Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 15 de 15
Filter
Add more filters










Publication year range
1.
J Neuromuscul Dis ; 11(2): 275-284, 2024.
Article in English | MEDLINE | ID: mdl-38277301

ABSTRACT

Dystroglycanopathies are a group of muscle degenerative diseases characterized with significant reduction in matriglycan expression critical in disease pathogenesis. Missense point mutations in the Fukutin-related protein (FKRP) gene cause variable reduction in the synthesis of matriglycan on alpha-dystroglycan (α-DG) and a wide range of disease severity. Data analyses of muscle biopsies from patients fail to show consistent correlation between the levels of matriglycan and clinical phenotypes. By reviewing clinical reports in conjunction with analysis of clinically relevant mouse models, we identify likely causes for the confusion. Nearly all missense FKRP mutations retain variable, but sufficient function for the synthesis of matriglycan during the later stage of muscle development and periods of muscle regeneration. These factors lead to a highly heterogenous pattern of matriglycan expression in diseased muscles, depending on age and stages of muscle regeneration. The limited size in clinical biopsy samples from different parts of even a single muscle tissue at different time points of disease progression may well mis-represent the residual function (base-levels) of the mutated FKRPs and phenotypes. We propose to use a simple Multi Point tool from ImageJ to more accurately measure the signal intensity of matriglycan expression on fiber membrane for assessing mutant FKRP function and therapeutic efficacy. A robust and sensitive immunohistochemical protocol would further improve reliability and comparability for the detection of matriglycan.


Subject(s)
Dystroglycans , Pentosyltransferases , Animals , Humans , Mice , Dystroglycans/genetics , Dystroglycans/metabolism , Glycosylation , Pentosyltransferases/genetics , Pentosyltransferases/metabolism , Phenotype , Reproducibility of Results
2.
Mol Ther ; 31(12): 3478-3489, 2023 Dec 06.
Article in English | MEDLINE | ID: mdl-37919902

ABSTRACT

Mutations in the fukutin-related protein (FKRP) gene cause dystroglycanopathy, with disease severity ranging from mild LGMD2I to severe congenital muscular dystrophy. Recently, considerable progress has been made in developing experimental therapies, with adeno-associated virus (AAV) gene therapy and ribitol treatment demonstrating significant therapeutic effect. However, each treatment has its strengths and weaknesses. AAV gene therapy can achieve normal levels of transgene expression, but it requires high doses, with toxicity concerns and variable distribution. Ribitol relies on residual FKRP function and restores limited levels of matriglycan. We hypothesized that these two treatments can work synergistically to offer an optimized therapy with efficacy and safety unmatched by each treatment alone. The most effective treatment is the combination of high-dose (5e-13 vg/kg) AAV-FKRP with ribitol, whereas low dose (1e-13 vg/kg) AAV-FKRP combined with ribitol showed a 22.6% increase in positive matriglycan fibers and the greater improvement in pathology when compared to low-dose AAV-FKRP alone. Together, our results support the potential benefits of combining ribitol with AAV gene therapy for treating FKRP-related muscular dystrophy. The fact that ribitol is a metabolite in nature and has already been tested in animal models and clinical trials in humans without severe side effects provides a safety profile for it to be trialed in combination with AAV gene therapy.


Subject(s)
Muscular Dystrophies , Pentosyltransferases , Animals , Humans , Pentosyltransferases/genetics , Pentosyltransferases/metabolism , Pentosyltransferases/therapeutic use , Ribitol/metabolism , Ribitol/therapeutic use , Dependovirus/genetics , Dependovirus/metabolism , Dystroglycans/metabolism , Muscular Dystrophies/drug therapy , Genetic Therapy/methods , Mutation , Muscle, Skeletal/metabolism
3.
Mol Ther Methods Clin Dev ; 17: 271-280, 2020 Jun 12.
Article in English | MEDLINE | ID: mdl-31988979

