The dynamics of bax channel formation: influence of ionic strength

Mitochondrial outer membrane permeabilization (MOMP) is a complex multistep process. Studies of MOMP in vivo are limited by the stochastic variability of MOMP between cells and rapid completion of IMS protein release within single cells. In vitro models have provided useful insights into MOMP. We have investigated the dynamics of Bax-mediated MOMP in isolated mitochondria using ionic strength as a tool to control the rate of MOMP. We find that Bax can induce both transient permeabilization, detected by protein release, and more substantial long-lasting permeabilization, measured by the rate of oxidation of added cytochrome c. We found that higher ionic strength causes Bax to form small channels quickly but the expansion of these early channels is impeded. This inhibitory effect of ionic strength is independent of tBid. Channels formed under low ionic strength are not destabilized by raising the ionic strength. Increase in ionic strength also increases the ability of Bcl-xL to inhibit Bax-mediated MOMP. Ionic strength does not affect Bax insertion into mitochondria. Thus, ionic strength influences the assembly of Bax molecules already in membrane into channels. Ionic strength can be used as an effective biophysical tool to study Bax-mediated channel formation.     

Conserved Ser/Arg-rich motif in PPZ orthologs from fungi is important for its role in cation tolerance

PPZ1 orthologs, novel members of a phosphoprotein phosphatase family of phosphatases, are found only in fungi. They regulate diverse physiological processes in fungi e.g. ion homeostasis, cell size, cell integrity, etc. Although they are an important determinant of salt tolerance in fungi, their physiological role remained unexplored in any halotolerant species. In this context we report here molecular and functional characterization of DhPPZ1 from Debaryomyces hansenii, which is one of the most halotolerant and osmotolerant species of yeast. Our results showed that DhPPZ1 knock-out strain displayed higher tolerance to toxic cations, and unlike in Saccharomyces cerevisiae, Na(+)/H(+) antiporter appeared to have an important role in this process. Besides salt tolerance, DhPPZ1 also had role in cell wall integrity and growth in D. hansenii. We have also identified a short, serine-arginine-rich sequence motif in DhPpz1p that is essential for its role in salt tolerance but not in other physiological processes. Taken together, these results underscore a distinct role of DhPpz1p in D. hansenii and illustrate an example of how organisms utilize the same molecular tool box differently to garner adaptive fitness for their respective ecological niches.     

How cells process information: quantification of spatiotemporal signaling dynamics

Arguably, one of the foremost distinctions between life and non-living matter is the ability to sense environmental changes and respond appropriately—an ability that is invested in every living cell. Within a single cell, this function is largely carried out by networks of signaling molecules. However, the details of how signaling networks help cells make complicated decisions are still not clear. For instance, how do cells read graded, analog stress signals but convert them into digital live-or-die responses? The answer to such questions may originate from the fact that signaling molecules are not static but dynamic entities, changing in numbers and activity over time and space. In the past two decades, researchers have been able to experimentally monitor signaling dynamics and use mathematical techniques to quantify and abstract general principles of how cells process information. In this review, the authors first introduce and discuss various experimental and computational methodologies that have been used to study signaling dynamics. The authors then discuss the different types of temporal dynamics such as oscillations and bistability that can be exhibited by signaling systems and highlight studies that have investigated such dynamics in physiological settings. Finally, the authors illustrate the role of spatial compartmentalization in regulating cellular responses with examples of second-messenger signaling in cardiac myocytes.     

Studies on collagen-tannic acid-collagenase ternary system: Inhibition of collagenase against collagenolytic degradation of extracellular matrix component of collagen

We report the detailed studies on the inhibitory effect of tannic acid (TA) on Clostridium histolyticum collagenase (ChC) activity against degradation of extracellular matrix component of collagen. The TA treated collagen exhibited 64% resistance against collagenolytic hydrolysis by ChC, whereas direct interaction of TA with ChC exhibited 99% inhibition against degradation of collagen and the inhibition was found to be concentration dependant. The kinetic inhibition of ChC has been deduced from the extent of hydrolysis of N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA). This data provides a selective competitive mode of inhibition on ChC activity seems to be influenced strongly by the nature and structure of TA. TA showed inhibitor activity against the ChC by molecular docking method. This result demonstrated that TA containing digalloyl radical possess the ability to inhibit the ChC. The inhibition of ChC in gaining new insight into the mechanism of stabilization of collagen by TA is discussed.     

