Oral enzyme therapy for maple syrup urine disease (MSUD) suppresses plasma leucine levels in intermediate MSUD mice and healthy nonhuman primates.

Maple syrup urine disease (MSUD) is an inborn error of branched-chain amino acid metabolism affecting several thousand individuals worldwide. MSUD patients have elevated levels of plasma leucine and its metabolic product α-ketoisocaproate (KIC), which can lead to severe neurotoxicity, coma, and death. Patients must maintain a strict diet of protein restriction and medical formula, and periods of noncompliance or illness can lead to acute metabolic decompensation or cumulative neurological impairment. Given the lack of therapeutic options for MSUD patients, we sought to develop an oral enzyme therapy that can degrade leucine within the gastrointestinal tract prior to its systemic absorption and thus enable patients to maintain acceptable plasma leucine levels while broadening their access to natural protein. We identified a highly active leucine decarboxylase enzyme from Planctomycetaceae bacterium and used directed evolution to engineer the enzyme for stability to gastric and intestinal conditions. Following high-throughput screening of over 12 000 enzyme variants over 9 iterative rounds of evolution, we identified a lead variant, LDCv10, which retains activity following simulated gastric or intestinal conditions in vitro. In intermediate MSUD mice or healthy nonhuman primates given a whey protein meal, oral treatment with LDCv10 suppressed the spike in plasma leucine and KIC and reduced the leucine area under the curve in a dose-dependent manner. Reduction in plasma leucine correlated with decreased brain leucine levels following oral LDCv10 treatment. Collectively, these data support further development of LDCv10 as a potential new therapy for MSUD patients.

A chemoenzymatic strategy for site-selective functionalization of native peptides and proteins.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

Blood serum alpha fetoprotein enhancer binding protein, a tumor suppressor, decreases in chronic HBV hepatitis patients as hepatocellular cancer appears.

Chronic hepatitis increases the risk of hepatocellular carcinoma (HCC). To test whether circulating proteins reflect hepatic carcinogenesis, sera from patients and controls were albumin depleted, enriched for glycoproteins, digested with trypsin, and subjected to reverse phase chromatography and tandem mass spectrometry. Alpha-fetoprotein enhancer binding protein (AFPebp), a tumor suppressor, was repeatedly identified in sera from chronic HBV hepatitis patients. We independently identified and quantified AFPebp with a deuterated, phenylisocyanate-labeled synthetic peptide standard. Elevated AFPebp levels in sera from chronic HBV hepatitis patients decreased as cancer developed. These data suggest that rising AFPebp levels in chronic HBV hepatitis may be protective, while falling levels may contribute to HCC development.

Covalent adduction of human serum albumin by 4-hydroxy-2-nonenal: kinetic analysis of competing alkylation reactions.

The electrophilic lipid oxidation product 4-hydroxy-2-nonenal (HNE) reacts with proteins to form covalent adducts, and this damage has been implicated in pathologies associated with oxidative stress. HNE adduction of blood proteins, such as human serum albumin (HSA), yields adducts that may serve as markers of oxidative stress in vivo. We used liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the P-Mod algorithm to map the sites of 10 adducts formed by reaction of HNE with HSA in vitro. The detected adducts included Michael adducts formed at histidine and lysine residues. The selectivity of HNE in competing adduction reactions was evaluated by analysis of kinetics for HNE Michael adduction at six targeted HSA histidine residues. Reaction kinetics were analyzed by selected reaction monitoring in LC-MS-MS using stable isotope tagging with phenyl isocyanate. Rate constants ranged over 4 orders of magnitude, with the order of reactivity being H242 > H510 > H67 > H367 > H247 approximately K233. The most reactive target, H242, is located in a fatty acid- and drug binding cavity in subdomain IIa of HSA and appears to be a hot-spot for HNE modification. Analysis of adduction kinetics together with HSA structure and target residue pK(a) values suggest that location in the hydrophobic binding cavity and low predicted pK(a) of H242 account for its high reactivity toward HNE. H242 adducts may be preferred products of adduction by lipophilic electrophiles and may comprise a family of biomarkers for oxidative stress.

Enhanced sequence coverage of proteins in human cerebrospinal fluid using multiple enzymatic digestion and linear ion trap LC-MS/MS.