ABSTRACT

Dystroglycanopathy, a subgroup of muscular dystrophies, is characterized by hypoglycosylation of α-dystroglycan (α-DG), which reduces its laminin-binding activity to extracellular matrix proteins, causing progressive loss of muscle integrity and function. Mutations in the fukutin-related protein (FKRP) gene are the most common causes of dystroglycanopathy. FKRP transfers ribitol-5-phosphate to the O-mannosyl glycan on α-DG from substrate cytidine diphosphate (CDP)-ribitol, which is synthesized by isoprenoid synthase domain-containing protein (ISPD). We previously reported that oral administration of ribitol restores therapeutic levels of functional glycosylation of α-DG (F-α-DG) in a FKRP mutant mouse model. Here we examine the contribution of adeno-associated virus (AAV)-mediated overexpression of ISPD to the levels of CDP-ribitol and F-α-DG with and without ribitol supplementation in the disease model. ISPD overexpression alone and in combination with ribitol improves dystrophic phenotype. Furthermore, the combined approach of ribitol and ISPD acts synergistically, increasing F-α-DG up to 40% of normal levels in cardiac muscle and more than 20% in limb and diaphragm. The results suggest that low levels of substrate limit production of CDP-ribitol, and endogenous ISPD also becomes a limiting factor in the presence of a supraphysiological concentration of ribitol. Our data support further investigation of the regulatory pathway for enhancing efficacy of ribitol supplement to FKRP-related dystroglycanopathy.

4.
Nat Commun ; 9(1): 3448, 2018 08 27.
Article in English | MEDLINE | ID: mdl-30150693

ABSTRACT

O-mannosylated α-dystroglycan (α-DG) serves as receptors for cell-cell and cell-extracellular matrix adhesion and signaling. Hypoglycosylation of α-DG is involved in cancer progression and underlies dystroglycanopathy with aberrant neuronal development. Here we report that ribitol, a pentose alcohol with previously unknown function in mammalian cells, partially restores functional O-mannosylation of α-DG (F-α-DG) in the dystroglycanopathy model containing a P448L mutation in fukutin-related protein (FKRP) gene, which is clinically associated with severe congenital muscular dystrophy. Oral administration of ribitol increases levels of ribitol-5-phosphate and CDP-ribitol and restores therapeutic levels of F-α-DG in skeletal and cardiac muscles. Furthermore, ribitol, given before and after the onset of disease phenotype, reduces skeletal muscle pathology, significantly decreases cardiac fibrosis and improves skeletal and respiratory functions in the FKRP mutant mice. Ribitol treatment presents a new class, low risk, and easy to administer experimental therapy to restore F-α-DG in FKRP-related muscular dystrophy.


Subject(s)
Dystroglycans/metabolism , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscular Dystrophies/drug therapy , Muscular Dystrophies/metabolism , Proteins/metabolism , Ribitol/therapeutic use , Administration, Oral , Animals , Blotting, Western , Cell Line , Female , Glycosylation/drug effects , Immunohistochemistry , Mice , Myocardium/metabolism , Pentosephosphates/metabolism , Pentosyltransferases , Plethysmography , Proteins/genetics , Transferases
5.
Oncotarget ; 7(38): 61601-61618, 2016 Sep 20.
Article in English | MEDLINE | ID: mdl-27533247

