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PURPOSE OF REVIEW: In Alport syndrome, over 1,700 genetic variants in the COL4A3, COL4A4, and COL4A5 genes cause the absence or malfunctioning of the collagen IV(α345) scaffold – an essential component of the glomerular basement membrane (GBM). Therapies are limited to treatment with ACE inhibitors to slow progression of the disease. Here, we review recent progress in therapy development to replace the scaffold or restore its function. RECENT FINDINGS: Multiple approaches emerged recently for development of therapies that target different stages of production and assembly of the collagen IV(α345) scaffold in the GBM. These approaches are based on 1) recent advances in technologies allowing to decipher pathogenic mechanisms that underlie scaffold assembly and dysfunction, 2) development of DNA editing tools for gene therapy, 3) RNA splicing interference, and 4) control of mRNA translation. SUMMARY: There is a growing confidence that these approaches will ultimately provide cure for Alport patients. Development of therapy will be accelerated by studies that provide a deeper understanding of mechanisms that underlie folding, assembly, and function of the collagen IV(α345) scaffold.

Collagen IV scaffold is a principal component of the basement membrane (BM), a specialized extracellular matrix that is essential for animal multicellularity and tissue evolution. Scaffold assembly begins with the trimerization of α-chains into protomers inside the cell, which then are secreted and undergo oligomerization outside the cell. For the ubiquitous scaffold composed of α1- and α2-chains, both intracellular and extracellular stages are mediated by the noncollagenous domain (NC1). The association of protomers is chloride-dependent, whereby chloride ions induce interactions of the protomers' trimeric NC1 domains leading to NC1 hexamer formation. Here, we investigated the mechanisms, kinetics, and functionality of the chloride ion-mediated protomer assembly by using a single-chain technology to produce a stable NC1 trimer comprising α1, α2, and α1 NC1 monomers. We observed that in the presence of chloride, the single-chain NC1-trimer self-assembles into a hexamer, for which the crystal structure was determined. We discovered that a chloride ring, comprising 12 ions, induces the assembly of and stabilizes the NC1 hexamer. Furthermore, we found that the chloride ring is evolutionarily conserved across all animals, first appearing in cnidarians. These findings reveal a fundamental role for the chloride ring in the assembly of collagen IV scaffolds of BMs, a critical event enabling tissue evolution and development. Moreover, the single-chain technology is foundational for generating trimeric NC1 domains of other α-chain compositions to investigate the α121, α345, and α565 collagen IV scaffolds and to develop therapies for managing Alport syndrome, Goodpasture's disease, and cancerous tumor growth.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood with a propensity to metastasize. Current treatment for patients with RMS includes conventional systemic chemotherapy, radiation therapy, and surgical resection; nevertheless, little to no improvement in long term survival has been achieved in decades—underlining the need for target discovery and new therapeutic approaches to targeting tumor cells or the tumor microenvironment. To evaluate cross-species sarcoma extracellular matrix production, we have used murine models which feature knowledge of the myogenic cell-of-origin. With focus on the RMS/undifferentiated pleomorphic sarcoma (UPS) continuum, we have constructed tissue microarrays of 48 murine and four human sarcomas to analyze expression of seven different collagens, fibrillins, and collagen-modifying proteins, with cross-correlation to RNA deep sequencing. We have uncovered that RMS produces increased expression of type XVIII collagen alpha 1 (COL18A1), which is clinically associated with decreased long-term survival. We have also identified significantly increased RNA expression of COL4A1, FBN2, PLOD1, and PLOD2 in human RMS relative to normal skeletal muscle. These results complement recent studies investigating whether soft tissue sarcomas utilize collagens, fibrillins, and collagen-modifying enzymes to alter the structural integrity of surrounding host extracellular matrix/collagen quaternary structure resulting in improved ability to improve the ability to invade regionally and metastasize, for which therapeutic targeting is possible.

