Siddhartha Roy

Small cationic protein from a marine turtle has β‐defensin‐like fold and antibacterial and antiviral activity

Egg white of marine turtle Caretta caretta contains a small cationic protein but lacks lysozyme. The protein was sequenced by a combination of sequential Edman degradation, carboxypeptidase digestion, nuclear magnetic resonance (NMR) and electrospray ionization tandem mass spectrometry. The protein contains 36 amino acid residues of which six are half‐cysteines. The three‐dimensional structure of the protein was deduced from two‐dimensional NMR experiments and was observed to be similar to vertebrate β‐defensins. However, disulfide connectivity is C1–C6/C2–C5/C3–C4; different from that of the vertebrate β‐defensins. The protein showed strong antibacterial activity against Escherichia coli and Salmonella typhimurium. The protein also showed significant antiviral activity against an enveloped rhabdovirus, Chandipura virus, which is an emerging human pathogen. This virus is also closely related to the vesicular stomatitis virus, whose growth was also inhibited. This small cationic protein is part of the innate immunity of this organism and replaces lysozyme in the egg. It has the potential to be developed as an antibacterial and antiviral agent. Proteins 2006.

Multifunctional enzymes and evolution of biosynthetic pathways: Retro‐evolution by jumps

A likely scenario of evolution of biosynthetic pathways is believed to have occurred by retro‐evolution through recruitment of existing enzymes rather than generation of de novo classes. It had been proposed that such retro‐evolution occurred in steps as a response to depletion of an essential metabolite and availability of another related substance in the environment. In this article, I argue that because of instability of many such extant intermediates, it is unlikely that retro‐evolution had occurred in steps. I further propose that such evolution in many cases has taken place by jumps, i.e., by recruitment of a multifunctional enzyme capable of catalyzing several steps at a time, albeit inefficiently. I further speculate that in some cases one primordial multienzyme may have catalyzed the whole sequence of reaction of a biosynthetic pathway, i.e., the pathway may have evolved by a single leap. Gene duplications and further evolution to more efficient enzymes led to extant pathways. Such a mechanism predicts that some or all enzymes of a pathway must have descended from a common ancestor. Sequence and structural homologies among extant enzymes of a biosynthetic pathway have been examined. Proteins 1999;37:303–309. ©1999 Wiley‐Liss, Inc.

Hydroxychavicol, a Piper betle leaf component, induces apoptosis of CML cells through mitochondrial reactive oxygen species-dependent JNK and endothelial nitric oxide synthase activation and overrides imatinib resistance.

Alcoholic extract of Piperbetle (Piper betle L.) leaves was recently found to induce apoptosis of CML cells expressing wild type and mutated Bcr‐Abl with imatinib resistance phenotype. Hydroxychavicol (HCH), a constituent of the alcoholic extract of Piper betle leaves, was evaluated for anti‐CML activity. Here, we report that HCH and its analogues induce killing of primary cells in CML patients and leukemic cell lines expressing wild type and mutated Bcr‐Abl, including the T315I mutation, with minimal toxicity to normal human peripheral blood mononuclear cells. HCH causes early but transient increase of mitochondria‐derived reactive oxygen species. Reactive oxygen species‐dependent persistent activation of JNK leads to an increase in endothelial nitric oxide synthase‐mediated nitric oxide generation. This causes loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, cleavage of caspase 9, 3 and poly‐adenosine diphosphate‐ribose polymerase leading to apoptosis. One HCH analogue was also effective in vivo in SCID mice against grafts expressing the T315I mutation, although to a lesser extent than grafts expressing wild type Bcr‐Abl, without showing significant bodyweight loss. Our data describe the role of JNK‐dependent endothelial nitric oxide synthase‐mediated nitric oxide for anti‐CML activity of HCH and this molecule merits further testing in pre‐clinical and clinical settings. (Cancer Sci 2012; 103: 88–99)

Unique pyrimidine 2D-COSY aromatic cross-peaks as monitors of pyrimidine environments and mobility in oligo- and polynucleotides☆

Only cytosine contains two adjacent aromatic protons that give rise to cross-peaks in the aromatic region of 2D-COSY spectra of oligodeoxynucleotides. In two GC-containing sequences several such cross-peaks were resolved. The intensity of these cross-peaks is a sensitive monitor of local mobility, and upon the addition of the intercalating drug daunomycin selective intensity losses were observed, indicating binding to specific GC base pairs. We have also monitored the 2D-COSY cross-peaks from mobile pyrimidine bases in tRNA (Phe) as a function of temperature.

