Exploration of Potential Broad-Spectrum Antiviral Targets in the Enterovirus Replication Element: Identification of Six Distinct 5' Cloverleaves.

genomic replication initiates at a predicted RNA cloverleaf (5'CL) at the 5' end of the RNA genome. The 5'CL contains one stem (SA) and three stem-loops (SLB, SLC, SLD). Here, we present an analysis of 5'CL conservation and divergence for 209 human health-related serotypes from the genus, including enterovirus and rhinovirus species.

Evidence of direct interaction between cisplatin and the caspase-cleaved prostate apoptosis response-4 tumor suppressor.

Prostate apoptosis response-4 (Par-4) tumor suppressor protein has gained attention as a potential therapeutic target owing to its unique ability to selectively induce apoptosis in cancer cells, sensitize them to chemotherapy and radiotherapy, and mitigate drug resistance. It has recently been reported that Par-4 interacts synergistically with cisplatin, a widely used anticancer drug. However, the mechanistic details underlying this relationship remain elusive.

Enhancing the Conformational Stability of the cl-Par-4 Tumor Suppressor via Site-Directed Mutagenesis.

Intrinsically disordered proteins play important roles in cell signaling, and dysregulation of these proteins is associated with several diseases. Prostate apoptosis response-4 (Par-4), an approximately 40 kilodalton proapoptotic tumor suppressor, is a predominantly intrinsically disordered protein whose downregulation has been observed in various cancers. The caspase-cleaved fragment of Par-4 (cl-Par-4) is active and plays a role in tumor suppression by inhibiting cell survival pathways.

Structural Analysis of the cl-Par-4 Tumor Suppressor as a Function of Ionic Environment.

Prostate apoptosis response-4 (Par-4) is a proapoptotic tumor suppressor protein that has been linked to a large number of cancers. This 38 kilodalton (kDa) protein has been shown to be predominantly intrinsically disordered in vitro. In vivo, Par-4 is cleaved by caspase-3 at Asp-131 to generate the 25 kDa functionally active cleaved Par-4 protein (cl-Par-4) that inhibits NF-κB-mediated cell survival pathways and causes selective apoptosis in tumor cells.

Structural Biology of the Enterovirus Replication-Linked 5'-Cloverleaf RNA and Associated Virus Proteins.

Although enteroviruses are associated with a wide variety of diseases and conditions, their mode of replication is well conserved. Their genome is carried as a single, positive-sense RNA strand. At the 5' end of the strand is an approximately 90-nucleotide self-complementary region called the 5' cloverleaf, or the oriL.

A synthetic Pur-based peptide binds and alters G-quadruplex secondary structure present in the expanded RNA repeat of C9orf72 ALS/FTD.

Increased Pur-alpha (Pura) protein levels in animal models alleviate certain cellular symptoms of the disease spectrum amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). Pura is a member of the Pur family of evolutionarily conserved guanine-rich polynucleotide binding proteins containing a repeated signature PUR domain of 60-80 amino acids. Here we have employed a synthetic peptide, TZIP, similar to a Pur domain, but with sequence alterations based on a consensus of evolutionarily conserved Pur family binding domains and having an added transporter sequence.

Tetramer formation by the caspase-activated fragment of the Par-4 tumor suppressor.

The prostate apoptosis response-4 (Par-4) tumor suppressor can selectively kill cancer cells via apoptosis while leaving healthy cells unharmed. Full length Par-4 has been shown to be predominantly intrinsically disordered in vitro under neutral conditions. As part of the apoptotic process, cellular Par-4 is cleaved at D131 by caspase-3, which generates a 24 kDa C-terminal activated fragment (cl-Par-4) that enters the nucleus and inhibits pro-survival genes, thereby preventing cancer cell proliferation.

Conformational flexibility in the enterovirus RNA replication platform.