Background: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood with a propensity to metastasize. Current treatment for patients with RMS includes conventional systemic chemotherapy, radiation therapy and surgical resection; nevertheless, little to no improvement in long term survival has been achieved in decades – underlining the need for target discovery and new therapeutic approaches to targeting tumor cells or the tumor microenvironment.Methods:To evaluate cross-species sarcoma extracellular matrix production, we have used murine models which feature knowledge of the myogenic cell-of-origin. With focus on the RMS/undifferentiated pleomorphic sarcoma (UPS) continuum, we have constructed tissue microarrays of 48 murine and 4 human sarcomas to analyze expression of 7 different collagens, fibrillins and collagen-modifying proteins, with cross-correlation to RNA deep sequencing.Results:We have uncovered that RMS produces increased expression of type XVIII collagen alpha 1 (COL18A1), which is clinically associated with decreased long-term survival. We have also identified significantly increased RNA expression of COL4A1, FBN2, PLOD1 and PLOD2 in human RMS relative to normal skeletal muscle.Conclusion:These results complement recent studies investigating whether soft tissue sarcomas utilize collagens, fibrillins and collagen-modifying enzymes to alter the structural integrity of surrounding host extracellular matrix/collagen quaternary structure resulting in improved ability to improve the ability to invade regionally and metastasize, for which therapeutic targeting is possible.

Collagen molecules are crucial extracellular players in animal tissue development and in functions ranging from ultrafiltration to organism locomotion. Among the 28 types of collagen found in human, type IV collagen stands out as a primordial type found in all species of the animal kingdom. Collagen IV forms smart scaffolds for basement membranes, sheet-like acellular structures that isolate, coordinate, and direct cells during morphogenesis. Collagen IV is also involved in multiple functions in developed tissues. As part of the basement membrane, collagen IV scaffolds provide mechanical strength, spatially tether extracellular macromolecules and directly signal to cells via receptor binding sites. Proper assembly and structure of the scaffolds are critical for development and function of multiple types of basement membranes. Within last 5 years it was established that Cl− concentration is a key factor for initiating collagen IV scaffold assembly. The biological role of Cl− in multiple physiological processes and detailed mechanisms for its signaling and structural impacts are well established. Cl− gradients are generated across the plasma and intracellular organelle membranes. As collagen IV molecules are secreted outside the cell, they experience a switch from low to high Cl− concentration. This transition works as a trigger for collagen IV scaffold assembly. Within the scaffold, collagen IV remains to be a Cl− sensor as its structural integrity continues to depend on Cl− concentration. Here, we review recent findings and set future directions for studies on the role of Cl− in type IV collagen assembly, function, and disease.

Type IV collagen is a major component of the basement membrane and interacts with numerous other basement membrane proteins. Many of these interactions are poorly characterized. Type IV collagen is abundantly post-translationally modified with 3-hydroxyproline (3-Hyp), but 3-Hyp's biochemical role in type IV collagen's interactions with other proteins is not well established. In this work, we present binding data consistent with a major role of 3-Hyp in interactions of collagen IV with glycoprotein VI and nidogens 1 and 2. The increased binding interaction between type IV collagen without 3-Hyp and glycoprotein VI has been the subject of some controversy, which we sought to explore, whereas the lack of binding of nidogens to type IV collagen without 3-Hyp is novel. Using tandem MS, we show that the putative glycoprotein VI-binding site is 3-Hyp–modified in WT PFHR-9 type IV collagen, but not in PFHR-9 cells in which prolyl-3-hydroxylase 2 (P3H2) has been knocked out (KO). Moreover, we observed altered 3-Hyp occupancy across many other sites. Using amino acid analysis of type IV collagen from the WT and P3H2 KO cell lines, we confirm that P3H2 is the major, but not the only 3-Hyp–modifying enzyme of type IV collagen. These findings underscore the importance of post-translational modifications of type IV collagen for interactions with other proteins.

