https://www.cell.com/cell/pdf/S0092-8674(09)00083-X.pdf

Origins and Mechanisms of miRNAs and siRNAs

MicroRNAs (miRNAs) are a large family of post-transcriptional regulators of gene expression that are ~21 nucleotides in length and control many developmental and cellular processes in eukaryotic organisms. Research during the past decade has identified major factors participating in miRNA biogenesis and has established basic principles of miRNA function. More recently, it has become apparent that miRNA regulators themselves are subject to sophisticated control. Many reports over the past few years have reported the regulation of miRNA metabolism and function by a range of mechanisms involving numerous protein-protein and protein-RNA interactions. Such regulation has an important role in the context-specific functions of miRNAs.

http://m.docente.unife.it/franco.pagani/lezione-1101a-mirna/articoli-smallrna/2010%20FIlipowic%20The%20widespread%20regulation%20of%20miRNA%20%20copy.pdf

The widespread regulation of microRNA biogenesis, function and decay.

MicroRNAs are important regulators of gene expression that control both physiological and pathological processes such as development and cancer. Although their mode of action has attracted great attention, the principles governing their expression and activity are only beginning to emerge. Recent studies have introduced a paradigm shift in our understanding of the microRNA biogenesis pathway, which was previously believed to be universal to all microRNAs. Maturation steps specific to individual microRNAs have been uncovered, and these offer a plethora of regulatory options after transcription with multiple proteins affecting microRNA processing efficiency. Here we review the recent advances in knowledge of the microRNA biosynthesis pathways and discuss their impact on post-transcriptional microRNA regulation during tumour development.

/pdf/many-roads-to-maturity-microrna-biogenesis-pathways-and-xwvxbe5xac.pdf

Many roads to maturity: microRNA biogenesis pathways and their regulation

The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.

https://science.sciencemag.org/content/sci/253/5022/872.full.pdf

Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein

http://www.bio.brandeis.edu/classes/biochem104/Bogan&Thorn.1998.pdf

Anatomy of hot spots in protein interfaces.

Pyridoxal phosphate (PLP)-dependent enzymes are unrivaled in the diversity of reactions that they catalyze. New structural data have paved the way for targeted mutagenesis and mechanistic studies and have provided a framework for interpretation of those results. Together, these complementary approaches yield new insight into function, particularly in understanding the origins of substrate and reaction type specificity. The combination of new sequences and structures enables better reconstruction of their evolutionary heritage and illuminates unrecognized similarities within this diverse group of enzymes. The important metabolic roles of many PLP-dependent enzymes drive efforts to design specific inhibitors, which are now guided by the availability of comprehensive structural and functional databases. Better understanding of the function of this important group of enzymes is crucial not only for inhibitor design, but also for the design of improved protein-based catalysts.

Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations.

Autoinhibition of human dicer by its internal helicase domain.

THE controversy surrounding the idea that neutral mutations dominate protein evolution1 is attributable in part to the inadequacy of the tools available to evolutionary investigators. With a few exceptions2, most investigations into the force driving protein evolution3 have relied on indirect criteria for distinguishing neutral and non-neutral variants. To investigate a particular pathway of molecular evolution, we have reconstructed by site-directed mutagenesis likely ancestral variants of the lysozymes of modern game birds (order Galliformes), tested their activity and thermostability and determined their three-dimensional structure. We focused on amino acids at three positions that are occupied in all known game birds either by the triplet Thr 40, He 55, Ser 91, or by the triplet Ser 40, Val 55, Thr 91. We have synthesized proteins representing intermediates along the possible three-step evolutionary pathways between these triplets. Although all of these are active and stable, none of these intermediates is found in known lysozymes. A comparison of the stuctures and thermostabilities of the variants reveals a linear correlation between the side-chain volume of the triplet and the thermostability of the protein. Each pathway connecting the two extant triplet sequences includes a variant with a thermostability outside the range of the extant proteins. This observation is consistent with a non-neutral evolutionary pathway. The existence of variants that are more stable than the extant proteins suggests that selection for maximum thermostability may not have been an important factor in the evolution of this enzyme.

Ancestral lysozymes reconstructed, neutrality tested, and thermostability linked to hydrocarbon packing.

A true Bronsted analysis of proton transfer in an enzyme mechanism is made possible by the chemical rescue of an inactive mutant of aspartate aminotransferase, where the endogenous general base, Lys258, is replaced with Ala by site-directed mutagenesis. Catalytic activity is restored to this inactive mutant by exogenous amines. The eleven amines studied generate a Bronsted correlation with beta of 0.4 for the transamination of cysteine sulfinate, when steric effects are included in the regression analysis. Localized mutagenesis thus allows the classical Bronsted analysis of transition-state structure to be applied to enzyme-catalyzed reactions.

https://science.sciencemag.org/content/sci/243/4897/1485.full.pdf

Direct Bronsted analysis of the restoration of activity to a mutant enzyme by exogenous amines

The possibility that the rates of acylation of chymotrypsin by certain highly reactive substrates approach the diffusion-controlled limits was investigated by measuring the values of kcat/Km for three substrates as a function of increasing viscosity with sucrose and ficoll as the viscosogenic reagents. The values of Kcat/Km (pH 8.0, 25 degrees C) representing the acylation rate constants are the following: N-(methoxycarbonyl)-L-tryptophan p-nitrophenyl ester, 3.5 x 10(7) M-1 s-1; N-acetyl-L-tryptophan methyl ester, 8 x 10(5) M-1 s-1; N-acetyl-L-tryptophan p-nitroanilide, 300 M-1 s-1. The rate constants decrease significantly with increasing viscosity for the first compound, decrease slightly for the second, and are insensitive to this perturbation for the third. The p-nitroanilide results taken together with the observation that the high concentrations of sucrose or ficoll used produce insignificant changes in kcat for the ester substrates argue against a general nonspecific perturbation in the enzyme structure effected by these reagents. The values of the association rate constants calculated from these results are 9 x 10(7) and 1 x 10(7) M-1 s-1 for the p-nitrophenyl and methyl esters, respectively. The values of kcat/Km divided by the association rate constants show that the rates of acylation by the p-nitrophenyl ester occur at ca. 40% and by the methyl ester at ca. 10% of the diffusion limits. Possibilities involving reorientation of a nonproductively bound substrate within the ES complex or desolvation of part of the active site of the enzyme are considered to account for the lower association rate constant for the methyl as compared to the p-nitrophenyl ester.

Jack F. Kirsch

Papers

Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations.

Autoinhibition of human dicer by its internal helicase domain.

Ancestral lysozymes reconstructed, neutrality tested, and thermostability linked to hydrocarbon packing.

Direct Bronsted analysis of the restoration of activity to a mutant enzyme by exogenous amines

Investigation of diffusion-limited rates of chymotrypsin reactions by viscosity variation.