ABSTRACT

Vesicular stomatitis virus (VSV) based recombinant viruses (such as VSV-ΔM51) are effective oncolytic viruses (OVs) against a majority of pancreatic ductal adenocarcinoma (PDAC) cell lines. However, some PDAC cell lines are highly resistant to VSV-ΔM51. We recently showed that treatment of VSV-resistant PDAC cells with ruxolitinib (JAK1/2 inhibitor) or TPCA-1 (IKK-ß inhibitor) breaks their resistance to VSV-ΔM51. Here we compared the global effect of ruxolitinib or TPCA-1 treatment on cellular gene expression in PDAC cell lines highly resistant to VSV-ΔM51. Our study identified a distinct subset of 22 interferon-stimulated genes (ISGs) downregulated by both ruxolitinib and TPCA-1. Further RNA and protein analyses demonstrated that 4 of these genes (MX1, EPSTI1, XAF1, and GBP1) are constitutively co-expressed in VSV-resistant, but not in VSV-permissive PDACs, thus serving as potential biomarkers to predict OV therapy success. Moreover, shRNA-mediated knockdown of one of such ISG, MX1, showed a positive effect on VSV-ΔM51 replication in resistant PDAC cells, suggesting that at least some of the identified ISGs contribute to resistance of PDACs to VSV-ΔM51. As certain oncogene and tumor suppressor gene variants are often associated with increased tropism of OVs to cancer cells, we also analyzed genomic DNA in a set of PDAC cell lines for frequently occurring cancer associated mutations. While no clear correlation was found between such mutations and resistance of PDACs to VSV-ΔM51, the analysis generated valuable genotypic data for future studies.


Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Pancreatic Ductal/therapy , Oncolytic Virotherapy/methods , Oncolytic Viruses/physiology , Pancreatic Neoplasms/therapy , Protein Kinase Inhibitors/pharmacology , Vesiculovirus/physiology , Adaptor Proteins, Signal Transducing , Amides/pharmacology , Apoptosis Regulatory Proteins , Carcinoma, Pancreatic Ductal/genetics , Cell Line, Tumor , DNA Mutational Analysis , Down-Regulation , GTP-Binding Proteins/metabolism , Gene Expression/drug effects , Gene Expression Profiling , Humans , I-kappa B Kinase/antagonists & inhibitors , Interferon Type I/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 2/antagonists & inhibitors , Mutation , Myxovirus Resistance Proteins/genetics , Myxovirus Resistance Proteins/metabolism , Neoplasm Proteins/metabolism , Nitriles , Pancreatic Neoplasms/genetics , Pyrazoles/pharmacology , Pyrimidines , RNA Interference , RNA, Small Interfering/metabolism , Thiophenes/pharmacology , Transcriptome/drug effects , Virus Replication/drug effects
6.
Mol Ther Methods Clin Dev ; 3: 16036, 2016.
Article in English | MEDLINE | ID: mdl-27331076

ABSTRACT

The reversibility of neuropathic lysosomal storage diseases, including MPS IIIA, is a major goal in therapeutic development, due to typically late diagnoses and a large population of untreated patients. We used self-complementary adeno-associated virus (scAAV) serotype 9 vector expressing human N-sulfoglucosamine sulfohydrolase (SGSH) to test the efficacy of treatment at later stages of the disease. We treated MPS IIIA mice at 1, 2, 3, 6, and 9 months of age with an intravenous injection of scAAV9-U1a-hSGSH vector, leading to restoration of SGSH activity and reduction of glycosaminoglycans (GAG) throughout the central nervous system (CNS) and somatic tissues at a dose of 5E12 vg/kg. Treatment up to 3 months age improved learning ability in the Morris water maze at 7.5 months, and lifespan was normalized. In mice treated at 6 months age, behavioral performance was impaired at 7.5 months, but did not decline further when retested at 12 months, and lifespan was increased, but not normalized. Treatment at 9 months did not increase life-span, though the GAG storage pathology in the CNS was improved. The study suggests that there is potential for gene therapy intervention in MPS IIIA at intermediate stages of the disease, and extends the clinical relevance of our systemic scAAV9-hSGSH gene delivery approach.

7.
Virology ; 485: 340-54, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26331681

ABSTRACT

Vesicular stomatitis virus (VSV) is an effective oncolytic virus against most human pancreatic ductal adenocarcinoma (PDAC) cell lines. However, some PDAC cell lines are highly resistant to oncolytic VSV-ΔM51 infection. To better understand the mechanism of resistance, we tested a panel of 16 small molecule inhibitors of different cellular signaling pathways, and identified TPCA-1 (IKK-ß inhibitor) and ruxolitinib (JAK1/2 inhibitor), as strong enhancers of VSV-ΔM51 replication and virus-mediated oncolysis in all VSV-resistant PDAC cell lines. Both TPCA-1 and ruxolitinib similarly inhibited STAT1 and STAT2 phosphorylation and decreased expression of antiviral genes MxA and OAS. Moreover, an in situ kinase assay provided biochemical evidence that TPCA-1 directly inhibits JAK1 kinase activity. Together, our data demonstrate that TPCA-1 is a unique dual inhibitor of IKK-ß and JAK1 kinase, and provide a new evidence that upregulated type I interferon signaling plays a major role in resistance of pancreatic cancer cells to oncolytic viruses.