Diseases of the glomerular basement membrane (GBM), such as Goodpasture’s disease (GP) and Alport syndrome (AS), are a major cause of chronic kidney failure and an unmet medical need. Collagen IVα345 is an important architectural element of the GBM that was discovered in previous research on GP and AS. How this collagen enables GBM to function as a permselective filter and how structural defects cause renal failure remain an enigma. We found a distinctive genetic variant of collagen IVα345 in both a familial GP case and four AS kindreds that provided insights into these mechanisms. The variant is an 8-residue appendage at the C-terminus of the α3 subunit of the α345 hexamer. A knock-in mouse harboring the variant displayed GBM abnormalities and proteinuria. This pathology phenocopied AS, which pinpointed the α345 hexamer as a focal point in GBM function and dysfunction. Crystallography and assembly studies revealed underlying hexamer mechanisms, as described in Boudko et al. and Pedchenko et al. Bioactive sites on the hexamer surface were identified where pathogenic pathways of GP and AS converge and, potentially, that of diabetic nephropathy (DN). We conclude that the hexamer functions include signaling and organizing macromolecular complexes, which enable GBM assembly and function. Therapeutic modulation or replacement of α345 hexamer could therefore be a potential treatment for GBM diseases, and this knock-in mouse model is suitable for developing gene therapies.

Collagens constitute nearly 30% of all proteins in our body. Type IV collagen is a major and crucial component of basement membranes. Collagen chains undergo several posttranslational modifications that are indispensable for proper collagen function. One of these modifications, prolyl 3-hydroxylation, is accomplished by a family of prolyl 3-hydroxylases (P3H1, P3H2, and P3H3). The present study shows that P3H2-null mice are embryonic-lethal by embryonic day 8.5. The mechanism of the unexpectedly early lethality involves the interaction of non-3-hydroxylated embryonic type IV collagen with the maternal platelet-specific glycoprotein VI (GPVI). This interaction results in maternal platelet aggregation, thrombosis of the maternal blood, and death of the embryo. The phenotype is completely rescued by producing double KOs of P3H2 and GPVI. Double nulls are viable and fertile. Under normal conditions, subendothelial collagens bear the GPVI-binding sites that initiate platelet aggregation upon blood exposure during injuries. In type IV collagen, these sites are normally 3-hydroxylated. Thus, prolyl 3-hydroxylation of type IV collagen has an important function preventing maternal platelet aggregation in response to the early developing embryo. A unique link between blood coagulation and the ECM is established. The newly described mechanism may elucidate some unexplained fetal losses in humans, where thrombosis is often observed at the maternal/fetal interface. Moreover, epigenetic silencing of P3H2 in breast cancers implies that the interaction between GPVI and non-3-hydroxylated type IV collagen might also play a role in the progression of malignant tumors and metastasis.

The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.

SummaryDengue virus (DENV) is a significant human pathogen that causes millions of infections and results in about 24,000 deaths each year. Dendritic cell-specific ICAM3 grabbing nonintegrin (DC-SIGN), abundant in immature dendritic cells, was previously reported as being an ancillary receptor interacting with the surface of DENV. The structure of DENV in complex with the carbohydrate recognition domain (CRD) of DC-SIGN was determined by cryo-electron microscopy at 25 Å resolution. One CRD monomer was found to bind to two glycosylation sites at Asn67 of two neighboring glycoproteins in each icosahedral asymmetric unit, leaving the third Asn67 residue vacant. The vacancy at the third Asn67 site is a result of the nonequivalence of the glycoprotein environments, leaving space for the primary receptor binding to domain III of E. The use of carbohydrate moieties for receptor binding sites suggests a mechanism for avoiding immune surveillance.

The complex biosynthesis of type I procollagen involves a large number of post-translational modifications and a highly choreographed folding mechanism. This folding machinery resides primarily in the rough endoplasmic reticulum and consists of many enzymes and molecular chaperones which act as interacting cogs in this process. The importance of this machinery for normal bone formation has recently been demonstrated by the discovery of recessive forms of osteogenesis imperfecta that are due to mutations in the proteins that are part of this machinery. In this chapter we present the current knowledge of the folding machinery and the consequences of the absence of some of these cogs on the structure and function of type I procollagen.

The role that alkyl chain packing density in a self-assembled monolayer (SAM) plays in the adsorption of protein to the SAM surface was investigated using in situ neutron reflectivity (NR) and tota...

