The 5' untranslated region of Rhopalosiphum padi virus contains an internal ribosome entry site which functions efficiently in mammalian, plant, and insect translation systems

Rhopalosiphum padi virus (RhPV) is one of several picorna-like viruses that infect insects; sequence analysis has revealed distinct differences between these agents and mammalian picornaviruses. RhPV has a single-stranded positive-sense RNA genome of about 10 kb; unlike the genomes of Picornaviridae, however, this genome contains two long open reading frames (ORFs). ORF1 encodes the virus nonstructural proteins, while the downstream ORF, ORF2, specifies the structural proteins. Both ORFs are preceded by long untranslated regions (UTRs). The intergenic UTR is known to contain an internal ribosome entry site (IRES) which directs non-AUG-initiated translation of ORF2. We have examined the 5' UTR of RhPV for IRES activity by translating synthetic dicistronic mRNAs containing this sequence in a variety of systems. We now report that the 5' UTR contains an element which directs internal initiation of protein synthesis from an AUG codon in mammalian, plant, and Drosophila in vitro translation systems. In contrast, the encephalomyocarditis virus IRES functions only in the mammalian system. The RhPV 5' IRES element has features in common with picornavirus IRES elements, in that no coding sequence is required for IRES function, but also with cellular IRES elements, as deletion analysis indicates that this IRES element does not have sharply defined boundaries.     

Effective energy function for proteins in solution

A Gaussian solvent-exclusion model for the solvation free energy is developed. It is based on theoretical considerations and parametrized with experimental data. When combined with the CHARMM 19 polar hydrogen energy function, it provides an effective energy function (EEF1) for proteins in solution. The solvation model assumes that the solvation free energy of a protein molecule is a sum of group contributions, which are determined from values for small model compounds. For charged groups, the self-energy contribution is accounted for primarily by the exclusion model. Ionic side-chains are neutralized, and a distance-dependent dielectric constant is used to approximate the charge-charge interactions in solution. The resulting EEF1 is subjected to a number of tests. Molecular dynamics simulations at room temperature of several proteins in their native conformation are performed, and stable trajectories are obtained. The deviations from the experimental structures are similar to those observed in explicit water simulations. The calculated enthalpy of unfolding of a polyalanine helix is found to be in good agreement with experimental data. Results reported elsewhere show that EEF1 clearly distinguishes correctly from incorrectly folded proteins, both in static energy evaluations and in molecular dynamics simulations and that unfolding pathways obtained by high-temperature molecular dynamics simulations agree with those obtained by explicit water simulations. Thus, this energy function appears to provide a realistic first approximation to the effective energy hypersurface of proteins.     

Heat capacity and compactness of denatured proteins

One of the striking results of protein thermodynamics is that the heat capacity change upon denaturation is large and positive. This change is generally ascribed to the exposure of non-polar groups to water on denaturation, in analogy to the large heat capacity change for the transfer of small non-polar molecules from hydrocarbons to water. Calculations of the heat capacity based on the exposed surface area of the completely unfolded denatured state give good agreement with experimental data. This result is difficult to reconcile with evidence that the heat denatured state in the absence of denaturants is reasonably compact. In this work, sample conformations for the denatured state of truncated CI2 are obtained by use of an effective energy function for proteins in solution. The energy function gives denatured conformations that are compact with radii of gyration that are slightly larger than that of the native state. The model is used to estimate the heat capacity, as well as that of the native state, at 300 and 350 K via finite enthalpy differences. The calculations show that the heat capacity of denaturation can have large positive contributions from non-covalent intraprotein interactions because these interactions change more with temperature in non-native conformations than in the native state. Including this contribution, which has been neglected in empirical surface area models, leads to heat capacities of unfolding for compact denatured states that are consistent with the experimental heat capacity data. Estimates of the stability curve of CI2 made with the effective energy function support the present model.     

A simple method to improve probe set estimates from oligonucleotide arrays

A popular commercially available oligonucleotide microarray technology employs sets of 25 base pair oligonucleotide probes for measurement of gene expression levels. A mathematical algorithm is required to compute an estimate of gene expression from the multiple probes. Previously proposed methods for summarizing gene expression data have either been substantially ad hoc or have relied on model assumptions that may be easily violated. Here we present a new algorithm for calculating gene expression from probe sets. Our approach is functionally related to leave-one-out cross-validation, a non-parametric statistical technique that is often applied in limited data situations. We illustrate this approach using data from our study seeking a molecular fingerprint of STAT3 regulated genes for early detection of human cancer.     

HSC70 interactions with SV40 viral proteins differ between permissive and nonpermissive mammalian cells

SV40 belongs to a group of DNA tumor viruses which induce the expression of the 70 Kd heat shock proteins, but the meaning of this induction remains unclear. Investigating the role of hsc70 in the SV40 life cycle, we found that the protein translocates to the nucleus late in infection of permissive CV1 cells, in contrast to infected nonpermissive BALB/3T3 and NIH/3T3 cells in which hsc70 remains cytoplasmic. Moreover, the pattern of hsc70 nuclear staining was diffused and clearly distinguishable from that observed after heat shock. In addition hsc70 late in infection coimmunoprecipitated with the viral capsid protein VP1, suggesting a role in the process of viral packaging. Interactions of hsc70 with the early viral oncoprotein T antigen were observed only in nonpermissive cells, indicating that the binding of the above proteins is specific to cells that do not support viral propagation. Finally, treatment of permissive CV1 cells with interferon gamma, a known antiviral cytokine, resulted in hsc70 binding to T antigen. Our results suggest that the role of hsc70 in the process of SV40 infection is directly related to the ability of the host cells to support viral propagation and is clearly different between permissive and nonpermissive cell lines.     