Role of the carboxy‐termini of tubulin on its chaperone‐like activity

Mutational analysis and the enzymatic digestion of many chaperones indicate the importance of both hydrophobic and hydrophilic residues for their unique property. Thus, the chaperone activity of α‐crystallin is lost due to the substitution of hydrophobic residues or upon enzymatic digestion of the negatively charged residues. Tubulin, an eukaryotic cytoskeletal protein, exhibits chaperone‐like activity as demonstrated by prevention of DTT‐induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase, βγ‐crystallin, and other proteins. We have shown that the tubulin lost its chaperone‐like activity upon digestion of its negatively charged C‐termini. In this article, the role of the C‐terminus of individual subunits has been investigated. We observe that the digestion of C‐terminus of β‐subunit with subtilisin causes loss of chaperone‐like activity of tubulin. The contribution of C‐terminus of α‐subunit is difficult to establish directly as subtilisin cleaves C‐terminus of β‐subunit first. This has been ascertained indirectly using a 14‐residue peptide P2 having the sequence corresponding to a conserved region of MHC class I molecules and that binds tightly to the C‐terminus of α‐subunit. We have shown that the binding of P2 peptide to αβ‐tubulin causes complete loss of its chaperone‐like activity. NMR and gel‐electrophoresis studies indicate that the P2 peptide has a significant higher binding affinity for the C‐terminus of α‐subunit compared to that of β‐subunit. Thus, we conclude that both the C‐termini are necessary for the chaperone‐like activity of tubulin. Implications for the chaperone functions in vivo have been discussed. Proteins 2001;44:262–269.

Dynamics of compact denatured states of glutaminyl-tRNA synthetase probed by bis-ANS binding kinetics.

Bis-ANS binds to native glutaminyl-tRNA synthetase (GlnRS) with a fast and a slow phase. The rate constant of the slow phase is independent of bis-ANS concentration suggesting a slow conformational change in the pathway of bis-ANS binding. Aging of GlnRS causes a large decrease of the slow phase amplitude with concomitant increase of the fast phase amplitude. Several other large, multi-domain proteins show similar patterns upon aging. The near UV-CD spectra of the native and the aged GlnRS remain similar. Significant changes in far UV-CD, acrylamide quenching and sulfhydryl reactivity, are seen upon aging, suggesting disruptions in native interactions. Refolding of GlnRS from the urea-denatured state rapidly produces a state that is very similar to the equilibrium molten globule state. Bis-ANS binds to the molten globule state with kinetics similar to that of the aged state and unlike that of the native state. This suggests that the slow binding phase of bis-ANS, seen in native proteins, originate from relatively high energy barriers between the native and the more open states. Thus bis-ANS can be used as a powerful probe for large amplitude, low-frequency motions of proteins.

Ligand specificity and ligand‐induced conformational change in gal repressor

Gal repressor (GalR) binds D‐galactose, which is responsible for lifting of repression of the gal operon. Proton T1 measurements of α‐ and β‐anomers of galactose as a function of gal repressor show preferential binding of the β‐anomer. The β‐anomer was isolated by high‐performance liquid chromatography and was shown to bind tightly to GalR. Calorimetry was used to determine enthalpy changes at several temperatures. Heat capacity change was found to be positive, indicating that a significant amount of hydrophobic surface area was exposed upon galactose binding. Bis‐ANS binding to GalR is significantly enhanced in the presence of a saturating amount of galactose, indicating additional exposure of hydrophobic surfaces. We propose that the galactose‐induced conformational change involves the opening of the two subdomains, which may disrupt protein–protein interactions responsible for repression. Proteins 2002;49:554–559.

