.

Bacterial collagenases are important virulence factors, secreted by several pathogenic Clostridium, Bacillus, Spirochaetes, and Vibrio species. Yet, the mechanism by which these enzymes cleave collagen is not well understood. Based on biochemical and mutational studies we reveal that collagenase G (ColG) from Hathewaya histolytica recognizes and processes collagen substrates differently depending on their nature (fibrillar vs. soluble collagen); distinct dynamic interactions between the activator and peptidase domain are required based on the substrate type. Using biochemical and circular dichroism studies, we identify the presumed noncatalytic activator domain as the single-domain triple helicase that unwinds collagen locally, transiently, and reversibly.

Nanocolloidal hydrogel mimics the structure and nonlinear mechanical properties of biological fibrous networks.

Fibrous networks formed by biological polymers such as collagen or fibrin exhibit nonlinear mechanical behavior. They undergo strong stiffening in response to weak shear and elongational strains, but soften under compressional strain, in striking difference with the response to the deformation of flexible-strand networks formed by molecules. The nonlinear properties of fibrous networks are attributed to the mechanical asymmetry of the constituent filaments, for which a stretching modulus is significantly larger than the bending modulus. Studies of the nonlinear mechanical behavior are generally performed on hydrogels formed by biological polymers, which offers limited control over network architecture. Here, we report an engineered covalently cross-linked nanofibrillar hydrogel derived from cellulose nanocrystals and gelatin. The variation in hydrogel composition provided a broad-range change in its shear modulus. The hydrogel exhibited both shear-stiffening and compression-induced softening, in agreement with the predictions of the affine model. The threshold nonlinear stress and strain were universal for the hydrogels with different compositions, which suggested that nonlinear mechanical properties are general for networks formed by rigid filaments. The experimental results were in agreement with an affine model describing deformation of the network formed by rigid filaments. Our results lend insight into the structural features that govern the nonlinear biomechanics of fibrous networks and provide a platform for future studies of the biological impact of nonlinear mechanical properties.

From Collagen Mimetics to Collagen Hybridization and Back.

ConspectusFacilitated by the unique triple-helical protein structure, fibrous collagens, the principal proteins in animals, demonstrate a dual function of serving as building blocks for tissue scaffolds and as a bioactive material capable of swift renewal in response to environmental changes. While studies of triple-helical collagen mimetic peptides (CMPs) have been instrumental in understanding the molecular forces responsible for the folding and assembly of triple helices, as well as identifying bioactive regions of fibrous collagen molecules, single-strand CMPs that can specifically target and hybridize to denatured collagens (i.e., collagen hybridizing peptides, CHPs) have proven useful in identifying the remodeling activity of collagen-rich tissues related to development, homeostasis, and pathology. Efforts to improve the utility of CHPs have resulted in the development of new skeletal structures, such as dimeric and cyclic CHPs, as well as the incorporation of artificial amino acids, including fluorinated proline and N-substituted glycines (peptoid residues). In particular, dimeric CHPs were used to capture collagen fragments from biological fluid for biomarker study, and the introduction of peptoid-based collagen mimetics has sparked renewed interest in peptidomimetic research because peptoids enable a stable triple-helical structure and the presentation of an extensive array of side chain structures offering a versatile platform for the development of new collagen mimetics.This Account will cover the evolution of our research from CMPs as biomaterials to ongoing efforts in developing triple-helical peptides with practical theranostic potential in targeting denatured and damaged collagens. Our early efforts in functionalizing natural collagen scaffolds via noncovalent modifications led to the discovery of an entirely new use of CMPs. This discovery resulted in the development of CHPs that are now used by many different laboratories for the investigation of pathologies associated with changes in the structures of extracellular matrices including fibrosis, cancer, and mechanical damage to collagen-rich, load-bearing tissues. Here, we delve into the essential design features of CHPs contributing to their collagen binding properties and practical usage and explore the necessity for further mechanistic understanding of not only the binding processes (e.g., binding domain and stoichiometry of the hybridized complex) but also the biology of collagen degradation, from proteolytic digestion of fibrils to cellular processing of collagen fragments. We also discuss the strengths and weaknesses of peptoid-based triple-helical peptides as applied to collagen hybridization touching on thermodynamic and kinetic aspects of triple-helical folding. Finally, we highlight current limitations and future directions in the use of peptoid building blocks to develop bioactive collagen mimetics as new functional biomaterials.

