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M Sarmiento

Publications and source records attributed to M Sarmiento.

At least 19 recordsLinked to original sources
PubMed2000

Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants.

The binding of several phosphonodifluoromethyl phenylalanine (F(2)Pmp)-containing peptides to protein-tyrosine phosphatase 1B (PTP1B) and its substrate-trapping mutants (C215S and D181A) has been studied using isothermal titration calorimetry. The binding of a high affinity ligand, Ac-Asp-Ala-Asp-Glu-F(2)Pmp-Leu-NH(2), to PTP1B (K(d) = 0.24 microm) is favored by both enthalpic and entropic contributions. Disruption of ionic interactions between the side chain of Arg-47 and the N-terminal acidic residues reduces the binding affinity primarily through the reduction of the TDeltaS term. The role of Arg-47 may be to maximize surface contact between PTP1B and the peptide, which contributes to high affinity binding. The active site Cys-215 --> Ser mutant PTP1B binds ligands with the same affinity as the wild-type enzyme. However, unlike wild-type PTP1B, peptide binding to C215S is predominantly driven by enthalpy change, which likely results from the elimination of the electrostatic repulsion between the thiolate anion and the phosphonate group. The increased enthalpic contribution is offset by reduction in the binding entropy, which may be the result of increased entropy of the unbound protein caused by this mutation. The general acid-deficient mutant D181A binds the peptide 5-fold tighter than the C215S mutant, consistent with the observation that the Asp to Ala mutant is a better "substrate-trapping" reagent than C215S. The increased binding affinity for D181A as compared with the wild-type PTP1B results primarily from an increase in the DeltaH of binding in the mutant, which may be related to decreased electrostatic repulsion between the phosphate moiety and PTP1B. These results have important implications for the design of high affinity PTP1B inhibitors.

Amino Acid Sequence↗
PubMed2000

Structural basis of plasticity in protein tyrosine phosphatase 1B substrate recognition.

Protein tyrosine phosphatase 1B (PTP1B) displays a preference for peptides containing acidic as well as aromatic/aliphatic residues immediately NH(2)-terminal to phosphotyrosine. The structure of PTP1B bound with DADEpYL-NH(2) (EGFR(988)(-)(993)) offers a structural explanation for PTP1B's preference for acidic residues [Jia, Z., Barford, D., Flint, A. J., and Tonks, N. K. (1995) Science 268, 1754-1758]. We report here the crystal structures of PTP1B in complex with Ac-ELEFpYMDYE-NH(2) (PTP1B.Con) and Ac-DAD(Bpa)pYLIPQQG (PTP1B.Bpa) determined to 1.8 and 1.9 A resolution, respectively. A structural analysis of PTP1B.Con and PTP1B.Bpa shows how aromatic/aliphatic residues at the -1 and -3 positions of peptide substrates are accommodated by PTP1B. A comparison of the structures of PTP1B.Con and PTP1B.Bpa with that of PTP1B.EGFR(988)(-)(993) reveals the structural basis for the plasticity of PTP1B substrate recognition. PTP1B is able to bind phosphopeptides by utilizing common interactions involving the aromatic ring and phosphate moiety of phosphotyrosine itself, two conserved hydrogen bonds between the Asp48 carboxylate side chain and the main chain nitrogens of the pTyr and residue 1, and a third between the main chain nitrogen of Arg47 and the main chain carbonyl of residue -2. The ability of PTP1B to accommodate both acidic and hydrophobic residues immediately NH(2)-terminal to pTyr appears to be conferred upon PTP1B by a single residue, Arg47. Depending on the nature of the NH(2)-terminal amino acids, the side chain of Arg47 can adopt one of two different conformations, generating two sets of distinct peptide binding surfaces. When an acidic residue is positioned at position -1, a preference for a second acidic residue is also observed at position -2. However, when a large hydrophobic group occupies position -1, Arg47 adopts a new conformation so that it can participate in hydrophobic interactions with both positions -1 and -3.

Arginine↗
PubMed2000

Structure-based discovery of small molecule inhibitors targeted to protein tyrosine phosphatase 1B.