A presumed RNA cloverleaf (5'CL), located at the 5'-most end of the noncoding region of the enterovirus genome, is the primary established site for initiation of genomic replication. Stem-loop B (SLB) and stem-loop D (SLD), the two largest stem-loops within the 5'CL, serve as recognition sites for protein interactions that are essential for replication. Here we present the solution structure of rhinovirus serotype 14 5'CL using a combination of nuclear magnetic resonance spectroscopy and small-angle X-ray scattering.

pH-Induced Folding of the Caspase-Cleaved Par-4 Tumor Suppressor: Evidence of Structure Outside of the Coiled Coil Domain.

Prostate apoptosis response-4 (Par-4) is a 38 kDa largely intrinsically disordered tumor suppressor protein that functions in cancer cell apoptosis. Par-4 down-regulation is often observed in cancer while up-regulation is characteristic of neurodegenerative conditions such as Alzheimer's disease. Cleavage of Par-4 by caspase-3 activates tumor suppression via formation of an approximately 25 kDa fragment (cl-Par-4) that enters the nucleus and inhibits Bcl-2 and NF-ƙB, which function in pro-survival pathways.

Conformational Clusters of Phosphorylated Tyrosine.

Tyrosine phosphorylation plays an important role in many cellular and intercellular processes including signal transduction, subcellular localization, and regulation of enzymatic activity. In 1999, Blom et al., using the limited number of protein data bank (PDB) structures available at that time, reported that the side chain structures of phosphorylated tyrosine (pY) are partitioned into two conserved conformational clusters ( Blom, N.

Structure of RNA Stem Loop B from the Picornavirus Replication Platform.

The presumptive RNA cloverleaf at the start of the 5'-untranslated region of the picornavirus genome is an essential element in replication. Stem loop B (SLB) of the cloverleaf is a recognition site for the host polyC-binding protein, which initiates a switch from translation to replication. Here we present the solution structure of human rhinovirus isotype 14 SLB using nuclear magnetic resonance spectroscopy.

Structural, dynamic, and chemical characterization of a novel S-glycosylated bacteriocin.

Bacteriocins are bacterial peptides with specific activity against competing species. They hold great potential as natural preservatives and for their probiotic effects. We show here nuclear magnetic resonance-based evidence that glycocin F, a 43-amino acid bacteriocin from Lactobacillus plantarum, contains two β-linked N-acetylglucosamine moieties, attached via side chain linkages to a serine via oxygen, and to a cysteine via sulfur.

Two-state conformational equilibrium in the Par-4 leucine zipper domain.

Prostate apoptosis response factor-4 (Par-4) is a pro-apoptotic and tumor-suppressive protein. A highly conserved heptad repeat sequence at the Par-4 C-terminus suggests the presence of a leucine zipper (LZ). This C-terminal region is essential for Par-4 self-association and interaction with various effector proteins.

Solution structure of the squash aspartic acid proteinase inhibitor (SQAPI) and mutational analysis of pepsin inhibition.

The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor.

Rheo-NMR studies of an enzymatic reaction: evidence of a shear-stable macromolecular system.

Understanding the effects of shear forces on biopolymers is key to understanding how biological systems function. Although currently there is good agreement between theoretical predictions and experimental measurements of the behavior of DNA and large multimeric proteins under shear flow, applying the same arguments to globular proteins leads to the prediction that they should only exhibit shear-induced conformational changes at extremely large shear rates. Nevertheless, contradictory experimental evidence continues to appear, and the effect of shear on these biopolymers remains contentious.

N-terminal domains of DELLA proteins are intrinsically unstructured in the absence of interaction with GID1/gibberellic acid receptors.

The plant growth-repressing DELLA proteins (DELLAs) are known to represent a convergence point in integration of multiple developmental and environmental signals in planta, one of which is hormone gibberellic acid (GA). Binding of the liganded GA receptor (GID1/GA) to the N-terminal domain of DELLAs is required for GA-induced degradation of DELLAs via the ubiquitin-proteasome pathway, thus derepressing plant growth. However, the conformational changes of DELLAs upon binding to GID1/GA, which are the key to understanding the precise mechanism of GID1/GA-mediated degradation of DELLAs, remain unclear.