Subject(s)
Amides/pharmacology , I-kappa B Kinase/antagonists & inhibitors , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/virology , Protein Kinase Inhibitors/pharmacology , Thiophenes/pharmacology , Vesicular stomatitis Indiana virus/physiology , Cell Line, Tumor , Cytopathogenic Effect, Viral/drug effects , Humans , Interferon Type I/metabolism , Janus Kinases/antagonists & inhibitors , Janus Kinases/metabolism , Myxovirus Resistance Proteins/genetics , Nitriles , Oncolytic Viruses/drug effects , Oncolytic Viruses/physiology , Pyrazoles/pharmacology , Pyrimidines , STAT Transcription Factors/metabolism , Sendai virus/drug effects , Sendai virus/physiology , Signal Transduction/drug effects , Vesicular stomatitis Indiana virus/drug effects , Virus Replication/drug effects
8.
Virology ; 483: 126-40, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25965802

ABSTRACT

Virus-encoded tumor suppressor p53 transgene expression has been successfully used in vesicular stomatitis virus (VSV) and other oncolytic viruses (OVs) to enhance their anticancer activities. However, p53 is also known to inhibit virus replication via enhanced type I interferon (IFN) antiviral responses. To examine whether p53 transgenes enhance antiviral signaling in human pancreatic ductal adenocarcinoma (PDAC) cells, we engineered novel VSV recombinants encoding human p53 or the previously described chimeric p53-CC, which contains the coiled-coil (CC) domain from breakpoint cluster region (BCR) protein and evades the dominant-negative activities of endogenously expressed mutant p53. Contrary to an expected enhancement of antiviral signaling by p53, our global analysis of gene expression in PDAC cells showed that both p53 and p53-CC dramatically inhibited type I IFN responses. Our data suggest that this occurs through p53-mediated inhibition of the NF-κB pathway. Importantly, VSV-encoded p53 or p53-CC did not inhibit antiviral signaling in non-malignant human pancreatic ductal cells, which retained their resistance to all tested VSV recombinants. To the best of our knowledge, this is the first report of p53-mediated inhibition of antiviral signaling, and it suggests that OV-encoded p53 can simultaneously produce anticancer activities while assisting, rather than inhibiting, virus replication in cancer cells.


Subject(s)
Interferon Type I/antagonists & inhibitors , Signal Transduction , Transgenes , Tumor Suppressor Protein p53/metabolism , Vesiculovirus/physiology , Virus Replication , Cell Line, Tumor , Host-Pathogen Interactions , Humans , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tumor Suppressor Protein p53/genetics , Vesiculovirus/genetics , Vesiculovirus/immunology , Viral Proteins/genetics , Viral Proteins/metabolism
9.
Virus Res ; 176(1-2): 16-32, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23796410

ABSTRACT

Vesicular stomatitis virus (VSV) is a prototypic nonsegmented negative-strand RNA virus. VSV's broad cell tropism makes it a popular model virus for many basic research applications. In addition, a lack of preexisting human immunity against VSV, inherent oncotropism and other features make VSV a widely used platform for vaccine and oncolytic vectors. However, VSV's neurotropism that can result in viral encephalitis in experimental animals needs to be addressed for the use of the virus as a safe vector. Therefore, it is very important to understand the determinants of VSV tropism and develop strategies to alter it. VSV glycoprotein (G) and matrix (M) protein play major roles in its cell tropism. VSV G protein is responsible for VSV broad cell tropism and is often used for pseudotyping other viruses. VSV M affects cell tropism via evasion of antiviral responses, and M mutants can be used to limit cell tropism to cell types defective in interferon signaling. In addition, other VSV proteins and host proteins may function as determinants of VSV cell tropism. Various approaches have been successfully used to alter VSV tropism to benefit basic research and clinically relevant applications.


