Self-acylation properties of type II fatty acid biosynthesis acyl carrier protein

Acyl carrier protein (ACIP) plays a central role in many metabolic processes inside the cell, and almost 4% of the total enzymes inside the cell require it as a cofactor. Here, we report self-acylation properties in ACPs from Plasmodium falciparum and Brassica napus that are essential components of type II fatty acid biosynthesis (FAS II), disproving the existing notion that this phenomenon is restricted only to ACPs involved in polyketide biosynthesis. We also provide strong evidence to suggest that catalytic self-acylation is intrinsic to the individual ACP. Mutational analysis of these ACPs revealed the key residue(s) involved in this phenomenon. We also demonstrate that these FAS 11 ACPs exhibit a high degree of selectivity for self-acylation employing only dicarboxylic acids as substrates. A plausible mechanism for the self-acylation reaction is also proposed.

Three-dimensional structure of heat shock protein 90 from Plasmodium falciparum: molecular modelling approach to rational drug design against malaria

We have recently implicated heat shock protein 90 from Plasmodium falciparum (PfHsp90) as a potential drug target against malaria. Using inhibitors specific to the nucleotide binding domain of Hsp90, we have shown potent growth inhibitory effects on development of malarial parasite in human erythrocytes. To gain better understanding of the vital role played by PfHsp90 in parasite growth, we have modeled its three dimensional structure using recently described full length structure of yeast Hsp90. Sequence similarity found between PfHsp90 and yeast Hsp90 allowed us to model the core structure with high confidence. The superimposition of the predicted structure with that of the template yeast Hsp90 structure reveals an RMSD of 3.31 angstrom. The N-terminal and middle domains showed the least RMSD (1.76 angstrom) while the more divergent C-terminus showed a greater RMSD (2.84 angstrom) with respect to the template. The structure shows overall conservation of domains involved in nucleotide binding, ATPase activity, co-chaperone binding as well as inter-subunit interactions. Important co-chaperones known to modulate Hsp90 function in other eukaryotes are conserved in malarial parasite as well. An acidic stretch of amino acids found in the linker region, which is uniquely extended in PfHsp90 could not be modeled in this structure suggesting a flexible conformation. Our results provide a basis to compare the overall structure and functional pathways dependent on PfHsp90 in malarial parasite. Further analysis of differences found between human and parasite Hsp90 may make it possible to design inhibitors targeted specifically against malaria.

Evolution of domain combinations in protein kinases and its implications for functional diversity

Protein kinases phosphorylating Ser/Thr/Tyr residues in several cellular proteins exert tight control over their biological functions. They constitute the largest protein family in most eukaryotic species. Protein kinases classified based on sequence similarity in their catalytic domains, cluster into subfamilies, which share gross functional properties. Many protein kinases are associated or tethered covalently to domains that serve as adapter or regulatory modules,naiding substrate recruitment, specificity, and also serve as scaffolds. Hence the modular organisation of the protein kinases serves as guidelines to their functional and molecular properties. Analysis of genomic repertoires of protein kinases in eukaryotes have revealed wide spectrum of domain organisation across various subfamilies of kinases. Occurrence of organism-specific novel domain combinations suggests functional diversity achieved by protein kinases in order to regulate variety of biological processes. In addition, domain architecture of protein kinases revealed existence of hybrid protein kinase subfamilies and their emerging roles in the signaling of eukaryotic organisms. In this review we discuss the repertoire of non-kinase domains tethered to multi-domain kinases in the metazoans. Similarities and differences in the domain architectures of protein kinases in these organisms indicate conserved and unique features that are critical to functional specialization. (C) 2009 Elsevier Ltd. All rights reserved.

Stimulating immune responses to fight cancer: Basic biology and mechanisms

Chronic inflammation is now recognized as a major cause of malignant disease. In concert with various mechanisms (including DNA instability), hypoxia and activation of inflammatory bioactive lipid pathways and pro-inflammatory cytokines open the doorway to malignant transformation and proliferation, angiogenesis, and metastasis in many cancers. A balance between stimulatory and inhibitory signals regulates the immune response to cancer. These include inhibitory checkpoints that modulate the extent and duration of the immune response and may be activated by tumor cells. This contributes to immune resistance, especially against tumor antigen-specific T-cells. Targeting these checkpoints is an evolving approach to cancer immunotherapy, designed to foster an immune response. The current focus of these trials is on the programmed cell death protein 1 (PD-1) receptor and its ligands (PD-L1, PD-L2) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Researchers have developed anti-PD-1 and anti-PDL-1 antibodies that interfere with the ligands and receptor and allow the tumor cell to be recognized and attacked by tumor-infiltrating T-cells. These are currently being studied in lung cancer. Likewise, CTLA-4 inhibitors, which have had success treating advanced melanoma, are being studied in lung cancer with encouraging results.