Gene regulatory networks and epigenetic modifications in cell differentiation

It is becoming increasingly clear that the functionalities of an organism are mostly derived from regulation of its gene repertoire. Specialized cell types are created from pluripotent stem cells by regulating expression of genes. In eukaryotes, genes are primarily regulated by gene regulatory networks consisting of highly sequence‐specific transcription factors and epigenetic modifications. The former mode of regulation is more readily reversible and non‐heritable across cell generations, whereas the latter mode is less reversible and heritable. In this article, we explore the relationship between cell differentiation and the two modes of regulation of gene expression, focusing primarily on pluripotent and multipotent stem cells. Recent studies suggest that stem cells execute different gene expression programs, probably driven by one or more gene regulatory network(s). It is now also evident that as stem cells differentiate to more specialized progeny cells, rewriting of epigenetic marks occurs in parallel with the change in the pattern of gene expression. A conceptual framework is put forward in which it is proposed that the cell fate determining gene regulatory network in a pluripotent or multipotent cell has the capability to exist in multiple stationary states with each stationary state dictating a particular pattern of gene expression. We also propose that the broad pattern of gene expression in each stationary state, termed the lineage biased state or LIBS, resembles that of a more differentiated progeny cell. The differentiation process leading to a particular progeny cell involves rewriting of epigenetic marks that result in upregulation of genes in a LIBS and silencing of genes involved in alternative LIBS; thus selecting a particular pattern of gene expression and making a lineage commitment.

Antagonists of Hsp16.3, a Low-Molecular-Weight Mycobacterial Chaperone and Virulence Factor, Derived from Phage-Displayed Peptide Libraries

ABSTRACT The persistence of Mycobacterium tuberculosis is a major cause of concern in tuberculosis (TB) therapy. In the persistent mode the pathogen can resist drug therapy, allowing the possibility of reactivation of the disease. Several protein factors have been identified that contribute to persistence, one of them being the 16-kDa low-molecular-weight mycobacterial heat shock protein Hsp16.3, a homologue of the mammalian eye lens protein alpha-crystallin. It is believed that Hsp16.3 plays a key role in the persistence phase by protecting essential proteins from being irreversibly denatured. Because of the close association of Hsp16.3 with persistence, an attempt has been made to develop inhibitors against it. Random peptide libraries displayed on bacteriophage M13 were screened for Hsp16.3 binding. Two phage clones were identified that bind to the Hsp16.3 protein. The corresponding synthetic peptides, an 11-mer and a 16-mer, were able to bind Hsp16.3 and inhibit its chaperone activity in vitro in a dose-dependent manner. Little or no effect of these peptides was observed on alphaB-crystallin, a homologous protein that is a key component of human eye lens, indicating that there is an element of specificity in the observed inhibition. Two histidine residues appear to be common to the selected peptides. Nuclear magnetic resonance studies performed with the 11-mer peptide indicate that in this case these two histidines may be the crucial binding determinants. The peptide inhibitors of Hsp16.3 thus obtained could serve as the basis for developing potent drugs against persistent TB.

Simultaneous Inhibition of Key Growth Pathways in Melanoma Cells and Tumor Regression by a Designed Bidentate Constrained Helical Peptide

Protein–protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium‐regulated protein, plays a crucial role in the proliferation of melanoma cells through protein–protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight‐binding peptide, TRTK‐12. The helical conformation of the peptide was constrained by the substitution of α‐amino isobutyric acid—an amino acid having high helical propensity—in positions which do not interact with S100B. A branched bidentate version of the peptide was bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell‐penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts antiproliferative action through simultaneous inhibition of key growth pathways, including reactivation of wild‐type p53 and inhibition of Akt and STAT3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein–protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein–protein interactions for de novo drug development.