Ivaylo Ivanov

Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences

In eukaryotes, it is generally assumed that translation initiation occurs at the AUG codon closest to the messenger RNA 5′ cap. However, in certain cases, initiation can occur at codons differing from AUG by a single nucleotide, especially the codons CUG, UUG, GUG, ACG, AUA and AUU. While non-AUG initiation has been experimentally verified for a handful of human genes, the full extent to which this phenomenon is utilized—both for increased coding capacity and potentially also for novel regulatory mechanisms—remains unclear. To address this issue, and hence to improve the quality of existing coding sequence annotations, we developed a methodology based on phylogenetic analysis of predicted 5′ untranslated regions from orthologous genes. We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences. Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human genes including VANGL2, FGFR1, KCNN4, TRPV6, HDGF, CITED2, EIF4G3 and NTF3, and also affirmed the conservation of known non-AUG-initiated extensions in 17 other genes. In several instances, we have been able to obtain independent experimental evidence of the expression of non-AUG-initiated products from the previously published literature and ribosome profiling data.

Characterizing Loop Dynamics and Ligand Recognition in Human- and Avian-Type Influenza Neuraminidases via Generalized Born Molecular Dynamics and End-Point Free Energy Calculations

The comparative dynamics and inhibitor binding free energies of group-1 and group-2 pathogenic influenza A subtype neuraminidase (NA) enzymes are of fundamental biological interest and relevant to structure-based drug design studies for antiviral compounds. In this work, we present seven generalized Born molecular dynamics simulations of avian (N1)- and human (N9)-type NAs in order to probe the comparative flexibility of the two subtypes, both with and without the inhibitor oseltamivir bound. The enhanced sampling obtained through the implicit solvent treatment suggests several provocative insights into the dynamics of the two subtypes, including that the group-2 enzymes may exhibit similar motion in the 430-binding site regions but different 150-loop motion. End-point free energy calculations elucidate the contributions to inhibitor binding free energies and suggest that entropic considerations cannot be neglected when comparing across the subtypes. We anticipate the findings presented here will have broad implications for the development of novel antiviral compounds against both seasonal and pandemic influenza strains.

Synthetic mimics of antimicrobial peptides

Infectious diseases and antibiotic resistance are now considered the most imperative global healthcare problem. In the search for new treatments, host defense, or antimicrobial, peptides have attracted considerable attention due to their various unique properties; however, attempts to develop in vivo therapies have been severely limited. Efforts to develop synthetic mimics of antimicrobial peptides (SMAMPs) have increased significantly in the last decade, and this review will focus primarily on the structural evolution of SMAMPs and their membrane activity. This review will attempt to make a bridge between the design of SMAMPs and the fundamentals of SMAMP-membrane interactions. In discussions regarding the membrane interaction of SMAMPs, close attention will be paid to the lipid composition of the bilayer. Despite many years of study, the exact conformational aspects responsible for the high selectivity of these AMPs and SMAMPs toward bacterial cells over mammalian cells are still not fully understood. The ability to design SMAMPs that are potently antimicrobial, yet nontoxic to mammalian cells has been demonstrated with a variety of molecular scaffolds. Initial animal studies show very good tissue distribution along with more than a 4-log reduction in bacterial counts. The results on SMAMPs are not only extremely promising for novel antibiotics, but also provide an optimistic picture for the greater challenge of general proteomimetics.

Conservation of polyamine regulation by translational frameshifting from yeast to mammals

Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. Here we show that a similar mechanism exists in the fission yeast Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. As shown here, the yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S.pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals.

An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum

The analysis of cell-free nucleic acids (cfNAs), which are present at significant levels in the blood of cancer patients, can reveal the mutational spectrum of a tumour without the need for invasive sampling of the tissue. However, this requires differentiation between the nucleic acids that originate from healthy cells and the mutated sequences shed by tumour cells. Here we report an electrochemical clamp assay that directly detects mutated sequences in patient serum. This is the first successful detection of cfNAs without the need for enzymatic amplification, a step that normally requires extensive sample processing and is prone to interference. The new chip-based assay reads out the presence of mutations within 15 minutes using a collection of oligonucleotides that sequester closely related sequences in solution, and thus allow only the mutated sequence to bind to a chip-based sensor. We demonstrate excellent levels of sensitivity and specificity and show that the clamp assay accurately detects mutated sequences in a collection of samples taken from lung cancer and melanoma patients.