Compressive instabilities enable cell-induced extreme densification patterns in the fibrous extracellular matrix: Discrete model predictions.

We present a new model and extensive computations that explain the dramatic remodelling undergone by a fibrous collagen extracellular matrix (ECM), when subjected to contractile mechanical forces from embedded cells or cell clusters. This remodelling creates complex patterns, comprising multiple narrow localised bands of severe densification and fiber alignment, extending far into the ECM, often joining distant cells or cell clusters (such as tumours). Most previous models cannot capture this behaviour, as they assume stable mechanical fiber response with stress an increasing function of fiber stretch, and a restriction to small displacements. Our fully nonlinear network model distinguishes between two types of single-fiber nonlinearity: fibers that undergo stable (supercritical) buckling (as in previous work) versus fibers that suffer unstable (subcritical) buckling collapse. The model allows unrestricted, arbitrarily large displacements (geometric nonlinearity). Our assumptions on single-fiber instability are supported by recent simulations and experiments on buckling of individual beams with a hierarchical microstructure, such as collagen fibers. We use simple scenarios to illustrate, for the first time, two distinct compressive-instability mechanisms at work in our model: unstable buckling collapse of single fibers, and snap-through of multiple-fiber groups. The latter is possible even when single fibers are stable. Through simulations of large fiber networks, we show how these instabilities lead to spatially extended patterns of densification, fiber alignment and ECM remodelling induced by cell contraction. Our model is simple, but describes a very complex, multi-stable energy landscape, using sophisticated numerical optimisation methods that overcome the difficulties caused by instabilities in large systems. Our work opens up new ways of understanding the unique biomechanics of fibrous-network ECM, by fully accounting for nonlinearity and associated loss of stability in fiber networks. Our results provide new insights on tumour invasion and metastasis.

Preparation and in vitro evaluation of cell adhesion and long-term proliferation of stem cells cultured on silibinin co-embedded PLGA/Collagen electrospun composite nanofibers.

The present research aims to evaluate the efficacy of Silibinin-loaded mesoporous silica nanoparticles (Sil@MSNs) immobilized into polylactic-co-glycolic acid/Collagen (PLGA/Col) nanofibers on the in vitro proliferation of adipose-derived stem cells (ASCs) and cellular senescence. Here, the fabricated electrospun PLGA/Col composite scaffolds were coated with Sil@MSNs and their physicochemical properties were examined by FTIR, FE-SEM, and TGA. The growth, viability and proliferation of ASCs were investigated using various biological assays including PicoGreen, MTT, and RT-PCR after 21 days. The proliferation and adhesion of ASCs were supported by the biological and mechanical characteristics of the Sil@MSNs PLGA/Col composite scaffolds, according to FE- SEM. PicoGreen and cytotoxicity analysis showed an increase in the rate of proliferation and metabolic activity of hADSCs after 14 and 21 days, confirming the initial and controlled release of Sil from nanofibers. Gene expression analysis further confirmed the increased expression of stemness markers as well as hTERT and telomerase in ASCs seeded on Sil@MSNs PLGA/Col nanofibers compared to the control group. Ultimately, the findings of the present study introduced Sil@MSNs PLGA/Col composite scaffolds as an efficient platform for long-term proliferation of ASCs in tissue engineering.

Breast cancer cells rely on environmental pyruvate to shape the metastatic niche.