Abnormal levels of heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) in tumour tissue and blood samples from patients diagnosed with lung cancer

Lung cancer is the second most common type of cancer in the world and is the most common cause of cancer-related death in both men and women. Research into causes, prevention and treatment of lung cancer is ongoing and much progress has been made recently in these areas, however survival rates have not significantly improved. Therefore, it is essential to develop biomarkers for early diagnosis of lung cancer, prediction of metastasis and evaluation of treatment efficiency, as well as using these molecules to provide some understanding about tumour biology and translate highly promising findings in basic science research to clinical application. In this investigation, two-dimensional difference gel electrophoresis and mass spectrometry were initially used to analyse conditioned media from a panel of lung cancer and normal bronchial epithelial cell lines. Significant proteins were identified with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), pyruvate kinase M2 isoform (PKM2), Hsc-70 interacting protein and lactate dehydrogenase A (LDHA) selected for analysis in serum from healthy individuals and lung cancer patients. hnRNPA2B1, PKM2 and LDHA were found to be statistically significant in all comparisons. Tissue analysis and knockdown of hnRNPA2B1 using siRNA subsequently demonstrated both the overexpression and potential role for this molecule in lung tumorigenesis. The data presented highlights a number of in vitro derived candidate biomarkers subsequently verified in patient samples and also provides some insight into their roles in the complex intracellular mechanisms associated with tumour progression.

Role of Magmas in protein transport and human mitochondria biogenesis

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of delta pam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process.

New measures for estimating surface complementarity and packing at protein-protein interfaces

A number of methods exist that use different approaches to assess geometric properties like the surface complementarity and atom packing at the protein-protein interface. We have developed two new and conceptually different measures using the Delaunay tessellation and interface slice selection to compute the surface complementarity and atom packing at the protein-protein interface in a straightforward manner. Our measures show a strong correlation among themselves and with other existing measures, and can be calculated in a highly time-efficient manner. The measures are discriminative for evaluating biological, as well as non-biological protein-protein contacts, especially from large protein complexes and large-scale structural studies(http://pallab.serc. iisc.ernet.in/nip_nsc). (C) 201 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Rotavirus nonstructural proteins: a structural perspective

Rotavirus is a major cause of acute infantile diarrhoea worldwide. The virus genome consists of 11 segments of double-stranded RNA that codesfor six structural proteins (VP1-6) and six non-structural proteins(NSP1-6). NSPs are proteins expressed from the virus genome in the infected cell, but are not incorporated into the mature virus article. NSPs play an essential role in virus replication, morphogenesis and pathogenesis, and most of them exhibit multifunctional properties. Structure-function analysis of the NSPs is essential for understanding the molecular mechanisms by which the virus circumvents host innate immune responses, inhibits cellular protein synthesis, hijacks the protein synthetic machinery for its own propagation and manifests the disease process. Because of their essential roles in virus biology, NSPs represent potential targets for the development of antiviral agents. Determination of the three-dimensional structure of NSPs has been hindered due to low-level expression and aggregation. To date, the complete three-dimensional structure of only NSP2 has been determined. The structures of the N- and C-terminal domains of NSP3 and the diarrhoea-inducing domain of NSP4 have also been determined. This review primarily covers the structural and biological functions of the NSPs whose three-dimensional structural aspects have been fully or partially understood, but provides a brief account of other NSPs and the structural features of the mature virion as determined by electron cryomicroscopy.