Subject(s)
Vesiculovirus/physiology , Viral Tropism , Animals , Humans , Vesiculovirus/genetics , Viral Proteins/genetics , Viral Proteins/metabolism
10.
J Virol ; 84(17): 8673-82, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20538857

ABSTRACT

The linear DNA genomes of recombinant adeno-associated virus (rAAV) gene delivery vectors are acted upon by multiple DNA repair and recombination pathways upon release into the host nucleus, resulting in circularization, concatemer formation, or chromosomal integration. We have compared the fates of single-strand rAAV (ssAAV) and self-complementary AAV (scAAV) genomes in cell lines deficient in each of three signaling factors, ATM, ATR, and DNA-PK(CS), orchestrating major DNA double-strand break (DSB) repair pathways. In cells deficient in ATM, transduction as scored by green fluorescent protein (GFP) expression is increased relative to that in wild-type (wt) cells by 2.6-fold for ssAAV and 6.6-fold for scAAV vectors, arguing against a mechanism related to second-strand synthesis. The augmented transduction is not reflected in Southern blots of nuclear vector DNA, suggesting that interactions with ATM lead to silencing in normal cells. The additional functional genomes in ATM(-/-) cells remain linear, and the number of circularized genomes is not affected by the mutation, consistent with compartmentalization of genomes into different DNA repair pathways. A similar effect is observed in ATR-deficient cells but is specific for ssAAV vector. Conversely, a large decrease in transduction is observed in cells deficient in DNA-PK(CS), which is involved in DSB repair by nonhomologous end joining rather than homologous recombination. The mutations also have differential effects on chromosomal integration of ssAAV versus scAAV vector genomes. Integration of ssAAV was specifically reduced in ATM(-/-) cells, while scAAV integration was more profoundly inhibited in DNA-PK(CS)(-/-) cells. Taken together, the results suggest that productive rAAV genome circularization is mediated primarily by nonhomologous end joining.


Subject(s)
DNA Breaks, Double-Stranded , DNA Repair , Dependovirus/genetics , Gene Targeting/instrumentation , Genetic Vectors/genetics , Genome, Viral , Cell Line, Tumor , DNA, Single-Stranded/genetics , Dependovirus/physiology , Genetic Vectors/physiology , Humans , Recombination, Genetic , Virus Integration
11.
Magn Reson Chem ; 45 Suppl 1: S73-83, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18157811

ABSTRACT

Differentially isotopically enriched 1-73((13)C,(15)N)/74-108((15)N) and 1-73((15)N)/74-108((13)C,(15)N) Escherichia coli thioredoxin reassemblies prepared by fragment complementation were investigated by high-resolution magic angle spinning solid-state NMR spectroscopy. Nearly complete resonance assignments, secondary and tertiary structure analysis are reported for 1-73((13)C,(15)N)/74-108((15)N) reassembled thioredoxin. Temperature dependence of the dipolar-assisted rotational resonance (DARR) spectra reveals the residues undergoing intermediate timescale motions at temperatures below - 15 degrees C. Analysis of the DARR intensity buildups as a function of mixing time in these reassemblies indicates that at long mixing times medium- and long-range cross-peaks do not experience dipolar truncation, suggesting that isotopic dilution is not required for gaining nontrivial distance restraints for structure calculations.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Thioredoxins/chemistry , Carbon Isotopes , Escherichia coli Proteins , Motion , Nitrogen Isotopes , Protein Conformation , Temperature
12.
Biophys J ; 89(5): 3293-302, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16113108