The extracellular matrix is a major component of the local environment-that is, the niche-that determines cell behaviour1. During metastatic growth, cancer cells shape the extracellular matrix of the metastatic niche by hydroxylating collagen to promote their own metastatic growth2,3. However, only particular nutrients might support the ability of cancer cells to hydroxylate collagen, because nutrients dictate which enzymatic reactions are active in cancer cells4,5. Here we show that breast cancer cells rely on the nutrient pyruvate to drive collagen-based remodelling of the extracellular matrix in the lung metastatic niche. Specifically, we discovered that pyruvate uptake induces the production of α-ketoglutarate. This metabolite in turn activates collagen hydroxylation by increasing the activity of the enzyme collagen prolyl-4-hydroxylase (P4HA). Inhibition of pyruvate metabolism was sufficient to impair collagen hydroxylation and consequently the growth of breast-cancer-derived lung metastases in different mouse models. In summary, we provide a mechanistic understanding of the link between collagen remodelling and the nutrient environment in the metastatic niche.

Transmembrane collagen receptors.

Collagen, the most abundant protein in animals, is a key component of extracellular matrices. Not only do collagens provide essential structural support for connective tissues, but they are also intimately involved in controlling a spectrum of cellular functions such as growth, differentiation, and morphogenesis. All collagens possess triple-helical regions through which they interact with a host of other proteins including cell surface receptors. A structurally diverse group of transmembrane receptors mediates the recognition of the collagen triple helix: integrins, discoidin domain receptors, glycoprotein VI, and leukocyte-associated immunoglobulin-like receptor-1. These collagen receptors regulate a wide range of behaviors including cell adhesion and migration, hemostasis, and immune function. Here these collagen receptors are discussed in terms of their molecular basis of collagen recognition, their signaling and developmental functions, and their roles in disease.

Discovering design principles of collagen molecular stability using a genetic algorithm, deep learning, and experimental validation.

Collagen is the most abundant structural protein in humans, providing crucial mechanical properties, including high strength and toughness, in tissues. Collagen-based biomaterials are, therefore, used for tissue repair and regeneration. Utilizing collagen effectively during materials processing ex vivo and subsequent function in vivo requires stability over wide temperature ranges to avoid denaturation and loss of structure, measured as melting temperature (Tm). Although significant research has been conducted on understanding how collagen primary amino acid sequences correspond to Tm values, a robust framework to facilitate the design of collagen sequences with specific Tm remains a challenge. Here, we develop a general model using a genetic algorithm within a deep learning framework to design collagen sequences with specific Tm values. We report 1,000 de novo collagen sequences, and we show that we can efficiently use this model to generate collagen sequences and verify their Tm values using both experimental and computational methods. We find that the model accurately predicts Tm values within a few degrees centigrade. Further, using this model, we conduct a high-throughput study to identify the most frequently occurring collagen triplets that can be directly incorporated into collagen. We further discovered that the number of hydrogen bonds within collagen calculated with molecular dynamics (MD) is directly correlated to the experimental measurement of triple-helical quality. Ultimately, we see this work as a critical step to helping researchers develop collagen sequences with specific Tm values for intended materials manufacturing methods and biomedical applications, realizing a mechanistic materials by design paradigm.

A primer for ZooMS applications in archaeology.

Collagen peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, also known as zooarchaeology by mass spectrometry (ZooMS), is a rapidly growing analytical technique in the fields of archaeology, ecology, and cultural heritage. Minimally destructive and cost effective, ZooMS enables rapid taxonomic identification of large bone assemblages, cultural heritage objects, and other organic materials of animal origin. As its importance grows as both a research and a conservation tool, it is critical to ensure that its expanding body of users understands its fundamental principles, strengths, and limitations. Here, we outline the basic functionality of ZooMS and provide guidance on interpreting collagen spectra from archaeological bones. We further examine the growing potential of applying ZooMS to nonmammalian assemblages, discuss available options for minimally and nondestructive analyses, and explore the potential for peptide mass fingerprinting to be expanded to noncollagenous proteins. We describe the current limitations of the method regarding accessibility, and we propose solutions for the future. Finally, we review the explosive growth of ZooMS over the past decade and highlight the remarkably diverse applications for which the technique is suited.

Simple synthesis of soft, tough, and cytocompatible biohybrid composites.