Protein tyrosine phosphatases (PTPases) are involved in the control of tyrosine phosphorylation levels in the cell and are believed to be crucial for the regulation of a multitude of cellular functions. A detailed understanding of the role played by PTPases in various signaling pathways has not yet been achieved, and potent and selective PTPase inhibitors are essential in the quest to determine the functionality of individual PTPases. Using the DOCK methodology, we have carried out a structure-based, computer-assisted search of an available chemical database in order to identify low molecular weight, nonpeptidic PTP1B inhibitors. We have identified several organic molecules that not only possess inhibitory activity against PTP1B but which also display significant selectivity for PTP1B. This indicates that although structural features important for pTyr recognition are conserved among different PTPases, it is possible to generate selective inhibitors targeted primarily to the catalytic site. Kinetic analysis and molecular modeling experiments suggest that the PTP1B active site possesses significant plasticity such that substituted and extended aromatic systems can be accommodated. The newly identified molecules provide a molecular framework upon which therapeutically useful compounds can ultimately be based, and systematic optimization of these lead compounds is likely to further enhance their potency and selectivity.

Catalytic Domain↗
PubMed2000

Dup(12)(q13-q22) and 13q14 deletion in a case of B-cell chronic lymphocytic leukemia.

Cases with partial trisomy 12 have rarely been found in B-cell chronic lymphocytic leukemia (CLL). We report our clinical, cytogenetic and fluorescence in situ hybridization (FISH) findings in a CLL patient with a duplication of part of the long arm of chromosome 12 between bands q13-q22. This patient was the only case with this duplication among the 112 cases (0.9%) of CLL cytogenetically analyzed in our laboratory. FISH studies using unique-sequence specific probes for the RB-1 (retinoblastoma) gene and the D13S319 locus at the 13q14 band showed a monoallelic loss for the D13S319 locus (20% of cells) with a diploid RB-1 gene. Our case showed an atypical morphology (35% prolymphocytes), a high proliferation rate and progression of the disease, indicating that the duplication of this region may be equivalent to complete trisomy 12 in CLL patients.

Aged↗
PubMed2000

[Hairy cell leukemia. An alternative method for the detection of minimal residual disease using flow cytometry].

In patients with hairy cell leukemia (HCL) who received chemotherapeutic treatment and achieved complete remission (CR), minimal residual disease (MRD) can be detected in bone marrow biopsies using immunohistochemical (IHC) techniques. In this study, we investigated the value of flow cytometry (FCM) and IHC to detect MRD and to establish whether MRD+ could predict relapse. A total of 15 HCL patients in CR were studied. Samples of bone marrow (BM) and peripheral blood (PB) were processed by FCM with triple staining of the following monoclonal antibodies (mAbs): CD20, CD22, CD11c, CD103, CD25, anti-Kappa and anti-Lambda light chains. Reference values were obtained from normal samples of peripheral blood and bone marrow. FCM detected MRD in 64% of the patients. BM samples were more demonstrative than peripheral blood for MRD detection in HCL. IHC was performed in paraffin-embedded BM biopsies using CD20 and DBA44 mAbs. MRD+ was detected in 46% of patients. Although not statistically significant, FCM appeared more sensitive compared with IHC. Detection of MRD by either of these methods in our series did not predict hematological relapse. The results show that FCM is a useful alternative method to detect MRD in HCL and that a longer, follow-up is required to establish the predictive outcome of MRD+ patients.

Antibodies, Monoclonal↗
PubMed1999

Immune complexes inhibit apoptosis of chronic lymphocytic leukaemia B cells.

