Aspartic Acid Residues (aspartic + acid_residue)

Distribution by Scientific Domains

Selected Abstracts

Role of DNA polymerase , in tolerance of endogenous and exogenous DNA damage in mouse B cells

GENES TO CELLS, Issue 2 2006
Akiko Ukai
DNA polymerase , (Pol,) is a family A polymerase that contains an intrinsic helicase domain. To investigate the function of Pol, in mammalian cells, we have inactivated its polymerase activity in CH12 mouse B lymphoma cells by targeted deletion of the polymerase core domain that contains the catalytic aspartic acid residue. Compared to parental CH12 cells, mutant cells devoid of Pol, polymerase activity exhibited a slightly reduced growth rate, accompanied by increased spontaneous cell death. In addition, mutant cells showed elevated sensitivity to mitomycin C, cisplatin, etoposide, ,-irradiation and ultraviolet (UV) radiation. Interestingly, mutant cells were more sensitive to the alkylating agent methyl methanesulfonate (MMS) than parental cells. This elevated MMS sensitivity relative to WT cells persisted in the presence of methoxyamine, an inhibitor of the major base excision repair (BER) pathway, suggesting that Pol, is involved in tolerance of MMS through a mechanism that appears to be different from BER. These results reveal an important role for Pol, in preventing spontaneous cell death and in tolerance of not only DNA interstrand cross-links and double strand breaks but also UV adducts and alkylation damage in mammalian lymphocytes. [source]

The exceptionally tight affinity of DnaA for ATP/ADP requires a unique aspartic acid residue in the AAA+ sensor 1 motif

Hironori Kawakami
Summary Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp. [source]

Single-step affinity purification of recombinant proteins using a self-excising module from Neisseria meningitidis FrpC

PROTEIN SCIENCE, Issue 10 2008
Lenka Sadilkova
Abstract Purification of recombinant proteins is often a challenging process involving several chromatographic steps that must be optimized for each target protein. Here, we developed a self-excising module allowing single-step affinity chromatography purification of untagged recombinant proteins. It consists of a 250-residue-long self-processing module of the Neisseria meningitidis FrpC protein with a C-terminal affinity tag. The N terminus of the module is fused to the C terminus of a target protein of interest. Upon binding of the fusion protein to an affinity matrix from cell lysate and washing out contaminating proteins, site-specific cleavage of the Asp,Pro bond linking the target protein to the self-excising module is induced by calcium ions. This results in the release of the target protein with only a single aspartic acid residue added at the C terminus, while the self-excising affinity module remains trapped on the affinity matrix. The system was successfully tested with several target proteins, including glutathione-S-transferase, maltose-binding protein, ,-galactosidase, chloramphenicol acetyltransferase, and adenylate cyclase, and two different affinity tags, chitin-binding domain or poly-His. Moreover, it was demonstrated that it can be applied as an alternative to two currently existing systems, based on the self-splicing intein of Saccharomyces cerevisiae and sortase A of Staphylococcus aureus. [source]

A new mutation in the linker 12 domain of keratin 5 in a Chinese family with Weber,Cockayne epidermolysis bullosa simplex

J.-G. Li
Summary A previously undescribed missense mutation was detected in the L12 domain of keratin 5 (K5) in a Chinese family with Weber,Cockayne epidermolysis bullosa simplex. Direct sequencing of the PCR products identified a single base substitution (983A,G) that changes the aspartic acid residue at codon 328 to glycine in all affected family members, while no mutation was observed either in the healthy individual or 50 unrelated control samples. Asp328 of K5 is remarkably conserved among all type II keratins. D328G is the fourth mutation found to affect this residue in K5-related epidermolysis bullosa simplex, indicating the importance of Asp328 for K5 structure and the dramatic effect that fine changes can have on keratin intermediate filament integrity. [source]

Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene

FEBS JOURNAL, Issue 16 2000
Alberto Alape-Girón
Clostridium perfringens phospholipase C (PLC), also called ,-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered ,-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103 -fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11,73% of the hemolytic activity and exhibited a cytotoxic potency 102 - to 105 -fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level. [source]

Effect of Soluble Soybean Protein Hydrolysate-Calcium Complexes on Calcium Uptake by Caco-2 Cells

Y. Lv
ABSTRACT:, Soybean protein hydrolysates (SPHs) bind with calcium, forming soluble SPH-calcium complexes via the carboxyl groups of glutamic and aspartic acid residues. However, their effect on calcium uptake is still unclear. In this study, Caco-2 cells were used to estimate the effect of SPH-calcium complexes with different molecular weights on calcium uptake in vitro. The changes in intracellular calcium ion concentration were measured by Fura-2 loading and expressed in fluorescence intensity. SPH-calcium complexes could promote calcium uptake. Improved fluorescence intensity was significantly different in SPH-calcium complexes (10 to 30 kDa), SPH-calcium complexes (3 to 10 kDa), and SPH-calcium complexes (1 to 3 kDa). The maximum levels of relative fluorescence intensity (18.3) occurred with SPH-calcium complexes (10 to 30 kDa). The effect of SPH-calcium complexes (10 to 30 kDa) on Ca2+ increase was determined to be concentration dependent in the range of 0.5 to 4 mg/mL. Our results indicate that soybean protein itself might be responsible for promoting calcium absorption. [source]