Collagen is the most abundant component of mammalian extracellular matrices. As such, the development of materials that mimic the biological and mechanical properties of collagenous tissues is an enduring goal of the biomaterials community. Despite the development of molded and 3D printed collagen hydrogel platforms, their use as biomaterials and tissue engineering scaffolds is hindered by either low stiffness and toughness or processing complexity. Here, we demonstrate the development of stiff and tough biohybrid composites by combining collagen with a zwitterionic hydrogel through simple mixing. This combination led to the self-assembly of a nanostructured fibrillar network of collagen that was ionically linked to the surrounding zwitterionic hydrogel matrix, leading to a composite microstructure reminiscent of soft biological tissues. The addition of 5-15 mg mL-1 collagen and the formation of nanostructured fibrils increased the elastic modulus of the composite system by 40% compared to the base zwitterionic matrix. Most notably, the addition of collagen increased the fracture energy nearly 11-fold ([Formula: see text] 180 J m-2) and clearly delayed crack initiation and propagation. These composites exhibit elastic modulus ([Formula: see text] 0.180 MJ) and toughness ([Formula: see text]0.617 MJ m-3) approaching that of biological tissues such as articular cartilage. Maintenance of the fibrillar structure of collagen also greatly enhanced cytocompatibility, improving cell adhesion more than 100-fold with >90% cell viability.

Collagen structure and stability.

Collagen is the most abundant protein in animals. This fibrous, structural protein comprises a right-handed bundle of three parallel, left-handed polyproline II-type helices. Much progress has been made in elucidating the structure of collagen triple helices and the physicochemical basis for their stability. New evidence demonstrates that stereoelectronic effects and preorganization play a key role in that stability. The fibrillar structure of type I collagen-the prototypical collagen fibril-has been revealed in detail. Artificial collagen fibrils that display some properties of natural collagen fibrils are now accessible using chemical synthesis and self-assembly. A rapidly emerging understanding of the mechanical and structural properties of native collagen fibrils will guide further development of artificial collagenous materials for biomedicine and nanotechnology.

Sustained Secretion of CCL21 via an Implantable Cell Reservoir Hydrogel Enhances the Systemic Antitumor Effect of Radiotherapy.

The combination of radiotherapy (RT) and immunotherapy shows promise in improving the clinical treatment of solid tumors; however, it faces challenges of low response rates and systemic toxicity. Herein, an implantable alginate/collagen hydrogel encapsulating C-C motif ligand 21 (CCL21)-expressing dendritic cells (CCL21-DCs@gel) was developed to potentiate the systemic antitumor effects of RT. The hydrogel functioned as a suitable reservoir for in vivo culture and proliferation of CCL21-DCs, thereby enabling sustained CCL21 release. The local CCL21 gradient induced by CCL21-DCs@gel significantly enhanced the efficacy of RT in suppressing primary tumor growth and inhibiting distant metastasis across several mouse models. Furthermore, the combination of RT with CCL21-DCs@gel provided complete prophylactic protection to mice. Mechanistic investigations revealed that CCL21-DCs@gel potentiated RT by promoting tumor lymphangiogenesis and attracting immune cell infiltration into the tumor. Collectively, these results suggest that CCL21-DCs@gel is a promising adjunct to RT for effectively eradicating tumors and preventing tumor recurrence.

Tooth acellular extrinsic fibre cementum incremental lines in humans are formed by parallel branched Sharpey's fibres and not by its mineral phase.