Structural and functional analysis of HtrA1 and its subdomains

1. Download : Download high-res image (216KB)
2. Download : Download full-size image

Highlights? IGFBP-like domain structure suggests that it is incompetent for IGF binding ? Kazal-like domain structure shows distorted “P1” loop incompetent for inhibition ? Protease domain shows catalytic readiness in apo form ? SAXS envelope for full-length HtrA1     

Predicting protein structural class by SVM with class-wise optimized features and decision probabilities

Determination of protein structural class solely from sequence information is a challenging task. Several attempts to solve this problem using various methods can be found in literature. We present support vector machine (SVM) approach where probability-based decision is used along with class-wise optimized feature sets. This approach has two distinguishing characteristics from earlier attempts: (1) it uses class-wise optimized features and (2) decisions of different SVM classifiers are coupled with probability estimates to make the final prediction. The algorithm was tested on three datasets, containing 498 domains, 1092 domains and 5261 domains. Ten-fold external cross-validation was performed to assess the performance of the algorithm. Significantly high accuracy of 92.89% was obtained for the 498-dataset. We achieved 54.67% accuracy for the dataset with 1092 domains, which is better than the previously reported best accuracy of 53.8%. We obtained 59.43% prediction accuracy for the larger and less redundant 5261-dataset. We also investigated the advantage of using class-wise features over union of these features (conventional approach) in one-vs.-all SVM framework. Our results clearly show the advantage of using class-wise optimized features. Brief analysis of the selected class-wise features indicates their biological significance.     

Superior quality agar from <Emphasis Type="Italic">Gracilaria</Emphasis> species (Gracilariales,Rhodophyta) collected from the Gulf of Mannar,India

Gracilaria edulis, G. crassa, G. foliifera, and G. corticata are naturally occurring agarophytes of Indian waters. These agarophytes were evaluated for their agar contents using an improved process recently reported by us (US Patent 2005/0267296A1). The effect of different concentrations of NaOH in the alkali treatment was studied for optimizing the extraction conditions. These Gracilaria species of Indian waters produced agars, both native and alkali treated, with different properties confirming the heterogeneity of the agar polymers in this genera, as one would expect. Among these, G. edulis and G. crassa produced agar polymers having high gel strengths of 490 ± 8.16 and 800 ± 15.4 g cm⁻², respectively, with 8% NaOH treatment as opposed the low gel strength agars that have been reported in the literature to date.     

Fatty Acid Production from Amino Acids and α-Keto Acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and α-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of α-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and α-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from α-keto acids only. BL2 also converted α-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and α-keto acids and that carbon metabolism is important in regulating this event.     

Cloning and characterization of thermotolerant xylitol dehydrogenases from yeast Pichia angusta

Pichia angusta (syn. Hansenula polymorpha) represents one of the rare yeast that can grow and ferment both xylose and glucose at higher temperature (50°C). However, little is known about the enzymes involved in xylose utilization from this species. Previous studies indicated the presence of one xylose reductase and two xylitol dehydrogenase genes in P. angusta. In this study, we have expressed both xylitol dehydrogenases (PaXdh1p and PaXdh2p) in Escherichia coli and purified them as 6X-Histidine-tagged proteins. Biochemical characterization of the recombinant proteins reveals that both PaXdh1p and PaXdh2p are thermotolerant enzymes. PaXdh2p contains a catalytic and a structural Zn atom. However, the structural Zn atom is not present in PaXdh1p. Both enzymes also differ in their affinity for the substrate as well as in the catalytic efficiency. Through mutagenesis and modeling approaches, we have also identified residues important for catalysis and substrate binding.