In the present study we examined the effect of immune complexes (IC) on the survival of chronic lymphocytic leukaemia (B-CLL) B cells. Our results showed that either precipitating IC (pIC), Ab-coated erythrocytes (E-IgG) or heat-aggregated IgG (aIgG) significantly inhibited spontaneous apoptosis of B-CLL cells, as well as that induced by fludarabine, chlorambucil or dexamethasone. After depletion of T lymphocytes, monocytes and NK cells, incubation with IC was no longer able to delay B-CLL cells apoptosis, suggesting that prevention of apoptosis depends on IC interaction with accessory leucocytes. The release of IFNgamma by non-malignant cells upon activation with IC was responsible, to some extent, for IC effects as shown by the fact that neutralizing anti-IFNgamma MoAb partially prevented their ability to inhibit B-CLL cells apoptosis. The observation that treatment with IC resulted in increased expression of HLA-DR on B-CLL cells suggests that inhibition of apoptosis is associated with cellular activation.

Antigen-Antibody Complex↗
PubMed1999

CD8 expression in a case of chronic lymphocytic leukemia with trisomy 12.

We report a case of B-cell chronic lymphocytic leukemia (B-CLL) with aberrant expression of the T-cell-associated antigen CD8, as revealed by two-color flow-cytometric analysis. DNA studies showed immunoglobulin heavy-chain gene rearrangement, but not of gamma-chain T-cell receptor, confirming the B-cell origin of the neoplastic cells. Ploidy analysis showed a tetraploid population and high S-phase fraction. B-CLL cells also carried trisomy 12, detected by fluorescence in situ hybridization. The identification of more cases with the same features would be necessary to establish the prognosis of this subtype of B-CLL.

Aged↗
PubMed1998

Molecular basis for substrate specificity of protein-tyrosine phosphatase 1B.

Protein-tyrosine phosphatases can exhibit stringent substrate specificity in vivo, although the molecular basis for this is not well understood. The three-dimensional structure of the catalytically inactive protein-tyrosine phosphate 1B (PTP1B)/C215S complexed with an optimal substrate, DADEpYL-NH2, reveals specific interactions between amino acid residues in the substrate and PTP1B. The goal of this work is to rigorously evaluate the functional significance of Tyr46, Arg47, Asp48, Phe182, and Gln262 in substrate binding and catalysis, using site-directed mutagenesis. Combined with structural information, kinetic analysis of the wild type and mutant PTP1B using p-nitrophenyl phosphate and phosphotyrosine-containing peptides has yielded further insight into PTP1B residues, which recognize general features, as well as specific properties, in peptide substrates. In addition, the kinetic results suggest roles of these residues in E-P hydrolysis, which are not obvious from the structure of PTP1B/peptide complex. Thus, Tyr46 and Asp48 recognize common features of peptide substrates and are important for peptide substrate binding and/or E-P formation. Arg47 acts as a determinant of substrate specificity and is responsible for the modest preference of PTP1B for acidic residues NH2-terminal to phosphotyrosine. Phe182 and the invariant Gln262 are not only important for substrate binding and/or E-P formation but also important for the E-P hydrolysis step.

Amino Acid Sequence↗
PubMed1995

High resolution, standard format two-dimensional protein electrophoresis using disposable glass micropipettes and non-dedicated equipment.

A method for high resolution, two-dimensional polyacrylamide gel electrophoresis (PAGE) of proteins on a standard format that utilizes non-dedicated electrophoretic equipment is described. This method combines key features of various previous protocols into an improved approach which can readily be applied by persons experienced only in sodium dodecyl sulfate (SDS)-PAGE. The method yields high-quality two-dimensional protein separations that are reliably reproducible and uniform between different analyses. High protein resolution is attained and preserved primarily by facilitating the execution of the first isoelectric focusing (IEF) dimension and streamlining the initiation of the second (SDS-PAGE) dimension. Important features of this method include the use of piperazine diacrylate cross-linker, disposable glass micropipettes, and conical sample chambers in the IEF dimension. In addition, the equilibration of the first-dimensional gels is accomplished at the same time the gels are loaded onto the second dimension. Loss of protein resolution due to diffusion after the end of the first dimension is reduced by minimizing the time required to load the SDS-PAGE dimension. The method is versatile and adaptable to the analysis of one or many samples. The high degree of resolution, reproducibility and uniformity attained by this method are sufficient to abrogate the need for dedicated equipment in most applications.

Amnion↗