In humans, the growth pattern of the acellular extrinsic fibre cementum (AEFC) has been useful to estimate the age-at-death. However, the structural organization behind such a pattern remains poorly understood. In this study tooth cementum from seven individuals from a Mexican modern skeletal series were analyzed with the aim of unveiling the AEFC collagenous and mineral structure using multimodal imaging approaches. The organization of collagen fibres was first determined using: light microscopy, transmission electron microscopy (TEM), electron tomography, and plasma FIB scanning electron microscopy (PFIB-SEM) tomography. The mineral properties were then investigated using: synchrotron small-angle X-ray scattering (SAXS) for T-parameter (correlation length between mineral particles); synchrotron X-ray diffraction (XRD) for L-parameter (mineral crystalline domain size estimation), alignment parameter (crystals preferred orientation) and lattice parameters a and c; as well as synchrotron X-ray fluorescence for spatial distribution of calcium, phosphorus and zinc. Results show that Sharpey's fibres branched out fibres that cover and uncover other collagen bundles forming aligned arched structures that are joined by these same fibres but in a parallel fashion. The parallel fibres are not set as a continuum on the same plane and when they are superimposed project the AEFC incremental lines due to the collagen birefringence. The orientation of the apatite crystallites is subject to the arrangement of the collagen fibres, and the obtained parameter values along with the elemental distribution maps, revealed this mineral tissue as relatively homogeneous. Therefore, no intrinsic characteristics of the mineral phase could be associated with the alternating AEFC incremental pattern.

A solution structure analysis reveals a bent collagen triple helix in the complement activation recognition molecule mannan-binding lectin.

Collagen triple helices are critical in the function of mannan-binding lectin (MBL), an oligomeric recognition molecule in complement activation. The MBL collagen regions form complexes with the serine proteases MASP-1 and MASP-2 in order to activate complement, and mutations lead to common immunodeficiencies. To evaluate their structure-function properties, we studied the solution structures of four MBL-like collagen peptides. The thermal stability of the MBL collagen region was much reduced by the presence of a GQG interruption in the typical (X-Y-Gly)n repeat compared to controls. Experimental solution structural data were collected using analytical ultracentrifugation and small angle X-ray and neutron scattering. As controls, we included two standard Pro-Hyp-Gly collagen peptides (POG)10-13, as well as three more peptides with diverse (X-Y-Gly)n sequences that represented other collagen features. These data were quantitatively compared with atomistic linear collagen models derived from crystal structures and 12,000 conformations obtained from molecular dynamics simulations. All four MBL peptides were bent to varying degrees up to 85o in the best-fit molecular dynamics models. The best-fit benchmark peptides (POG)n were more linear but exhibited a degree of conformational flexibility. The remaining three peptides showed mostly linear solution structures. In conclusion, the collagen helix is not strictly linear, the degree of flexibility in the triple helix depends on its sequence, and the triple helix with the GQG interruption showed a pronounced bend. The bend in MBL GQG peptides resembles the bend in the collagen of complement C1q and may be key for lectin pathway activation.

Self-Sorting Collagen Heterotrimers.

Nature uses elaborate methods to control protein assembly, including that of heterotrimeric collagen. Here, we established design principles for the composition and register-selective assembly of synthetic collagen heterotrimers. The assembly code enabled the self-sorting of eight different strands into three─out of 512 possible─triple helices via complementary (4S)-aminoproline and aspartate residues. Native ESI-MS corroborated the specific assembly into coexisting heterotrimers.

Laminin and hyaluronan supplementation of collagen hydrogels enhances endothelial function and tight junction expression on three-dimensional cylindrical microvessel-on-a-chip.

Vasculature-on-chip (VoC) models have become a prominent tool in the study of microvasculature functions because of their cost-effective and ethical production process. These models typically use a hydrogel in which the three-dimensional (3D) microvascular structure is embedded. Thus, VoCs are directly impacted by the physical and chemical cues of the supporting hydrogel. Endothelial cell (EC) response in VoCs is critical, especially in organ-specific vasculature models, in which ECs exhibit specific traits and behaviors that vary between organs. Many studies customize the stimuli ECs perceive in different ways; however, customizing the hydrogel composition accordingly to the target organ's extracellular matrix (ECM), which we believe has great potential, has been rarely investigated. We explored this approach to organ-specific VoCs by fabricating microvessels (MVs) with either human umbilical vein ECs or human brain microvascular ECs in a 3D cylindrical VoC using a collagen hydrogel alone or one supplemented with laminin and hyaluronan, components found in the brain ECM. We characterized the physical properties of these hydrogels and analyzed the barrier properties of the MVs. Barrier function and tight junction (ZO-1) expression improved with the addition of laminin and hyaluronan in the composite hydrogel.