The cerebrospinal fluid (CSF) provides a ready access into the health state of the central nervous system, and alterations in some CSF proteins have been documented in brain disease. However, the complete variety of proteins is not known and methods to identify protein components are still being developed. The goal of this study was to examine the sequence coverage obtained from human CSF digests produced with different proteases. Enzymatic digests of CSF proteins were obtained with arginine-C endopeptidase (ArgC), glutamic acid endopeptidase (GluC), chymotrypsin, trypsin and their combinations, and then examined using reverse phase chromatography and a Finnigan LTQ linear ion trap mass spectrometer. Peptide sequences were identified with BioWorks 3.1 and sequence coverage calculated for the 38 most confidently identified proteins. Trypsin and GluC yielded greater coverage than chymotrypsin, while ArgC had the least sequence coverage. Protein sequence coverage was affected only slightly over four orders of magnitude dynamic range of abundance. Combining the peptides derived from different proteases further increased the coverage. Maximal sequence coverage was achieved by combining digest results from both GluC and trypsin. These results have implications for future studies to identify CSF proteins and their post-translational modifications.

Identification of disease markers in human cerebrospinal fluid using lipidomic and proteomic methods.

Lipids comprise the bulk of the dry mass of the brain. In addition to providing structural integrity to membranes, insulation to cells and acting as a source of energy, lipids can be rapidly converted to mediators of inflammation or to signaling molecules that control molecular and cellular events in the brain. The advent of soft ionization procedures such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) have made it possible for compositional studies of the diverse lipid structures that are present in brain. These include phospholipids, ceramides, sphingomyelin, cerebrosides, cholesterol and their oxidized derivatives. Lipid analyses have delineated metabolic defects in disease conditions including mental retardation, Parkinson's Disease (PD), schizophrenia, Alzheimer's Disease (AD), depression, brain development, and ischemic stroke. In this review, we examine the structure of the major lipid classes in the brain, describe methods used for their characterization, and evaluate their role in neurological diseases. The potential utility of characterizing lipid markers in the brain, with specific emphasis on disease mechanisms, will be discussed. Additionally, we describe several proteomic strategies for characterizing lipid-metabolizing proteins in human cerebrospinal fluid (CSF). These proteins may be potential therapeutic targets since they transport lipids required for neuronal growth or convert lipids into molecules that control brain physiology. Combining lipidomics and proteomics will enhance existing knowledge of disease pathology and increase the likelihood of discovering specific markers and biochemical mechanisms of brain diseases.

Differential DNA recognition and cleavage by EcoRI dependent on the dynamic equilibrium between the two forms of the malondialdehyde-deoxyguanosine adduct.

DNA damage may alter the outcome of protein-nucleic acid interactions. The malondialdehyde-deoxyguanosine adduct, 3-(2'-deoxy-beta-d-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10-(3H)-one (M(1)dG), miscodes in vivo and in vitro. M(1)dG is an exocyclic adduct that undergoes ring-opening in duplex DNA to form the acyclic adduct, N(2)-(3-oxo-1-propenyl)-deoxyguanosine (N(2)-OPdG). These two adducts have different effects on DNA polymerase bypass and may affect other DNA processing enzymes. We employed the EcoRI restriction endonuclease as a model for the interaction of DNA binding proteins with adducted DNA substrates. The presence of M(1)dG in the EcoRI recognition sequence impaired the ability of the enzyme to cleave DNA, resulting in only 60% cleavage of the adducted strand and 75% cleavage of the complementary strand. Three adducts of similar structure to M(1)dG that are unable to ring-open were cleaved poorly, or not at all, by EcoRI. None of the adducts appeared to inactivate or sequester EcoRI. Additional studies with BssHII and PauI confirmed these results and demonstrated a positional effect of M(1)dG on cleavage efficiency. These data suggest dissimilar modes of protein-nucleic acid interactions based on differences in adduct structure. Comparison of the solution structures of DNA adducts and the crystal structure of EcoRI complexed to substrate suggest a model to explain the functional differences.

Exocyclic DNA lesions stimulate DNA cleavage mediated by human topoisomerase II alpha in vitro and in cultured cells.