Proliferating cell nuclear antigen loaded onto double-stranded DNA: dynamics, minor groove interactions and functional implications

Proliferating cell nuclear antigen (PCNA) acts as a biologically essential processivity factor that encircles DNA and provides binding sites for polymerase, flap endonuclease-1 (FEN-1) and ligase during DNA replication and repair. We have computationally characterized the interactions of human and Archaeoglobus fulgidus PCNA trimer with double-stranded DNA (ds DNA) using multi-nanosecond classical molecular dynamics simulations. The results reveal the interactions of DNA passing through the PCNA trimeric ring including the contacts formed, overall orientation and motion with respect to the sliding clamp. Notably, we observe pronounced tilting of the axis of dsDNA with respect to the PCNA ring plane reflecting interactions between the DNA phosphodiester backbone and positively charged arginine and lysine residues lining the PCNA inner surface. Covariance matrix analysis revealed a pattern of correlated motions within and between the three equivalent subunits involving the PCNA C-terminal region and linker strand associated with partner protein binding sites. Additionally, principal component analysis identified low frequency global PCNA subunit motions suitable for translocation along duplex DNA. The PCNA motions and interactions with the DNA minor groove, identified here computationally, provide an unexpected basis for PCNA to act in the coordinated handoff of intermediates from polymerase to FEN-1 to ligase during DNA replication and repair.

Initiation context modulates autoregulation of eukaryotic translation initiation factor 1 (eIF1)

The central feature of standard eukaryotic translation initiation is small ribosome subunit loading at the 5′ cap followed by its 5′ to 3′ scanning for a start codon. The preferred start is an AUG codon in an optimal context. Elaborate cellular machinery exists to ensure the fidelity of start codon selection. Eukaryotic initiation factor 1 (eIF1) plays a central role in this process. Here we show that the translation of eIF1 homologs in eukaryotes from diverse taxa involves initiation from an AUG codon in a poor context. Using human eIF1 as a model, we show that this poor context is necessary for an autoregulatory negative feedback loop in which a high level of eIF1 inhibits its own translation, establishing that variability in the stringency of start codon selection is used for gene regulation in eukaryotes. We show that the stringency of start codon selection (preferential utilization of optimal start sites) is increased to a surprising degree by overexpressing eIF1. The capacity for the cellular level of eIF1 to impact initiation through the variable stringency of initiation codon selection likely has significant consequences for the proteome in eukaryotes.

Computational identification of putative programmed translational frameshift sites.

MOTIVATION In an effort to identify potential programmed frameshift sites by statistical analysis, we explore the hypothesis that selective pressure would have rendered such sites underabundant and underrepresented in protein-coding sequences. We developed a computer program to compare the frequencies of k-length subsequences of nucleotides with the frequencies predicted by a zero order Markov chain determined by the codon bias of the same set of sequences. The program was used to calculate and evaluate the distribution of 7-base oligonucleotides in the 6000+ putative protein-coding sequences of S. cerevisiae preliminary to the laboratory testing of the most highly underrepresented oligos for frameshifting efficiency. RESULTS Among the most significant results is the finding that the heptanucleotides CUU-AGG-C and CUU-AGU-U, sites of the programmed +1 translational frameshifts required for the production in yeast of actin filament-binding protein ABP140 and telomerase subunit EST3, respectively, rank among the least represented of phase I heptanucleotides in the coding sequences of S. cerevisiae. Laboratory experiments demonstrated that other underrepresented heptanucleotides identified by the program, for example GGU-CAG-A, are also prone to significant translational frameshifting, suggesting the possibility that genes containing other underrepresented heptamers may also encode transframe products. AVAILABILITY The program is available for download from http://www.gesteland.genetics.utah.edu/freqAnalysis SUPPLEMENTARY INFORMATION Complete results from the analysis of S. cerevisiae are available on http://www.gesteland.genetics.utah.edu/freqAnalysis

Unraveling the three-metal-ion catalytic mechanism of the DNA repair enzyme endonuclease IV

Endonuclease IV belongs to a class of important apurinic/apyrimidinic endonucleases involved in DNA repair. Although a structure-based mechanistic hypothesis has been put forth for this enzyme, the detailed catalytic mechanism has remained unknown. Using thermodynamic integration in the context of ab initio quantum mechanics/molecular mechanics molecular dynamics, we examined certain aspects of the phosphodiester cleavage step in the mechanism. We found the reaction proceeded through a synchronous bimolecular (ANDN) mechanism with reaction free energy and barrier of −3.5 and 20.6 kcal/mol, in agreement with experimental estimates. In the course of the reaction the trinuclear active site of endonuclease IV underwent dramatic local conformational changes: shifts in the mode of coordination of both substrate and first-shell ligands. This qualitative finding supports the notion that structural rearrangements in the active sites of multinuclear enzymes are integral to biological function.

A profusion of upstream open reading frame mechanisms in polyamine-responsive translational regulation

In many eukaryotic mRNAs one or more short ‘upstream’ open reading frames, uORFs, precede the initiator of the main coding sequence. Upstream ORFs are functionally diverse as illustrated by their variety of features in polyamine pathway biosynthetic mRNAs. Their propensity to act as sensors for regulatory circuits and to amplify the signals likely explains their occurrence in most polyamine pathway mRNAs. The uORF-mediated polyamine responsive autoregulatory circuits found in polyamine pathway mRNAs exemplify the translationally regulated dynamic interface between components of the proteome and metabolism.