Albumin degradation in the renal tubules is impaired in diabetic nephropathy such that levels of the resulting albumin fragments increase with the degree of renal injury. However, the mechanism of ...albumin degradation is unknown. In particular, fragmentation of the endogenous native albumin has not been demonstrated in the kidney and the enzymes that may contribute to fragmentation have not been identified. To explore this we utilized matrix-assisted laser desorption/ionization imaging mass spectrometry for molecular profiling of specific renal regions without disturbing distinct tissue morphology. Changes in protein expression were measured in kidney sections of eNOS-/-db/db mice, a model of diabetic nephropathy, by high spatial resolution imaging allowing molecular localizations at the level of single glomeruli and tubules. Significant increases were found in the relative abundances of several albumin fragments in the kidney of the mice with diabetic nephropathy compared with control nondiabetic mice. The relative abundance of fragments detected correlated positively with the degree of nephropathy. Furthermore, specific albumin fragments accumulating in the lumen of diabetic renal tubules were identified and predicted the enzymatic action of cathepsin D based on cleavage specificity and in vitro digestions. Importantly, this was demonstrated directly in the renal tissue with the endogenous nonlabeled murine albumin. Thus, our results provide molecular insights into the mechanism of albumin degradation in diabetic nephropathy.
Hyperglycemic conditions of diabetes accelerate protein modifications by glucose leading to the accumulation of advanced glycation end-products (AGEs). We have investigated the conversion of ...protein-Amadori intermediate to protein-AGE and the mechanism of its inhibition by pyridoxamine (PM), a potent AGE inhibitor that has been shown to prevent diabetic complications in animal models. During incubation of proteins with physiological diabetic concentrations of glucose, PM prevented the degradation of the protein glycation intermediate identified as fructosyllysine (Amadori) by 13C NMR using 2-13C-enriched glucose. Subsequent removal of glucose and PM led to conversion of protein-Amadori to AGE Nepsilon-carboxymethyllysine (CML). We utilized this inhibition of post-Amadori reactions by PM to isolate protein-Amadori intermediate and to study the inhibitory effect of PM on its degradation to protein-CML. We first tested the hypothesis that PM blocks Amadori-to-CML conversion by interfering with the catalytic role of redox metal ions that are required for this glycoxidative reaction. Support for this hypothesis was obtained by examining structural analogs of PM in which its known bidentate metal ion binding sites were modified and by determining the effect of endogenous metal ions on PM inhibition. We also tested the alternative hypothesis that the inhibitory mechanism involves formation of covalent adducts between PM and protein-Amadori. However, our 13C NMR studies demonstrated that PM does not react with the Amadori. Because the mechanism of interference with redox metal catalysis is operative under the conditions closely mimicking the diabetic state, it may contribute significantly to PM efficacy in preventing diabetic complications in vivo. Inhibition of protein-Amadori degradation by PM also provides a simple procedure for the isolation of protein-Amadori intermediate, prepared at physiological levels of glucose for relevancy, to study both the biological effects and the chemistry of post-Amadori pathways of AGE formation.
: Pyridoxamine (PM) is one of three natural forms of vitamin B6. It is a critical transient intermediate in catalysis of transamination reactions by vitamin B6‐dependent enzymes. The discovery eight ...years ago that PM can inhibit the Maillard reaction stimulated new interest in this B6 vitamer as a prospective pharmacological agent for treatment of complications of diabetes. PM application in diabetic nephropathy has now progressed to a phase III clinical trial. Investigation of the PM mechanism of action demonstrated that PM inhibits post‐Amadori steps of the Maillard reaction by sequestering catalytic metal ions and blocking oxidative degradation of Amadori intermediate. PM also has the capacity to scavenge toxic carbonyl products of sugar and lipid degradation, and to inhibit reactive oxygen species. These multiple activities position PM as a promising drug candidate for treatment of multifactorial chronic conditions in which oxidative reactions and/or carbonyl compounds confer pathogenicity.
Mechanism of radiosensitivity of normal tissues, a key factor in determining the toxic side effects of cancer radiotherapy, is not fully understood. We recently demonstrated that deficiency of ...mitochondrial tumor suppressor, Fus1, increases radiosensitivity at the organismal, tissue and cellular levels. Since Fus1-deficient mice and cells exhibit high levels of oxidative stress, we hypothesized that dysregulation of cellular antioxidant defenses may contribute to the increased radiosensitivity. To address this potential mechanism, we treated the Fus1 KO mice with an inhibitor of pathogenic oxidative reactions, pyridoxamine (PM). Treatment with PM ameliorated IR-induced damage to GI epithelium of Fus1 KO mice and significantly increased the survival of irradiated mice. In cultured Fus1 KO epithelial cells, IR-induced oxidative stress was enhanced because of inadequate cellular antioxidant defenses, such as low levels and/or activities of cytochrome C, Sod 2 and STAT3. This resulted in dysregulation of IR-induced DNA-damage response and DNA synthesis. Treatment of Fus1 KO cells with PM or Sod 2 mimetic Tempol normalized the oxidative stress response, thus compensating to a significant degree for inadequate antioxidant response. Our findings using Fus1 KO radiosensitive mice suggest that radiosensitivity is mediated via dysregulation of antioxidant response and defective redox homeostasis.