DNA adducts are mutagenic and clastogenic. Because of their harmful nature, lesions are recognized by many proteins involved in DNA repair. However, mounting evidence suggests that lesions also are recognized by proteins with no obvious role in repair processes. One such protein is topoisomerase II, an essential enzyme that removes knots and tangles from the DNA. Because topoisomerase II generates a protein-linked double-stranded DNA break during its catalytic cycle, it has the potential to fragment the genome. Previous studies indicate that abasic sites and other lesions that distort the double helix stimulate topoisomerase II-mediated DNA cleavage. Therefore, to further explore interactions between DNA lesions and the enzyme, the effects of exocyclic adducts on DNA cleavage mediated by human topoisomerase IIalpha were determined. When located within the four-base overhang of a topoisomerase II cleavage site (at the +2 or +3 position 3' relative to the scissile bond), 3,N(4)-ethenodeoxycytidine, 3,N(4)-etheno-2'-ribocytidine, 1,N(2)-ethenodeoxyguanosine, pyrimido[1,2-a]purin-10(3H)-one deoxyribose (M(1)dG), and 1,N(2)-propanodeoxyguanosine increased DNA scission approximately 5-17-fold. Enhanced cleavage did not result from an increased affinity of topoisomerase IIalpha for adducted DNA or a decreased rate of religation. Therefore, it is concluded that these exocyclic lesions act by accelerating the forward rate of enzyme-mediated DNA scission. Finally, treatment of cultured human cells with 2-chloroacetaldehyde, a reactive metabolite of vinyl chloride that generates etheno adducts, increased cellular levels of DNA cleavage by topoisomerase IIalpha. This finding suggests that type II topoisomerases interact with exocyclic DNA lesions in physiological systems.

In vitro bypass of malondialdehyde-deoxyguanosine adducts: differential base selection during extension by the Klenow fragment of DNA polymerase I is the critical determinant of replication outcome.

The major malondialdehyde-derived adduct in DNA is 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M(1)dG). M(1)dG undergoes hydrolytic ring opening in duplex DNA to 9-(2'-deoxy-beta-D-erythro-pentofuranosyl)-N(2)-(3-oxo-1-propenyl)guanine (N(2)OPdG). Template-primers were constructed containing M(1)dG or N(2)OPdG in a (CpG)(4) repeat sequence and replicated with the Klenow fragment of DNA polymerase I (Kf). Incorporation opposite the lesion and replication beyond the adduct sites by Kf was reduced compared to unadducted controls. The amount of bypass to full-length products was significantly greater with the acyclic adduct, N(2)OPdG, than with the cyclic adduct, M(1)dG. Sequence analysis indicated that the fully extended primers contained dC opposite both adducts when replication was conducted with Kf exo(+). In contrast, with Kf exo(-), primers extended past M(1)dG contained T opposite the adduct, but primers extended past N(2)OPdG contained dC opposite the adduct. Single nucleotide incorporation experiments indicated that Kf exo(-) incorporates all four nucleotides opposite M(1)dG or N(2)OPdG. Kf exo(+) removed dA, dG, and T opposite M(1)dG and N(2)OPdG but was much less active when dC was opposite the adduct. NMR studies on duplex DNA indicated that N(2)OPdG hydrogen bonds with dC in the complementary strand. The fact that base pairing can occur for the acyclic adduct may explain why N(2)OPdG is less blocking than M(1)dG. These results support in vivo findings that the ring-closed adduct, M(1)dG, is more mutagenic than the ring-opened adduct, N(2)OPdG. They also provide a detailed picture of in vitro replication in which the outcome is determined primarily by the selectivity of template-primer extension beyond rather than insertion opposite the adducts.

Kinetics and mechanism of the general-acid-catalyzed ring-closure of the malondialdehyde-DNA adduct, N2-(3-oxo-1-propenyl)deoxyguanosine (N2OPdG-), to 3-(2'-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin- 10(3H)-one (M1dG).

