A computational framework for modeling cell–matrix interactions in soft biological tissues

Biomechanics and Modeling in Mechanobiology - Living soft tissues appear to promote the development and maintenance of a preferred mechanical state within a defined tolerance around a so-called set...     

W323S variant of Xiap-Bir3 binds to SMAC but not caspase-9

The ability of the wild-type XIAP BIR3 domain as well as its Trp323Ser variant in inhibition of human caspase-9, binding to AVPFVASLPN (SMAC-peptide), SMAC protein, and mature caspase-9 was investigated. In order to investigate the role of W323 on these interactions, this residue was mutated to Serine. Circular dichroism as well as thermal denaturation studies showed that W323S mutation did not hamper proper folding of the protein. The dissociation constants for the interaction of the wild type BIR3 as well as its mutant to Smac-type peptide were found to be 1.8 and 27 muM, respectively. The inhibition of and binding to caspase-9 by wild-type BIR3 and its mutant were also compared. While the wild-type protein potently inhibited the enzyme, the mutant failed to do so. The lack of caspase-9 inhibition was due to absence of interaction of the mutant BIR3 with mature caspase-9. These results indicate that Trp323 of BIR3 plays a pivotal role both in maintaining necessary conformation for caspase-9 interaction and to a lesser extent, recognition of Smac-type peptide. Moreover, decreased stability of the mutant compared with the wild type indicates that W323 is essential for maintaining the stability BIR3-Smac-peptide complex.     

Use of sulfhydryl reagents to investigate branched chain alpha-keto acid transport in mitochondria

The goal of this paper was to determine the contribution of the mitochondrial branched chain aminotransferase (BCATm) to branched chain alpha-keto acid transport within rat heart mitochondria. Isolated heart mitochondria were treated with sulfhydryl reagents of varying permeability, and the data suggest that essential cysteine residues in BCATm are accessible from the cytosolic face of the inner membrane. Treatment with 15 nmol/mg N-ethylmaleimide (NEM) inhibited initial rates of alpha-ketoisocaproate (KIC) uptake in reconstituted mitochondrial detergent extracts by 70% and in the intact organelle by 50%. KIC protected against inhibition suggesting that NEM labeled a cysteine residue that is inaccessible when substrate is bound to the enzyme. Additionally, the apparent mitochondrial equilibrium KIC concentration was decreased 50-60% after NEM labeling, and this difference could not be attributed to effects of NEM on matrix pH or KIC oxidation. In fact, NEM was a better inhibitor of KIC oxidation than rotenone. Measuring matrix aspartate and glutamate levels revealed that the effects of NEM on the steady-state KIC concentration resulted from inhibition of BCATm catalyzed transamination of KIC with matrix glutamate to form leucine. Furthermore, circular dichroism spectra of recombinant human BCATm with liposomes showed that the commercial lipids used in the reconstituted transport assay contain BCAT amino acid substrates. Thus BCATm is distinct from the branched chain alpha-keto acid carrier but may interact with the inner mitochondrial membrane, and it is necessary to inhibit or remove transaminase activity in both intact and reconstituted systems prior to quantifying transport of alpha-keto acids which are transaminase substrates.     

Mechanism of stabilization of Bacillus circulans xylanase upon the introduction of disulfide bonds

The introduction of disulfide bonds has been used as a strategy to enhance the stability of Bacillus circulans xylanase. The transition temperature of the S100C/N148C (DS1), V98C/A152C (DS2), and A1GC/G187,C188 (cXl) in comparison to the wild type was increased by 5.0, 4.1 and 3.8 degrees C, respectively. Interestingly, a combination of two disulfide bonds of DS1 and cXl (cDS1, circular disulfide 1) led to a 12 degrees C increase in the transition temperature. Importantly, an increase in the melting point and DeltaDeltaG values of the cDS1 mutant was cooperative. These results suggest that the mechanism of stabilization by disulfide bonds under irreversible denaturation condition is achieved through: (1) a change in the rate-limiting step on the denaturation pathway; (2) destabilizing the unfolded state without affecting the relative rate constants on the denaturation pathway (like cXl mutant); and (3) or combination of the two (cDS1 mutant).     

Neuronal apoptosis inhibitory protein,NAIP, is an inhibitor of procaspase-9

Ability of the full length NAIP and its BIR3 domain in inhibition of the proteases of the intrinsic apoptosis pathway was investigated. Activity of endogenous executioner caspases was drastically reduced by both recombinant NAIP-BIR3 (NBIR3) and the full length protein. Western blotting experiments showed that the full length NAIP and its BIR3 domain inhibited the cleavage of procaspase-3 by apoptosome activated caspase-9. Moreover, full length NAIP inhibited autocatalytic processing of procaspase-9 in the apoptosome complex indicating that unlike other inhibitor of apoptosis proteins (IAPs) human NAIP is an inhibitor of procaspase-9. Furthermore, inhibition of single-chain caspase-9 (human caspase-9, D315, D330/A point mutations that abrogate the proteolytic processing but not the catalytic activity of caspase-9) by the BIR3 domain indicated that the this domain is the caspase-9 interacting moiety. Consistently, pull-down experiments of single-chain capsase-9 in apoptosome complex by the NBIR3 but not the X-linked inhibitor of apoptosis protein (XIAP)-BIR3 domain confirmed that the protein can associate with procaspase-9 prior to its autoproteolysis upon apoptosome formation. Interaction studies revealed the association of C338W variant of the NBIR3, but not the wild type protein with both SMAC-peptide and the SMAC protein. These data indicate that mutation of C338 to Trp is sufficient to accommodate the interaction of NAIP-BIR3 with SMAC-peptide and protein. Taken together, these results demonstrate that NAIP is evolved to prevent apoptosis right at the initiation stage of apoptosome formation and this inhibition cannot be antagonized by SMAC-type proteins.     

Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.     

The time course of Akt and ERK activation on XIAP expression in HEK 293 cell line

The cell growth is controlled by the interaction of survival and cell growth arrest pathways as well as apoptosis mechanisms which determine the outcome of cell faith as proliferation or apoptosis. In this study, we have studied the activity of survival pathways, i.e., Akt and ERK1/2 with regard to XIAP (inhibitor of apoptosis) in serum starved and stimulated conditions. The HEK-293 cells were cultured in RPMI + 10% FBS. The cells were serum starved by switching to medium with 1% FBS for 24 h and serum stimulated by changing the medium to 10% FBS following serum starvation. The expression of p-Akt, p-ERK, Akt, ERK and XIAP was studied in various time points using western blot. The apoptosis was evaluated by DNA condensation using Hoechst 33258 and Caspase-3 assay. In serum starved condition expression of p-Akt and XIAP is very low. Serum stimulation increases p-Akt and p-ERK within 5 min and sustains a high level for 30 min. The expression of total Akt and ERK1/2 has not changed significantly for 24 h. XIAP expression starts at 6 h after serum stimulation, reaches to maximum level at 12 h and decreases to baseline within 24 h. Furthermore, serum starvation for 24 h does not induced apoptosis and DNA condensation. Taken together, the results indicate that serum activates Akt and ERK pathways earlier than XIAP expression. Furthermore, XIAP expression is low in serum starvation unlike p-ERK which suggests a survival role for ERK in serums starvation. The expression pattern of XIAP indicates induction by Akt and/or ERK activation which requires further studies.     

The effect of extracellular calcium on colonocytes: evidence for differential responsiveness based upon degree of cell differentiation

Calcium supplementation decreases the incidence of colon cancer in animal models and may prevent colon cancer in man. Potential mechanisms include binding of mitogens and direct effects of calcium on colonic epithelial cells. In this study, the effects of extracellular calcium on epithelial cell growth and differentiation were studied in three colon carcinoma and two colonic adenoma cell lines. The characteristics studied included morphology, cell cycle kinetics, [Ca²⁺]_IC (intracellular calcium concentration), proliferation, and expression of differentiation markers such as carcinoembryonic antigen (CEA) and alkaline phosphatase (AP). Sodium butyrate (NaB) and 1,25-dihydroxyvitamin D₃ were used as controls in the latter three assays as these two agents are known differentiating agents. Alteration of [Ca⁺²]_EC (extracellular calcium concentration) did not affect carcinoembryonic antigen (CEA) or alkaline phosphatase (AP) expression. NaB enhanced the expression of AP three-fold and CEA five-fold. This effect was augmented by increasing [Ca²⁺]_EC. The exposure of cells to 1,25-(OH)₂-Vitamin D₃ increased CEA but not AP. [Ca²⁺]_IC increased in response to 1,25-(OH)₂-vitamin D₃ and NaB but not with variation in [Ca²⁺]_EC. Increased [Ca²⁺]_EC inhibited proliferation of well-differentiated cells, but had no effect on poorly-differentiated cells. Morphological studies showed that extracellular calcium was necessary for normal cell—cell interactions. These studies have demonstrated direct effects of calcium on colonic epithelial cells which may contribute to the protective effects of dietary calcium against colon cancer. Loss of responsivess to the antiprotective effects of [Ca²⁺]_EC with de-differentiation suggests that calcium supplementation may be most beneficial prior to the development of neoplastic changes in colonic epithelium.     

Mgs1 and Rad18/Rad5/Mms2 are required for survival of Saccharomyces cerevisiae mutants with novel temperature/cold sensitive alleles of the DNA polymerase delta subunit, Pol31

Saccharomyces cerevisiae DNA polymerase delta (Pol delta) is a heterotrimeric enzyme consisting of Pol3 (the catalytic subunit), Pol31 and Pol32. New pol31 alleles were constructed by introducing mutations into conserved amino acid residues in all 10 identified regions of Pol31. Six novel temperature-sensitive (ts) or cold-sensitive (cs) alleles, carrying mutations in regions III, IV, VII, VIII or IX, conferred a range of defects in the response to replication stress or DNA damage. Deletion of SGS1, RAD52, SRS2, MRC1 or RAD24 had a deleterious effect only in combination with those pol31 alleles that had a phenotype as single mutants, suggesting a requirement for recombination and checkpoint functions in processing the DNA lesions or structures that form as a consequence of replication with a defective Pol delta. In contrast, deletion of POL32 negatively affected the growth of almost all pol31 mutants, suggesting an important role for all conserved amino acids of Pol31 in maintaining the integrity of Pol delta complex structurally, at least in the absence of the third subunit. Surprisingly, deletions of RAD18 and MGS1 aggravated the temperature sensitivity conferred by most ts or cs alleles and specifically suppressed the hys2-1 and hys2-1-like mutations of POL31. Deletion of RAD5 or MMS2 had an effect on pol31 ts/cs mutants similar to that of RAD18, whereas deletion of RAD30 or REV3 had no effect. We propose that Rad18/Rad5/Mms2 and Mgs1 are required to promote replication when forks are destabilized or stalled due to defects in Pol delta. These data are consistent with the biochemical activity of the human Mgs1 orthologue, which binds and stimulates Pol deltain vitro. We also demonstrate that Mgs1 interacts physically with Pol31 in vivo. Moreover, regions I and VII of Pol31, which are specifically sensitive to high levels of Mgs1 and PCNA, could be sites of interaction.