Pseudophosphorylation of tau at serine 422 inhibits caspase cleavage: in vitro evidence and implications for tangle formation in vivo

Angela L. Guillozet-Bongaarts
Abstract The tangles of Alzheimer's disease (AD) are comprised of the tau protein displaying numerous alterations, including phosphorylation at serine 422 (S422) and truncation at aspartic acid 421 (D421). Truncation at the latter site appears to result from activation of caspases, a class of proteases that cleave specifically at aspartic acid residues. It has been proposed that phosphorylation at or near caspase cleavage sites could regulate the ability of the protease to cleave at those sites. Here, we use tau pseudophosphorylated at S422 (S422E) to examine the effects of tau phosphorylation on its cleavage by caspase 3. We find that S422E tau is more resistant to proteolysis by caspase 3 than non-pseudophosphorylated tau. Additionally, we use antibodies directed against the phosphorylation site and against the truncation epitope to assess the presence of these epitopes in neurofibrillary tangles in the aged human brain. We show that phosphorylation precedes truncation during tangle maturation. Moreover, the distribution of the two epitopes suggests that a significant length of time (perhaps as much as two decades) elapses between S422 phosphorylation and cleavage at D421. We further conclude that tau phosphorylation at S422 may be a protective mechanism that inhibits cleavage in vivo. [source]

Therapeutic potential of sulfamides as enzyme inhibitors

Jean-Yves Winum
Abstract Sulfamide, a quite simple molecule incorporating the sulfonamide functionality, widely used by medicinal chemists for the design of a host of biologically active derivatives with pharmacological applications, may give rise to at least five types of derivatives, by substituting one to four hydrogen atoms present in it, which show specific biological activities. Recently, some of these compounds started to be exploited for the design of many types of therapeutic agents. Among the enzymes for which sulfamide-based inhibitors were designed, are the carbonic anhydrases (CAs), a large number of proteases belonging to the aspartic protease (HIV-1 protease, ,-secretase), serine protease (elastase, chymase, tryptase, and thrombin among others), and metalloprotease (carboxypeptidase A (CPA) and matrix metalloproteinases (MMP)) families. Some steroid sulfatase (STS) and protein tyrosine phosphatase inhibitors belonging to the sulfamide class of derivatives have also been reported. In all these compounds, many of which show low nanomolar affinity for the target enzymes for which they have been designed, the free or substituted sulfamide moiety plays important roles for the binding of the inhibitor to the active site cavity, either by directly coordinating to a metal ion found in some metalloenzymes (CAs, CPA, STS), usually by means of one of the nitrogen atoms present in the sulfamide motif, or as in the case of the cyclic sulfamides acting as HIV protease inhibitors, interacting with the catalytically critical aspartic acid residues of the active site by means of an oxygen atom belonging to the HNSO2NH motif, which substitutes a catalytically essential water molecule. In other cases, the sulfamide moiety is important for inducing desired physico-chemical properties to the drug-like compounds incorporating it, such as enhanced water solubility, better bioavailability, etc., because of the intrinsic properties of this highly polarized moiety when attached to an organic scaffold. This interesting motif is thus of great value for the design of pharmacological agents with a lot of applications. © 2006 Wiley Periodicals, Inc. Med Res Rev [source]

Factors contributing to decreased protein stability when aspartic acid residues are in ,-sheet regions

P.R. Pokkuluri
Abstract Asp residues are significantly under represented in ,-sheet regions of proteins, especially in the middle of ,-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a ,-domain. Two Gln residues of the immunoglobulin light-chain variable domain (VL) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a ,-strand, and that of Q89D, located in the middle of a ,-strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type VL -Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, VL -Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type VL -Len domain. The structures of mutants VL -Len Q38D and VL -Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 Ĺ resolution. We found no major perturbances in the structures of these Q,D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a ,-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a ,-strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo. [source]

Crystallographic evidence for noncoplanar catalytic aspartic acids in plasmepsin II resides in the Protein Data Bank

Arthur H. Robbins
The carboxylate atoms of the two catalytic aspartic acid residues in aspartic proteases are nearly coplanar and in the uncomplexed form share an in-plane nucleophilic water molecule that is central to the mechanism of these enzymes. This note reports that while reviewing the electron-density maps derived from the deposited data for uncomplexed plasmepsin II from Plasmodium falciparum [Asojo et al. (2003), J. Mol. Biol.327, 173,181; PDB code 1lf4], it was discovered that the aspartic acid residues in this structure should in fact be distinctly noncoplanar. The crystallographic model from the deposited coordinates has been re-refined against the 1.9,Ĺ resolution published diffraction data to an Rcryst of 21.2% and an Rfree of 22.2%. The catalytic water molecule is present, but the plane of the carboxylate group of Asp214 is rotated by 66° from its original position. [source]