3-(2'-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG) is the major product of the reaction of deoxyguanosine with malondialdehyde (MDA). M1dG blocks replication by DNA polymerases in vitro and is mutagenic in vivo. M1dG reacts with hydroxide to form the N2-(3-oxo-1-propenyl)deoxyguanosine anion (N2OPdG-). This reaction is pH-dependent and reverses under neutral and acidic conditions to form M1dG. Here we describe the kinetics and mechanism of the ring-closure reaction in both the nucleoside and oligonucleotides. Kinetic analysis of absorbance and fluorescence changes demonstrates that ring-closure is biphasic, leading to the rapid formation of an intermediate that slowly converts to M1dG in a general-acid-catalyzed reaction. The dependence of the rate of the rapid phase on pH reveals the pKa for protonated N2OPdG is 6.9. One-dimensional 1H NMR and DQF-COSY experiments identified two distinct intermediates, N2OPdG-H and 8-hydroxy-6,7-propenodeoxyguanosine (HO-Prene-dG), that are formed upon acidification of N2OPdG-. Characterization of ring-closure in single-stranded and in melted duplex oligonucleotides shows M1dG formation is also acid-catalyzed in single-stranded oligonucleotides and that the denaturation of an oligonucleotide duplex enhances ring-closure. This work details the complexity of ring-closure in the nucleoside and oligonucleotides and provides new insight into the role of duplex DNA in catalyzing ring-opening and ring-closing of M1dG and N2OPdG.

Kinetic and thermodynamic analysis of the hydrolytic ring-opening of the malondialdehyde-deoxyguanosine adduct, 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)- pyrimido[1,2-alpha]purin-10(3H)-one.

3-(2'-Deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M1dG) is the major reaction product of deoxyguanosine with malondialdehyde or base propenals. M1dG undergoes hydrolytic ring-opening to N2-oxopropenyl-deoxyguanosine (N2OPdG) under basic conditions. We report that ring-opening of M1dG as a nucleoside or in oligonucleotides is a reversible second-order reaction with hydroxide ion. NMR and UV analysis revealed N2OPdG(-) to be the only product of M1dG ring-opening in basic solution. The rate constant for reaction of M1dG with hydroxide is 3.8 M(-1) s(-1), and the equilibrium constant is calculated to be 2.1 +/- 0.3 x 10(4) M(-1) at 25 degrees C. Equilibrium constants determined by spectroscopic analysis of the reaction end-point or by thermodynamic analysis of rate constants determined over a range of temperatures yielded a value 2.5 +/- 0.2 x 10(4) M(-1). Kinetic analysis of ring-opening of M1dG in oligonucleotides indicated the rate constant for ring-opening is decreased 10-fold compared to that in the nucleoside. Flanking purines or pyrimidines did not significantly alter the rate constants for ring-opening, but purines flanking M1dG enhanced the rate constant for the reverse reaction. A mechanism is proposed for ring-opening of M1dG under basic conditions and a role is proposed for duplex DNA in accelerating the rate of ring-opening of M1dG at neutral pH.

Malondialdehyde adducts in DNA arrest transcription by T7 RNA polymerase and mammalian RNA polymerase II.

Malondialdehyde, a genotoxic byproduct of lipid peroxidation, reacts with guanine in DNA to form pyrimido[1,2-alpha]purin-10(3H)one (M(1)dG), the first endogenous DNA lesion found to be a target of nucleotide excision repair enzymes. A subpathway of nucleotide excision repair, transcription-coupled repair, is thought to occur when RNA polymerase (RNAP) is arrested at damage in transcribed DNA strands and might function for efficient removal of M(1)dG in active genes. Results presented here show that M(1)dG and its stable, exocyclic analog 1,N(2)-propanodeoxyguanine (PdG), arrest translocation of T7 RNAP and mammalian RNAPII when located in the transcribed strand of a DNA template. M(1)dG paired with thymine is exocyclic and poses a stronger block to transcription than the acyclic N(2)-(3-oxo-1-propenyl)-dG, formed upon cytosine-catalyzed opening of M(1)dG in duplex DNA. PdG is a complete block to RNAPII regardless of base pairing. The elongation factor TFIIS (SII) induces reversal and RNA transcript cleavage by RNAPII arrested at PdG. Thus, arrested RNAPII complexes may be stable at M(1)dG in cells and may resume transcription once the offending adduct is removed. The conclusion from this work is that malondialdehyde adducts in the transcribed strand of expressed genes are strong blocks to RNAPs and are targets for cellular transcription-coupled repair. If so, then M(1)dG, already known to be highly mutagenic in human cells, also may contribute to apoptosis in the developing tissues of individuals with Cockayne's syndrome, a hereditary disorder characterized by transcription-coupled repair deficiency.