Role of an RNA polymerase interacting protein, MsRbpA, from Mycobacterium smegmatis in phenotypic tolerance to rifampicin

Rifampicin and its derivatives are at the forefront of the current standard chemotherapeutic regimen for active tuberculosis; they act by inhibiting the transcription activity of prokaryotic RNA polymerase. Rifampicin is believed to interact with the beta subunit of RNA polymerase. However, it has been observed that protein-protein interactions with RNA polymerase core enzyme lead to its reduced susceptibility to rifampicin. This mechanism became more diversified with the discovery of RbpA, a novel RNA polymerase-binding protein, in Streptomyces coelicolor that could mitigate the effect of rifampicin on RNA polymerase activity. MsRbpA is a homologue of RbpA in Mycobacterium smegmatis. On deciphering the role of MsRbpA in M. smegmatis we found that it interacts with RNA polymerase and increases the rifampicin tolerance levels, both in vitro and in vivo. It interacts with the beta subunit of RNA polymerase. However, it was found to be incapable of rescuing rifampicin-resistant RNA polymerases in the presence of rifampicin at the respective IC50.

A network representation of protein structures: Implications for protein stability

This study views each protein structure as a network of noncovalent connections between amino acid side chains. Each amino acid in a protein structure is a node, and the strength of the noncovalent interactions between two amino acids is evaluated for edge determination. The protein structure graphs (PSGs) for 232 proteins have been constructed as a function of the cutoff of the amino acid interaction strength at a few carefully chosen values. Analysis of such PSGs constructed on the basis of edge weights has shown the following: 1), The PSGs exhibit a complex topological network behavior, which is dependent on the interaction cutoff chosen for PSG construction. 2), A transition is observed at a critical interaction cutoff, in all the proteins, as monitored by the size of the largest cluster (giant component) in the graph. Amazingly, this transition occurs within a narrow range of interaction cutoff for all the proteins, irrespective of the size or the fold topology. And 3), the amino acid preferences to be highly connected (hub frequency) have been evaluated as a function of the interaction cutoff. We observe that the aromatic residues along with arginine, histidine, and methionine act as strong hubs at high interaction cutoffs, whereas the hydrophobic leucine and isoleucine residues get added to these hubs at low interaction cutoffs, forming weak hubs. The hubs identified are found to play a role in bringing together different secondary structural elements in the tertiary structure of the proteins. They are also found to contribute to the additional stability of the thermophilic proteins when compared to their mesophilic counterparts and hence could be crucial for the folding and stability of the unique three-dimensional structure of proteins. Based on these results, we also predict a few residues in the thermophilic and mesophilic proteins that can be mutated to alter their thermal stability.

Identification of protein interaction regions of VINC/NEAT1/Men epsilon RNA

The virus inducible non-coding RNA (VINC) was detected initially in the brain of mice infected with Japanese encephalitis virus (JEV) and rabies virus. VINC is also known as NEAT1 or Men epsilon RNA. It is localized in the nuclear paraspeckles of several murine as well as human cell lines and is essential for paraspeckle formation. We demonstrate that VINC interacts with the paraspeckle protein, P54nrb through three different protein interaction regions (PIRs) one of which (PIR-1) is localized near the 50 end while the other two (PIR-2, PIR-3) are localized near the 30 region of VINC. Our studies suggest that VINC may interact with P54nrb through a novel mechanism which is different from that reported for protein coding RNAs. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Modular design of synthetic protein mimics. Crystal structures, assembly, and hydration of two 15- and 16-residue apolar, leucyl-rich helical peptides

Two IS- and 16-residue peptides containing a-aminoisobutyric acid (Aib) have been synthesized, as part of a strategy to construct stereochemically rigid peptide helices, in a modular approach to design of protein mimics. The peptides Boc-(Val-Ala-Leu-Aib),-OMe ( I ) and Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-(Val-Ala-Leu-Aib()11z)- OhaMvee been crystallized.Both crystals are stable only in the presence of mother liquor or water. The crystal data are as follows. I: C78H140N16019~2H20,P2,, a = 16.391 (3) A, b = 16.860 (3) A, c = 18.428 (3) A, p = 103.02 (I)O, Z = 2, R = 9.6% for 3445 data with lFol >30(F), resolution 0.93 A. 11: C7,Hl,,N,S018.7.5H,0, C2221, a = 18.348 ( 5 ) A, b = 47.382 (1 1) A, c = 24.157 ( 5 ) A, Z =8, R = l0,6%, for 3147 data with lFol > 3a(F), resolution 1.00 A. The 15-residue peptide (11) is entirely a helical, while the 16-residue peptide ( I ) has a short segment of 310 helix at the N terminus. The packing of the helices in the crystals is rather incfficicnt with no particular attractions between Leu-Leu side chains, or any other pair. Both crystals have fairly large voids, which are filled with water molecules in a disordered fashion. Water molecule sites near the polar head-to-tail regions are well detcrmined, those closer to the hydrophobic side chains less so and a number of possible water sites in the remaining "empty" space are not determined. No interdigitation of Leu side chains is observed in the crystal as is hypothesized in the "leucine zipper" class of DNA binding proteins.