The influence of surface energy and structural properties of carbon coated polyethylene (PE) on the human platelet adhesion was studied. Three types of amorphous carbon coating were obtained by plasma pulse discharge, with the number of pulses grading as 10, 50, 100. Human serum albumin adsorption experiments have been carried out with all samples in vitro. Platelet adhesion analysis by SEM included determination of total quantity of adherent platelets, and respective quantities of platelets at different stages of activation (single, spread, aggregates). Surface topographies ranged from bare PE and such (10 pulses), to globular 0.5 microm in size (50 pulses), and complex fibrillar 3-4 microm structures (100 pulses). Surface free energy varies from 31.7 +/- 0.6 to 40.4 +/- 0.6 mN/m for uncoated PE and 10 pulse coatings, respectively, as determined by contact angle techniques. All studied coatings demonstrate weaker platelet activation properties in comparison with untreated PE. Among all studied coatings, the 50 pulse coated surface seems to be the least suitable for contact with platelets, mainly due to its structural rather than to its energy properties. These data are related to a sharp decrease in the adsorbed protein level for the samples with 50 pulse coatings. The applied analysis of platelet activation enables more accurate characterization of platelet-biomaterial interaction.

1. The method of FTIR was used for monitoring protein adsorption to IOL. 2. Energy characteristics of adsorption of the other main proteins of the intraocular fluid (α-, β-, and γ-immunoglobulins) to the IOL surface should also be studied to elucidate the mechanisms of biocompatibility of IOL at the stage of protein adsorption.

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils. Background: 3-Hydroxylation of proline residues in type I collagen is rare but important. Results: 3-Hyp sites have been identified in both chains of mouse type I collagen in wild type and P3H1 null mice. Conclusion: The absence of 3-Hyp does not alter the D-period of collagen fibrils, but alters the lateral growth of the fibrils. Significance: Type I collagen prolyl 3-hydroxylation is tissue-specific.

The nematocyst is a unique extrusive organelle involved in the defense and capture of prey in cnidarians. Minicollagens and the glycoprotein NOWA are major components of the nematocyst capsule wall, which resists osmotic pressure of 15 MPa. Here we present the recombinant expression of NOWA, which spontaneously assembles to globular macromolecular particles that are sensitive to reduction as the native wall structure. Ultra-structural analysis showed that the Hydra nematocyst wall is composed of several layers of globular particles, which are interconnected via radiating rodlike protrusions. Evidence is presented that native wall particles contain NOWA and minicollagen, supposed to be linked via disulfide bonds between their homologous cysteine-rich domains. Our data suggest a continuous suprastructure of the nematocyst wall, assembled from wall proteins that share a common oligomerization motif.

The minicollagens found in the nematocysts of Hydra constitute a family of invertebrate collagens with unusual properties. They share a common modular architecture with a central collagen sequence ranging from 14 to 16 Gly-X-Y repeats flanked by polyproline/hydroxyproline stretches and short terminal domains that show a conserved cysteine pattern (CXXXCXXXCXXX-CXXXCC). The minicollagen cysteine-rich domains are believed to function in a switch of the disulfide connectivity from intra- to intermolecular bonds during maturation of the capsule wall. The solution structure of the C-terminal fragment including a minicollagen cysteine-rich domain of minicollagen-1 was determined in two independent groups by 1H NMR. The corresponding peptide comprising the last 24 residues of the molecule was produced synthetically and refolded by oxidation under low protein concentrations. Both presented structures are identical in their fold and disulfide connections (Cys2-Cys18, Cys6-Cys14, and Cys10-Cys19) revealing a robust structural motif that is supposed to serve as the polymerization module of the nematocyst capsule.

A high-precision solution structure of the C-terminal minicollagen cysteine rich domain of Hydra has been determined using modern heteronuclear and weak alignment NMR techniques at natural isotope abundance. The domain consists of only 24 amino acids, six of which are prolines and six are cysteines bonded in disulfide bridges that constrain the structure into a new fold. The redox equilibrium of the structure has been characterized from a titration with glutathione. No local native structures are detectable in the reduced form. Thus, oxidation and folding are tightly coupled.