Reactive carbonyl compounds are formed during autoxidation of carbohydrates and peroxidation of lipids. These compounds are intermediates in the formation of advanced glycation end products (AGE) and ...advanced lipoxidation end products (ALE) in tissue proteins during aging and in chronic disease. We studied the reaction of carbonyl compounds glyoxal (GO) and glycolaldehyde (GLA) with pyridoxamine (PM), a potent post-Amadori inhibitor of AGE formation in vitro and of development of renal and retinal pathology in diabetic animals. PM reacted rapidly with GO and GLA in neutral, aqueous buffer, forming a Schiff base intermediate that cyclized to a hemiaminal adduct by intramolecular reaction with the phenolic hydroxyl group of PM. This bicyclic intermediate dimerized to form a five-ring compound with a central piperazine ring, which was characterized by electrospray ionization-liquid chromatography/mass spectrometry, NMR, and x-ray crystallography. PM also inhibited the modification of lysine residues and loss of enzymatic activity of RNase in the presence of GO and GLA and inhibited formation of the AGE/ALENε-(carboxymethyl)lysine during reaction of GO and GLA with bovine serum albumin. Our data suggest that the AGE/ALE inhibitory activity and the therapeutic effects of PM observed in diabetic animal models depend, at least in part, on its ability to trap reactive carbonyl intermediates in AGE/ALE formation, thereby inhibiting the chemical modification of tissue proteins.
Mechanism of Perturbation of Integrin-Mediated Cell-Matrix Interactions by Reactive Carbonyl Compounds and Its Implication
for Pathogenesis of Diabetic Nephropathy
Vadim K. Pedchenko 1 ,
Sergei V. ...Chetyrkin 1 ,
Peale Chuang 1 ,
Amy-Joan L. Ham 2 3 ,
Moin A. Saleem 4 ,
Peter W. Mathieson 4 ,
Billy G. Hudson 1 2 and
Paul A. Voziyan 1
1 Department of Medicine, Division of Nephrology, Vanderbilt University Medical Center, Nashville, Tennessee
2 Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee
3 Mass Spectrometry Research Center, Vanderbilt University Medical Center, Nashville, Tennessee
4 Children’s and Academic Renal Unit, University of Bristol, Bristol, U.K
Address correspondence and reprint requests to Paul A. Voziyan, Division of Nephrology, Vanderbilt University Medical Center,
S-3223 MCN, 1161 21st Ave. S., Nashville, Tennessee 37232-2372. E-mail: paul.voziyan{at}vanderbilt.edu
Abstract
Perturbation of interactions between cells and the extracellular matrix (ECM) of renal glomeruli may contribute to characteristic
histopathological lesions found in the kidneys of patients with diabetic nephropathy. However, the mechanism by which the
diabetic conditions may affect cell-ECM interactions is unknown. Existing hypotheses suggest a role of glucose in direct modification
of ECM. Here, we have demonstrated that carbonyl compound methylglyoxal (MGO) completely inhibited endothelial cell adhesion
to recombinant α3 noncollagenous 1 domain of type IV collagen mediated via a short collagenous region containing RGD (Arg-Gly-Asp)
sequence as well as binding of purified α v β 3 integrin to this protein. Specific MGO adducts of the arginine residue were detected within RGD sequence using mass spectrometry.
Modification by carbonyl compounds glyoxal or glycolaldehyde had similar but smaller effects. MGO strongly inhibited adhesion
of renal glomerular cells, podocytes, and mesangial cells to native collagen IV and laminin-1 as well as binding of collagen
IV to its major receptor in glomerular cells, α 1 β 1 integrin. In contrast, modification of these proteins by glucose had no effect on cell adhesion. Pyridoxamine, a promising
drug for treatment of diabetic nephropathy, protected cell adhesion and integrin binding from inhibition by MGO. We suggest
that in diabetes, perturbation of integrin-mediated cell-matrix interactions occurs via the modification of critical arginine
residues in renal ECM by reactive carbonyl compounds. This mechanism may contribute to the development of diabetic nephropathy.
CML, carboxymethyl lysine
3-DG, 3-deoxyglucosone
ECM, extracellular matrix
ELISA, enzyme-linked immunosorbent assay
GBM, glomerular basement membrane
GLA, glycolaldehyde
MGO, methylglyoxal
MS/MS, tandem mass spectrometry
NC1, noncollagenous 1
Footnotes
Accepted June 30, 2005.
Received May 11, 2005.