The production of soluble and correctly folded recombinant bovine beta-lactoglobulin variants A and B in Escherichia coli for NMR studies.

The production of soluble and correctly folded eukaryotic proteins in prokaryotic systems has always been hampered by the difference in or lack of cell machinery responsible for folding, post-translation modification and secretion of the proteins involved. In the case of bovine beta-lactoglobulin (BLG), a major cow's milk allergen and a protein widely used for protein folding studies, a eukaryotic yeast expression system has been the preferred choice of many researchers, particularly for the production of isotopically labeled protein required for NMR studies. Although this system yields high amounts of recombinant protein, the BLG produced is usually associated with extracellular polysaccharides, which is problematic for NMR analysis.

Intrinsic disorder and coiled-coil formation in prostate apoptosis response factor 4.

Prostate apoptosis response factor-4 (Par-4) is an ubiquitously expressed pro-apoptotic and tumour suppressive protein that can both activate cell-death mechanisms and inhibit pro-survival factors. Par-4 contains a highly conserved coiled-coil region that serves as the primary recognition domain for a large number of binding partners. Par-4 is also tightly regulated by the aforementioned binding partners and by post-translational modifications.

A picornaviral loop-to-loop replication complex.

Picornaviruses replicate their RNA genomes through a highly conserved mechanism that involves an interaction between the principal viral protease (3C(pro)) and the 5'-UTR region of the viral genome. The 3C(pro) catalytic site is the target of numerous replication inhibitors. This paper describes the first structural model of a complex between a picornaviral 3C(pro) and a region of the 5'-UTR, stem-loop D (SLD).

15N relaxation studies of Apo-Mts1: a dynamic S100 protein.

Mts1 is a member of the S100 family of EF-hand calcium-binding proteins. Like most S100 proteins, Mts1 exists as a dimer in solution and contains one canonical and one pseudo-EF-hand motif per monomer, each of which consists of two alpha helices connected by a loop capable of coordinating a calcium ion. The backbone dynamics of murine apo-Mts1 homodimer have been examined by nuclear magnetic resonance spectroscopy.

NMR structure of stem-loop D from human rhinovirus-14.

The 5'-cloverleaf of the picornavirus RNA genome is essential for the assembly of a ribonucleoprotein replication complex. Stem-loop D (SLD) of the cloverleaf is the recognition site for the multifunctional viral protein 3Cpro. This protein is the principal viral protease, and its interaction with SLD also helps to position the viral RNA-dependent RNA polymerase (3Dpol) for replication.

General method for suppression of diagonal peaks in heteronuclear-edited NOESY spectroscopy.

A novel approach is proposed for suppression of diagonal peaks in 15N- and 13C-edited NOESY spectroscopy based on subtracting a spectrum with only diagonal peaks from the conventional NOESY spectrum. This method can be applied to most heteronuclear-edited NOESY experiments. It is far more sensitive than the TROSY-based approach for biomolecules with little TROSY effect, and nearly complete suppression of diagonal peaks can be achieved.

Stabilization of a pH-sensitive apoptosis-linked coiled coil through single point mutations.

The apoptosis-associated Par-4 protein has been implicated in cancers of the prostate, colon, and kidney, and in Alzheimer's and Huntington's diseases, among other neurodegenerative disorders. Previously, we have shown that a peptide from the Par-4 C-terminus, which is responsible for Par-4 self-association as well as interaction with all currently identified effector molecules, is natively unfolded at neutral pH, but forms a tightly associated coiled coil at acidic pH and low temperature. Here, we have alternately mutated the two acidic residues predicted to participate in repulsive electrostatic interactions at the coiled coil interhelical interface.