Globular (G) actin is among the most abundant proteins in the eukaryotic cell cytoskeleton and the major protein, together with myosin, found in muscle cells. In the presence of ions, G-actin assembles into filamentous (F) actin. Current textbooks on molecular cell biology describe two types of supramolecular assemblies of F-actin [1]. In one type, parallel F-actin rods are assembled into bundles in the presence of bundling proteins, which belong to a large family of actin-binding proteins. In a second type, F-actin rods cross each other at an angle and form, in the presence of specific crosslinking proteins, a network of single filaments. Bundles and networks of actin rods are assembled for a variety of cellular activities; F-actin networks are involved in cell shape, motility, and adhesion. In the fundamental biological process of cell division, leading to growth, F-actin bundles, myosin II motors, and associated biomolecules form a contractile ring, which splits cells into two during cytokinesis. In our work we studied mixtures of F-actin with the bundling protein � -actinin (from rabbit skeletal muscle, Cytoskeleton, Inc.). � -actinin forms a dimer with the monomers arranged in an antiparallel manner to form a rod-shaped molecule, with an actin-binding region at each end. The molecule has been characterized by electron microscopy as having a width of 3‐ 4 nm and a typical length of � 30 nm [2]. In the cell � -actinin is

Intraocular liquid, in contrast to blood, has no cellular components; therefore, proteins (human serum albumin [HSA], and [alpha, beta, gamma] globulins) are the major components that determine patients' response to the intraocular lens (IOL) surface. In addition to the amount of adsorbed proteins, the possibility of its conformational changes, including conformational changes of globulins C1 and C3 that respond for the activation of the complements system by the classical and alternative pathways, cannot be excluded. The interaction between IOLs and protein components of intraocular liquid directly influences the ocular exudative reaction in the early postoperational period, the intensity of cellular and pigmental scurf on the surface of the IOLs, and the state of endothelial cells of the cornea in the distant postoperational period. Our goal was to compare the interaction of commercial IOLs made from polymethylmethacrylate, silicone, poly-2-hydroxyethyl methacrylate (p-HEMA), and copolymer p-HEMA with collagen with HSA and the complement system. The total internal reflection fluorescence (TIRF) method and hemolytic assay were used for this task, respectively. It has been demonstrated that the probability of biocompatibility of commercially produced IOLs on the stage of protein adsorption can be evaluated using the kinetic of HSA-fluorescein isothiocyanate adsorption onto the IOL surface by the TIRF METHOD: In the case of IOLs from p-HEMA, a negative correlation was shown between the degree of irreversible adsorption of HSA and the minimum relative rate constant of the surface-induced complement activation. We did not find any correlation between hydrophilicity/hydrophobicity of lenses and their adsorptional properties including complement activation. From suggested adsorptional criteria in vitro for biocompatible surfaces, the hydrogel lens from p-HEMA has a lower probability of biocompatibility in comparison with other IOLs.

Osteogenesis imperfecta (OI) is a skeletal disorder primarily caused by mutations in the type I collagen genes. However, recent investigations have revealed that mutations in the genes encoding for cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1 (P3H1) can cause a severe, recessive form of OI. These reports show minimal 3-hydroxylation of key proline residues in type I collagen as a result of CRTAP or P3H1 deficiency and demonstrate the importance of P3H1 and CRTAP to bone structure and development. P3H1 and CRTAP have previously been shown to form a stable complex with cyclophilin B, and P3H1 was shown to catalyze the 3-hydroxylation of specific proline residues in procollagen I in vitro. Here we describe a mouse model in which the P3H1 gene has been inactivated. Our data demonstrate abnormalities in collagen fibril ultrastructure in tendons from P3H1 null mice by electron microscopy. Differences are also seen in skin architecture, as well as in developing limbs by histology. Additionally bone mass and strength were significantly lower in the P3H1 mice as compared with wild-type littermates. Altogether these investigations demonstrate disturbances of collagen fiber architecture in tissues rich in fibrillar collagen, including bone, tendon, and skin. This model system presents a good opportunity to study the underlying mechanisms of recessive OI and to better understand its effects in humans.