ABSTRACT

A growing number of natively disordered proteins undergo a folding/binding process that is essential for their biological function. An interesting question is whether these proteins have incompletely solvated regions that drive the folding/binding process. Although the presence of predominantly hydrophobic buried regions can be easily ascertained by high-sensitivity differential scanning calorimetry analysis, the identification of those residues implicated in the burial requires NMR analysis. We have selected a partially solvated natively disordered fragment of Escherichia coli, thioredoxin, C37 (38-108), for full NMR spectral assignment. The secondary chemical shifts, temperature coefficients, and relaxation rates (R(1) and R(2)) of this fragment indicate the presence of a flexible backbone without a stable hydrogen bond network near neutral pH. (1)H-(15)N heteronuclear single quantum coherence analysis of the pH dependence of amide chemical shifts in fragment C37 within pH 2.0 and 7.0 suggests the presence of interactions between nonionizable residues and the carboxylate groups of four Asp and four Glu residues. The pH midpoints (pH(m)) of the amides in the ionizable residues (Asp or Glu) and, consequently, the shifts in the pH(m) (DeltapH(m)) of these residues with respect to model tetrapeptides, are sequence-dependent; and the nonionizable residues that show pH dependence cluster around the ionizable ones. The same pH dependence has been observed in two fragments: M37 (38-73) and C73 (74-108), ruling out the participation of long-range interactions. Our studies indicate the presence of a 15-residue pH-dependent segment with the highest density of ionizable sites in the disordered ensembles of fragments C37 and M37. The observed correlations between ionizable and nonionizable residues in this segment suggest the organization of the backbone and side chains through local and medium-range interactions up to nine residues apart, in contrast to only a few interactions in fragment C73. These results agree qualitatively with the predominantly hydrophobic buried surface detected only in fragments C37 and M37 by highly sensitive differential scanning calorimetry analysis. This work offers a sensitive and rapid new tool to obtain clues about local and nonlocal interactions between ionizable and nonionizable residues in the growing family of natively disordered small proteins with full NMR assignments.


Subject(s)
Amides/chemistry , Peptides/chemistry , Thioredoxins/chemistry , Aspartic Acid/chemistry , Calorimetry, Differential Scanning , Escherichia coli/metabolism , Glutamic Acid/chemistry , Hydrogen Bonding , Hydrogen-Ion Concentration , Ions , Magnetic Resonance Spectroscopy/methods , Mutagenesis , Protein Binding , Protein Conformation , Protein Denaturation , Protein Folding , Protons , Temperature , Time Factors
13.
J Phys Chem B ; 109(38): 18135-45, 2005 Sep 29.
Article in English | MEDLINE | ID: mdl-16853329

ABSTRACT

De novo site-specific 13C and 15N backbone and sidechain resonance assignments are presented for uniformly enriched E. coli thioredoxin, established using two-dimensional homo- and heteronuclear solid-state magic angle spinning NMR correlation spectroscopy. Backbone dihedral angles and secondary structure were derived from the statistical analysis of the secondary chemical shifts, and are in good agreement with solution values for the intact full-length thioredoxin, with the exception of a small number of residues located at the termini of the individual secondary structure elements. A large number of cross-peaks observed in the DARR spectra with long mixing times correspond to the pairs of carbon atoms separated by 4-6 angstroms, suggesting that DARR could be efficiently employed for observation of medium- and long-range correlations. The 108 amino acid residue E. coli thioredoxin is the largest uniformly enriched protein assigned to this degree of completeness by solid-state NMR spectroscopy to date. It is anticipated that with a combination of two-dimensional correlation experiments and high magnetic fields, resonance assignments and secondary structure can be generally derived for other noncrystalline proteins.