DIABETES
FUS1/TUSC2 is a mitochondrial tumor suppressor with activity to regulate cellular oxidative stress by maintaining balanced ROS production and mitochondrial homeostasis. Fus1 expression is inhibited ...by ROS, suggesting that individuals with a high level of ROS may have lower Fus1 in normal tissues and, thus, may be more prone to oxidative stress-induced side effects of cancer treatment, including radiotherapy. As the role of Fus1 in the modulation of cellular radiosensitivity is unknown, we set out to determine molecular mechanisms of Fus1 involvement in the IR response in normal tissues. Mouse whole-body irradiation methodology was employed to determine the role for Fus1 in the radiation response and explore underlying molecular mechanisms. Fus1(-/-) mice were more susceptible to radiation compared with Fus1(+/+) mice, exhibiting increased mortality and accelerated apoptosis of the GI crypt epithelial cells. Following untimely reentrance into the cell cycle, the Fus1(-/-) GI crypt cells died at accelerated rate via mitotic catastrophe that resulted in diminished and/or delayed crypt regeneration after irradiation. At the molecular level, dysregulated dynamics of activation of main IR response proteins (p53, NFκB, and GSK-3β), as well as key signaling pathways involved in oxidative stress response (SOD2, PRDX1, and cytochrome c), apoptosis (BAX and PARP1), cell cycle (Cyclins B1 and D1), and DNA repair (γH2AX) were found in Fus1(-/-) cells after irradiation. Increased radiosensitivity of other tissues, such as immune cells and hair follicles was also detected in Fus1(-/-) mice. Our findings demonstrate a previously unknown radioprotective function of the mitochondrial tumor suppressor Fus1 in normal tissues and suggest new individualized therapeutic approaches based on Fus1 expression.
This study compared the conformation of the antibody epitopes in Goodpasture's disease and Alport's post-transplantation nephritis in search of the pathogenesis of anti–basement membrane ...glomerulonephritis. The authors report evidence that the initiation of such disease depends on the conformation involving perturbation of the quaternary structure of basement-membrane collagen, eliciting an autoimmune response.
Goodpasture's disease is an organ-specific autoimmune disorder characterized by rapidly progressive glomerulonephritis, pulmonary hemorrhage, and glomerular pathological findings that include linear deposits of antibodies along the glomerular basement membrane (GBM) (Figure 1A).
1
,
2
(For this article we have studied Goodpasture's disease, which describes the specific entity in which the cause of organ dysfunction is proven to be anti-GBM antibodies, in contrast with Goodpasture's syndrome, which is a clinical term used to describe rapidly progressive glomerulonephritis and pulmonary hemorrhage.) Lerner and colleagues
3
passively transferred Goodpasture anti-GBM antibodies in a primate model, inducing glomerulonephritis and thereby showing that an autoantibody itself can . . .
One of the proposed roles of the GroEL-GroES cavity
is to provide an “infinite dilution” folding
chamber where protein substrate can fold avoiding deleterious
off-pathway aggregation. Support for ...this hypothesis has
been strengthened by a number of studies that demonstrated
a mandatory GroES requirement under nonpermissive solution
conditions, i.e., the conditions where proteins cannot
spontaneously fold. We have found that the refolding of
glutamine synthetase (GS) does not follow this pattern.
In the presence of natural osmolytes trimethylamine N-oxide
(TMAO) or potassium glutamate, refolding GS monomers readily
aggregate into very large inactive complexes and fail to
reactivate even at low protein concentration. Surprisingly,
under these “nonpermissive” folding conditions,
GS can reactivate with GroEL and ATP alone and does not
require the encapsulation by GroES. In contrast, the chaperonin
dependent reactivation of GS under another nonpermissive
condition of low Mg2+ (<2 mM MgCl2)
shows an absolute requirement of GroES. High-performance
liquid chromatography gel filtration analysis and irreversible
misfolding kinetics show that a major species of the GS
folding intermediates, generated under these “low
Mg2+” conditions exist as long-lived metastable
monomers that can be reactivated after a significantly
delayed addition of the GroEL. Our results indicate that
the GroES requirement for refolding of GS is not simply
dictated by the aggregation propensity of this protein
substrate. Our data also suggest that the GroEL-GroES encapsulated
environment is not required under all nonpermissive folding
conditions.
Our recent work identified a genetic variant of the α345 hexamer of the collagen IV scaffold that is present in patients with glomerular basement membrane diseases, Goodpasture’s disease (GP) and ...Alport syndrome (AS), and phenocopies of AS in knock-in mice. To understand the context of this “Zurich” variant, an 8-amino acid appendage, we developed a construct of the WT α345 hexamer using the single-chain NC1 trimer technology, which allowed us to solve a crystal structure of this key connection module. The α345 hexamer structure revealed a ring of 12 chloride ions at the trimer–trimer interface, analogous to the collagen α121 hexamer, and the location of the 170 AS variants. The hexamer surface is marked by multiple pores and crevices that are potentially accessible to small molecules. Loop-crevice-loop features constitute bioactive sites, where pathogenic pathways converge that are linked to AS and GP, and, potentially, diabetic nephropathy. In Pedchenko et al., we demonstrate that these sites exhibit conformational plasticity, a dynamic property underlying assembly of bioactive sites and hexamer dysfunction. The α345 hexamer structure is a platform to decipher how variants cause AS and how hypoepitopes can be triggered, causing GP. Furthermore, the bioactive sites, along with the pores and crevices on the hexamer surface, are prospective targets for therapeutic interventions.
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