Modular design of synthetic protein mimics. Crystal structure of two seven-residue helical peptide segments linked by .epsilon.-aminocaproic acid

Two seven-residue helical segments, Val-Ala-Leu-Aib-Val-Ala-Leu, were linked synthetically with an epsilon-aminocaproic acid (Acp) linker with the intention of making a stable antiparallel helix-helix motif. The crystal structure of the linked peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Acp-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe (1) shows the two helices displaced laterally from each other by the linker, but the linker has not folded the molecule into a close-packed antiparallel conformation. Two strong intermolecular NH...O = C hydrogen bonds are formed between the top of the lower helix of one molecule and the bottom of the upper helix in a laterally adjacent molecule to give the appearance of an extended single helix. The composite peptide with Boc and OMe end groups, C76H137N15O18.H2O, crystallize in space group P2(1) with a = 8.802 (1) angstrom, b = 20.409 (4) angstrom, c = 26.315 (3) angstrom, and beta = 90.72 (1)degrees; overall agreement R = 7.86% for 5030 observed reflections (\F(o)\ > 3-sigma(F)); resolution = 0.93 angstrom. Limited evidence for a more compact conformation in solution consistent with an antiparallel helix arrangement is obtained by comparison of the HPLC retention times and CD spectra of peptide 1 with well-characterized continuous helices of similar length and sequence.

Disallowed Ramachandran Conformations of Amino Acid Residues in Protein Structures

An analysis of the nature and distribution of disallowed Ramachandran conformations of amino acid residues observed in high resolution protein crystal structures has been carried out. A data set consisting of 110 high resolution, non-homologous, protein crystal structures from the Brookhaven Protein Data Bank was examined. The data set consisted of a total of 18,708 non-Gly residues, which were characterized on the basis of their backbone dihedral angles (φ, ψ). Residues falling outside the defined “broad allowed limits” on the Ramachandran map were chosen and the reportedB-factor value of the α-carbon atom was used to further select well defined disallowed conformations. The conformations of the selected 66 disallowed residues clustered in distinct regions of the Ramachandran map indicating that specific φ, ψ angle distortions are preferred under compulsions imposed by local constraints. The distribution of various amino acid residues in the disallowed residue data set showed a predominance of small polar/charged residues, with bulky hydrophobic residues being infrequent. As a further check, for all the 66 cases non-hydrogen van der Waals short contacts in the protein structures were evaluated and compared with the ideal “Ala-dipeptide” constructed using disallowed dihedral angle (φ, ψ) values. The analysis reveals that short contacts are eliminated in most cases by local distortions of bond angles. An analysis of the conformation of the identified disallowed residues in related protein structures reveals instances of conservation of unusual stereochemistry.

Non-Protein Amino Acids in the Design of Secondary Structure Scaffolds

The use of stereochemically constrained amino acids permits the design of short peptides as models for protein secondary structures. Amino acid residues that are restrained to a limited range of backbone torsion angles (ϕ-ψ) may be used as folding nuclei in the design of helices and β-hairpins. α-Amino-isobutyric acid (Aib) and related Cαα dialkylated residues are strong promoters of helix formation, as exemplified by a large body of experimentally determined structures of helical peptides. DPro-Xxx sequences strongly favor type II’ turn conformations, which serve to nucleate registered β-hairpin formation. Appropriately positioned DPro-Xxx segments may be used to nucleate the formation of multistranded antiparallel β-sheet structures. Mixed (α/β) secondary structures can be generated by linking rigid modules of helices and β-hairpins. The approach of using stereochemically constrained residues promotes folding by limiting the local structural space at specific residues. Several aspects of secondary structure design are outlined in this chapter, along with commonly used methods of spectroscopic characterization.