A map of glycation and glycoxidation sites in collagen I of human cortical bone: Effects of sex and type 2 diabetes.

Complications of diabetes is a major health problem affecting multiple organs including bone, where the chronic disease increases the risk of fragility fractures. One hypothesis suggests a pathogenic role for hyperglycemia-induced modification of proteins, a.k.

Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.

Prolonged obstruction of the ureter, which leads to injury of the kidney collecting ducts, results in permanent structural damage, while early reversal allows for repair. Cell structure is defined by the actin cytoskeleton, which is dynamically organized by small Rho guanosine triphosphatases (GTPases). In this study, we identified the Rho GTPase, Rac1, as a driver of postobstructive kidney collecting duct repair.

The Collagen Receptor Discoidin Domain Receptor 1b Enhances Integrin β1-Mediated Cell Migration by Interacting With Talin and Promoting Rac1 Activation.

Integrins and discoidin domain receptors (DDRs) 1 and 2 promote cell adhesion and migration on both fibrillar and non fibrillar collagens. Collagen I contains DDR and integrin selective binding motifs; however, the relative contribution of these two receptors in regulating cell migration is unclear. DDR1 has five isoforms (DDR1a-e), with most cells expressing the DDR1a and DDR1b isoforms.

Rac1 promotes kidney collecting duct integrity by limiting actomyosin activity.

A polarized collecting duct (CD), formed from the branching ureteric bud (UB), is a prerequisite for an intact kidney. The small Rho GTPase Rac1 is critical for actin cytoskeletal regulation. We investigated the role of Rac1 in the kidney collecting system by selectively deleting it in mice at the initiation of UB development.

Post-translational modifications in collagen type I of bone in a mouse model of aging.

The fracture resistance of cortical bone and matrix hydration are known to decline with advanced aging. However, the underlying mechanisms remain poorly understood, and so we investigated levels of matrix proteins and post-translational modifications (PTM) of collagen I in extracts from the tibia of 6-mo. and 20-mo.

Halogens are key cofactors in building of collagen IV scaffolds outside the cell.

Purpose Of Review: The purpose of this review is to highlight recent advances in understanding the molecular assembly of basement membranes, as exemplified by the glomerular basement membrane (GBM) of the kidney filtration apparatus. In particular, an essential role of halogens in the basement membrane formation has been discovered.

Recent Findings: Extracellular chloride triggers a molecular switch within non collagenous domains of collagen IV that induces protomer oligomerization and scaffold assembly outside the cell.

Salt-bridge modulates differential calcium-mediated ligand binding to integrin α1- and α2-I domains.

Integrins are transmembrane cell-extracellular matrix adhesion receptors that impact many cellular functions. A subgroup of integrins contain an inserted (I) domain within the α-subunits (αI) that mediate ligand recognition where function is contingent on binding a divalent cation at the metal ion dependent adhesion site (MIDAS). Ca is reported to promote α1I but inhibit α2I ligand binding.

Building collagen IV smart scaffolds on the outside of cells.

Collagen IV scaffolds assemble through an intricate pathway that begins intracellularly and is completed extracellularly. Multiple intracellular enzymes act in concert to assemble collagen IV protomers, the building blocks of collagen IV scaffolds. After being secreted from cells, protomers are activated to initiate oligomerization, forming insoluble networks that are structurally reinforced with covalent crosslinks.

Collagen IV and basement membrane at the evolutionary dawn of metazoan tissues.

The role of the cellular microenvironment in enabling metazoan tissue genesis remains obscure. Ctenophora has recently emerged as one of the earliest-branching extant animal phyla, providing a unique opportunity to explore the evolutionary role of the cellular microenvironment in tissue genesis. Here, we characterized the extracellular matrix (ECM), with a focus on collagen IV and its variant, spongin short-chain collagens, of non-bilaterian animal phyla.

Extracellular chloride signals collagen IV network assembly during basement membrane formation.

Basement membranes are defining features of the cellular microenvironment; however, little is known regarding their assembly outside cells. We report that extracellular Cl(-) ions signal the assembly of collagen IV networks outside cells by triggering a conformational switch within collagen IV noncollagenous 1 (NC1) domains. Depletion of Cl(-) in cell culture perturbed collagen IV networks, disrupted matrix architecture, and repositioned basement membrane proteins.

Antibodies to α5 chain of collagen IV are pathogenic in Goodpasture's disease.

Autoantibody against glomerular basement membrane (GBM) plays a direct role in the initiation and development of Goodpasture's (GP) disease. The principal autoantigen is the non-collagenous domain 1 (NC1) of α3 chain of collagen IV, with two immunodominant epitopes, EA-α3 and EB-α3. We recently demonstrated that antibodies targeting α5NC1 are bound to kidneys in GP patients, suggesting their pathogenic relevance.

Hypohalous acids contribute to renal extracellular matrix damage in experimental diabetes.

In diabetes, toxic oxidative pathways are triggered by persistent hyperglycemia and contribute to diabetes complications. A major proposed pathogenic mechanism is the accumulation of protein modifications that are called advanced glycation end products. However, other nonenzymatic post-translational modifications may also contribute to pathogenic protein damage in diabetes.

DNA Sequence Modulates Geometrical Isomerism of the trans-8,9- Dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)- 9-hydroxy Aflatoxin B1 Adduct.

Aflatoxin B(1) (AFB(1)), a mycotoxin produced by Aspergillus flavus, is oxidized by cytochrome P450 enzymes to aflatoxin B(1)-8,9-epoxide, which alkylates DNA at N7-dG. Under basic conditions, this N7-dG adduct rearranges to yield the trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B(1) (AFB(1)−FAPY) adduct. The AFB(1)−FAPY adduct exhibits geometrical isomerism involving the formamide moiety.