Effective energy functions for protein structure prediction

Protein structure prediction, fold recognition, homology modeling and design rely mainly on statistical effective energy functions. Although the theoretical foundation of such functions is not clear, their usefulness has been demonstrated in many applications. Molecular mechanics force fields, particularly when augmented by implicit solvation models, provide physical effective energy functions that are beginning to play a role in this area.     

Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity

Nicotinic acetylcholine receptors (nAChRs) are integral membrane proteins and prototypic members of the ligand-gated ion-channel superfamily, which has precursors in the prokaryotic world. They are formed by the assembly of five transmembrane subunits, selected from a pool of 17 homologous polypeptides (alpha1-10, beta1-4, gamma, delta, and epsilon). There are many nAChR subtypes, each consisting of a specific combination of subunits, which mediate diverse physiological functions. They are widely expressed in the central nervous system, while, in the periphery, they mediate synaptic transmission at the neuromuscular junction and ganglia. nAChRs are also found in non-neuronal/nonmuscle cells (keratinocytes, epithelia, macrophages, etc.). Extensive research has determined the specific function of several nAChR subtypes. nAChRs are now important therapeutic targets for various diseases, including myasthenia gravis, Alzheimer's and Parkinson's diseases, and schizophrenia, as well as for the cessation of smoking. However, knowledge is still incomplete, largely because of a lack of high-resolution X-ray structures for these molecules. Nevertheless, electron microscopy studies on 2D crystals of nAChR from fish electric organs and the determination of the high-resolution X-ray structure of the acetylcholine binding protein (AChBP) from snails, a homolog of the extracellular domain of the nAChR, have been major steps forward and the data obtained have important implications for the design of subtype-specific drugs. Here, we review some of the latest advances in our understanding of nAChRs and their involvement in physiology and pathology.     

The Structure of a Melittin-Stabilized Pore

Melittin has been reported to form toroidal pores under certain conditions, but the atomic-resolution structure of these pores is unknown. A 9-μs all-atom molecular-dynamics simulation starting from a closely packed transmembrane melittin tetramer in DMPC shows formation of a toroidal pore after 1 μs. The pore remains stable with a roughly constant radius for the rest of the simulation. Surprisingly, one or two melittin monomers frequently transition between transmembrane and surface states. All four peptides are largely helical. A simulation in a DMPC/DMPG membrane did not lead to a stable pore, consistent with the experimentally observed lower activity of melittin on anionic membranes. The picture that emerges from this work is rather close to the classical toroidal pore, but more dynamic with respect to the configuration of the peptides.     

Multiplex cytokine analysis of dermal interstitial blister fluid defines local disease mechanisms in systemic sclerosis

IntroductionClinical diversity in systemic sclerosis (SSc) reflects multifaceted pathogenesis and the effect of key growth factors or cytokines operating within a disease-specific microenvironment. Dermal interstitial fluid sampling offers the potential to examine local mechanisms and identify proteins expressed within lesional tissue. We used multiplex cytokine analysis to profile the inflammatory and immune activity in the lesions of SSc patients.MethodsDermal interstitial fluid sample from the involved forearm skin, and synchronous plasma samples were collected from SSc patients (n = 26, diffuse cutaneous SSc (DcSSc) n = 20, limited cutaneous SSc (LcSSc) n = 6), and healthy controls (HC) (n = 10) and profiled by Luminex® array for inflammatory cytokines, chemokines, and growth factors.ResultsLuminex® profiling of the dermal blister fluid showed increased inflammatory cytokines (median interleukin ( IL)-6 in SSc 39.78 pg/ml, HC 5.51 pg/ml, p = 0.01, median IL-15 in SSc 6.27 pg/ml, HC 4.38 pg/ml, p = 0.03), chemokines (monocyte chemotactic protein (MCP)-3 9.81 pg/ml in SSc, 7.18 pg/ml HC, p = 0.04), and profibrotic growth factors (platelet derived growth factor (PDGF)-AA 10.38 pg/ml versus 6.94 pg/ml in HC, p = 0.03). In general dermal fluid and plasma cytokine levels did not correlate, consistent with predominantly local production of these factors within the dermal lesions, rather than leakage from the serum. In hierarchical clustering and network analysis IL-6 emerged as a key central mediator.ConclusionsOur data confirm that an immuno-inflammatory environment and aberrant vascular repair are intimately linked to fibroblast activation in lesional skin in SSc. This non-invasive method could be used to profile disease activity in the clinic, and identifies key inflammatory or pro-fibrotic proteins that might be targeted therapeutically. Distinct subgroups of SSc may be defined that show innate or adaptive immune cytokine signatures.