Subject(s)
Escherichia coli/chemistry , Magnetic Resonance Spectroscopy/methods , Thioredoxins/chemistry , Carbon Isotopes , Crystallization , Escherichia coli Proteins/chemistry , Models, Molecular , Nitrogen Isotopes , Polyethylene Glycols , Protein Conformation , Protein Structure, Secondary
14.
J Am Chem Soc ; 126(50): 16608-20, 2004 Dec 22.
Article in English | MEDLINE | ID: mdl-15600367

ABSTRACT

De novo site-specific backbone and side-chain resonance assignments are presented for U-15N(1-73)/U-13C,15N(74-108) reassembly of Escherichia coli thioredoxin by fragment complementation, determined using solid-state magic angle spinning NMR spectroscopy at 17.6 T. Backbone dihedral angles and secondary structure predicted from the statistical analysis of 13C and 15N chemical shifts are in general agreement with solution values for the intact full-length thioredoxin, confirming that the secondary structure is retained in the reassembled complex prepared as a poly(ethylene glycol) precipitate. The differential labeling of complementary thioredoxin fragments introduced in this work is expected to be beneficial for high-resolution structural studies of protein interfaces formed by protein assemblies by solid-state NMR spectroscopy.


Subject(s)
Escherichia coli Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Peptide Fragments/chemistry , Thioredoxins/chemistry , Amino Acid Sequence , Carbon Isotopes , Models, Molecular , Molecular Sequence Data , Nitrogen Isotopes , Polyethylene Glycols/chemistry
15.
Biochemistry ; 43(13): 3835-43, 2004 Apr 06.
Article in English | MEDLINE | ID: mdl-15049690

ABSTRACT

Recent studies of proteins with reversed charged residues have demonstrated that electrostatic interactions on the surface can contribute significantly to protein stability. We have used the approach of reversing negatively charged residues using Arg to evaluate the effect of the electrostatics context on the transition temperature (T(m)), the unfolding Gibbs free energy change (DeltaG), and the unfolding enthalpy change (DeltaH). We have reversed negatively charged residues at a pocket (Asp9) and protrusions (Asp10, Asp20, Glu85), all located in interconnecting segments between elements of secondary structure on the surface of Arg73Ala Escherichia coli thioredoxin. DSC measurements indicate that reversal of Asp in a pocket (Asp9Arg/Arg73Ala, DeltaT(m) = -7.3 degrees C) produces a larger effect in thermal stability than reversal at protrusions: Asp10Arg/Arg73Ala, DeltaT(m) = -3.1 degrees C, Asp20Arg/Arg73Ala, DeltaT(m) = 2.0 degrees C, Glu85Arg/Arg73Ala, DeltaT(m) = 3.9 degrees ). The 3D structure of thioredoxin indicates that Asp20 and Glu85 have no nearby charges within 8 A, while Asp9 does not only have Asp10 as sequential neighbor, but it also forms a 5-A long-range ion pair with the solvent-exposed Lys69. Further DSC measurements indicate that neutralization of the individual charges of the ion pair Asp9-Lys69 with nonpolar residues produces a significant decrease in stability in both cases: Asp9Ala/Arg73Ala, DeltaT(m) = -3.7 degrees C, Asp9Met/Arg73Ala, DeltaT(m) = -5.5 degrees C, Lys69Leu/Arg73Ala, DeltaT(m) = -5.1 degrees C. However, thermodynamic analysis shows that reversal or neutralization of Asp9 produces a 9-15% decrease in DeltaH, while both reversal of Asp at protrusions and neutralization of Lys69 produce negligible changes. These results correlate well with the NMR analysis, which demonstrates that only the substitution of Asp9 produces extensive conformational changes and these changes occur in the surroundings of Lys69. Our results led us to suggest that reversal of a negative charge at a pocket has a larger effect on stability than a similar reversal at a protrusion and that this difference arises largely from short-range interactions with polar groups within the pocket, rather than long-range interactions with solvent-exposed charged groups.


Subject(s)
Amino Acid Substitution , Escherichia coli Proteins/chemistry , Thioredoxins/chemistry , Alanine/genetics , Amino Acid Substitution/genetics , Arginine/genetics , Aspartic Acid/genetics , Calorimetry, Differential Scanning , Escherichia coli Proteins/genetics , Models, Chemical , Models, Molecular , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Protein Folding , Static Electricity , Structure-Activity Relationship , Surface Properties , Thermodynamics , Thioredoxins/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...