Site-specific AGE modifications in the extracellular matrix: a role for glyoxal in protein damage in diabetes.

Non-enzymatic modification of proteins in hyperglycemia is a major proposed mechanism of diabetic complications. Specifically, advanced glycation end products (AGEs) derived from hyperglycemia-induced reactive carbonyl species (RCS) can have pathogenic consequences when they target functionally critical protein residues. Modification of a small number of these critical residues, often undetectable by the methodologies relying on measurements of total AGE levels, can cause significant functional damage.

Chemistry and structural biology of DNA damage and biological consequences.

The formation of adducts by the reaction of chemicals with DNA is a critical step for the initiation of carcinogenesis. The structural analysis of various DNA adducts reveals that conformational and chemical rearrangements and interconversions are a common theme. Conformational changes are modulated both by the nature of adduct and the base sequences neighboring the lesion sites.

Bypass of aflatoxin B1 adducts by the Sulfolobus solfataricus DNA polymerase IV.

Aflatoxin B(1) (AFB(1)) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB(1)-N7-dG). The AFB(1)-N7-dG can rearrange to a formamidopyrimidine (AFB(1)-FAPY) derivative. Both AFB(1)-N7-dG and the β-anomer of the AFB(1)-FAPY adduct yield G→T transversions in Escherichia coli, but the latter is more mutagenic.

Binding of the human nucleotide excision repair proteins XPA and XPC/HR23B to the 5R-thymine glycol lesion and structure of the cis-(5R,6S) thymine glycol epimer in the 5'-GTgG-3' sequence: destabilization of two base pairs at the lesion site.

The 5R thymine glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5'-GTgG-3' sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior.

Structural perturbations induced by the alpha-anomer of the aflatoxin B(1) formamidopyrimidine adduct in duplex and single-strand DNA.

The guanine N7 adduct of aflatoxin B(1) exo-8,9-epoxide hydrolyzes to form the formamidopyrimidine (AFB-FAPY) adduct, which interconverts between alpha and beta anomers. The beta anomer is highly mutagenic in Escherichia coli, producing G --> T transversions; it thermally stabilizes the DNA duplex. The AFB-alpha-FAPY adduct blocks replication; it destabilizes the DNA duplex.

The cis-(5R,6S)-thymine glycol lesion occupies the wobble position when mismatched with deoxyguanosine in DNA.

Oxidative damage to 5-methylcytosine in DNA, followed by deamination, yields thymine glycol (Tg), 5,6-dihydroxy-5,6-dihydrothymine, mispaired with deoxyguanosine. The structure of the 5R Tg.G mismatch pair has been refined using a combination of simulated annealing and isothermal molecular dynamics calculations restrained by NMR-derived distance restraints and torsion angle restraints in 5'-d(G(1)T(2)G(3)C(4)G(5)Tg(6)G(7)T(8)T(9)T(10)G(11)T(12))-3'.

Inherent stereospecificity in the reaction of aflatoxin B(1) 8,9-epoxide with deoxyguanosine and efficiency of DNA catalysis.

Kinetic analysis of guanine alkylation by aflatoxin B(1) exo-8,9-epoxide, the reactive form of the hepatocarcinogen aflatoxin B(1), shows the reaction to be >2000 times more efficient in DNA than in aqueous solution, that is, with free 2'-deoxyguanosine. Thermodynamic analysis reveals AFB(1) exo-8,9-epoxide intercalation as the predominant source of the observed DNA catalytic effect. However, the known exo > endo epoxide stereospecificity of the DNA alkylation is observed even with free deoxyguanosine (ratio >20:1 determined by LC-MS and NMR measurements), as predicted by theoretical calculations [ Bren , U.

Site-specific synthesis and characterization of oligonucleotides containing an N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine lesion, the ring-opened product from N7-methylation of deoxyguanosine.

A phosphoramidite reagent of N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-1,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dGuo) lesions was synthesized in four steps from 2'-deoxyguanosine. Fapy nucleosides can rearrange to the pyranose form when the 5'-hydroxyl group is unprotected. The phosphoramidite was incorporated into oligonucleotides using solid-phase synthesis by adjusting the deprotection time for removal of the 5'-dimethoxytrityl group of the MeFapy-dGuo nucleotide, thereby minimizing its rearrangement to the ribopyranose.

Interconversion of the cis-5R,6S- and trans-5R,6R-thymine glycol lesions in duplex DNA.

Thymine glycol (Tg), 5,6-dihydroxy-5,6-dihydrothymine, is formed in DNA by the reaction of thymine with reactive oxygen species. The 5R Tg lesion was incorporated site-specifically into 5'-d(G(1)T(2)G(3)C(4)G(5)Tg(6)G(7)T(8)T(9)T(10)G(11)T(12))-3'; Tg = 5R Tg. The Tg-modified oligodeoxynucleotide was annealed with either 5'-d(A(13)C(14)A(15)A(16)A(17)C(18)A(19)C(20)G(21)C(22)A(23)C(24))-3', forming the Tg(6) x A(19) base pair, corresponding to the oxidative damage of thymine in DNA, or 5'-d(A(13)C(14)A(15)A(16)A(17)C(18)G(19)C(20)G(21)C(22)A(23)C(24))-3', forming the mismatched Tg(6) x G(19) base pair, corresponding to the formation of Tg following oxidative damage and deamination of 5-methylcytosine in DNA.

Unraveling the aflatoxin-FAPY conundrum: structural basis for differential replicative processing of isomeric forms of the formamidopyrimidine-type DNA adduct of aflatoxin B1.

Aflatoxin B1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers.