The scenario on the origin of translation in the RNA world: in principle of replication parsimony

Background  

It is now believed that in the origin of life, proteins should have been "invented" in an RNA world. However, due to the complexity of a possible RNA-based proto-translation system, this evolving process seems quite complicated and the associated scenario remains very blurry. Considering that RNA can bind amino acids with specificity, it has been reasonably supposed that initial peptides might have been synthesized on "RNA templates" containing multiple amino acid binding sites. This "Direct RNA Template (DRT)" mechanism is attractive because it should be the simplest mechanism for RNA to synthesize peptides, thus very likely to have been adopted initially in the RNA world. Then, how this mechanism could develop into a proto-translation system mechanism is an interesting problem.     

Role of glutamine-169 in the substrate recognition of human aminopeptidase B

Background

Aminopeptidase B (EC 3.4.11.6, APB) preferentially hydrolyzes N-terminal basic amino acids of synthetic and peptide substrates. APB is involved in the production and maturation of peptide hormones and neurotransmitters such as miniglucagon, cholecystokinin and enkephalin by cleaving N-terminal basic amino acids in extended precursor proteins. Therefore, the specificity for basic amino acids is crucial for the biological function of APB.

Methods

Site-directed mutagenesis and molecular modeling of the S1 site were used to identify amino acid residues of the human APB responsible for the basic amino acid preference and enzymatic efficiency.

Results

Substitution of Gln169 with Asn caused a significant decrease in hydrolytic activity toward the fluorescent substrate Lys-4-methylcoumaryl-7-amide (MCA). Substantial retardation of enzyme activity was observed toward Arg-MCA and substitution with Glu caused complete loss of enzymatic activity of APB. Substitution with Asn led to an increase in IC₅₀ values of inhibitors that interact with the catalytic pocket of APB. The EC₅₀ value of chloride ion binding was also found to increase with the Asn mutant. Gln169 was required for maximal cleavage of the peptide substrates. Molecular modeling suggested that interaction of Gln169 with the N-terminal Arg residue of the substrate could be bridged by a chloride anion.

Conclusion

Gln169 is crucial for obtaining optimal enzymatic activity and the unique basic amino acid preference of APB via maintaining the appropriate catalytic pocket structure and thus for its function as a processing enzyme of peptide hormones and neurotransmitters.     

Partition function and base pairing probabilities of RNA heterodimers

Background  

RNA has been recognized as a key player in cellular regulation in recent years. In many cases, non-coding RNAs exert their function by binding to other nucleic acids, as in the case of microRNAs and snoRNAs. The specificity of these interactions derives from the stability of inter-molecular base pairing. The accurate computational treatment of RNA-RNA binding therefore lies at the heart of target prediction algorithms.     

Generating quantitative models describing the sequence specificity of biological processes with the stabilized matrix method

Background  

Many processes in molecular biology involve the recognition of short sequences of nucleic-or amino acids, such as the binding of immunogenic peptides to major histocompatibility complex (MHC) molecules. From experimental data, a model of the sequence specificity of these processes can be constructed, such as a sequence motif, a scoring matrix or an artificial neural network. The purpose of these models is two-fold. First, they can provide a summary of experimental results, allowing for a deeper understanding of the mechanisms involved in sequence recognition. Second, such models can be used to predict the experimental outcome for yet untested sequences. In the past we reported the development of a method to generate such models called the Stabilized Matrix Method (SMM). This method has been successfully applied to predicting peptide binding to MHC molecules, peptide transport by the transporter associated with antigen presentation (TAP) and proteasomal cleavage of protein sequences.     

Examining the influence of specificity ligands and ATP-competitive ligands on the overall effectiveness of bivalent kinase inhibitors

Background

Identifying selective kinase inhibitors remains a major challenge. The design of bivalent inhibitors provides a rational strategy for accessing potent and selective inhibitors. While bivalent kinase inhibitors have been successfully designed, no comprehensive assessment of affinity and selectivity for a series of bivalent inhibitors has been performed. Here, we present an evaluation of the structure activity relationship for bivalent kinase inhibitors targeting ABL1.

Methods

Various SNAPtag constructs bearing different specificity ligands were expressed in vitro. Bivalent inhibitor formation was accomplished by synthesizing individual ATP-competitive kinase inhibitors containing a SNAPtag targeting moiety, enabling the spontaneous self-assembly of the bivalent inhibitor. Assembled bivalent inhibitors were incubated with K562 lysates, and then subjected to affinity enrichment using various ATP-competitive inhibitors immobilized to sepharose beads. Resulting eluents were analyzed using Tandem Mass Tag (TMT) labeling and two-dimensional liquid chromatography-tandem mass spectrometry (2D–LC-MS/MS). Relative binding affinity of the bivalent inhibitor was determined by calculating the concentration at which 50% of a given kinase remained bound to the affinity matrix.

Results

The profiling of three parental ATP-competitive inhibitors and nine SNAPtag conjugates led to the identification of 349 kinase proteins. In all cases, the bivalent inhibitors exhibited enhanced binding affinity and selectivity for ABL1 when compared to the parental compound conjugated to SNAPtag alone. While the rank order of binding affinity could be predicted by considering the binding affinities of the individual specificity ligands, the resulting affinity of the assembled bivalent inhibitor was not predictable. The results from this study suggest that as the potency of the ATP-competitive ligand increases, the contribution of the specificity ligand towards the overall binding affinity of the bivalent inhibitor decreases. However, the affinity of the specificity components in its interaction with the target is essential for achieving selectivity.

Conclusion

Through comprehensive chemical proteomic profiling, this work provides the first insight into the influence of ATP-competitive and specificity ligands binding to their intended target on a proteome-wide scale. The resulting data suggest a subtle interplay between the ATP-competitive and specificity ligands that cannot be accounted for by considering the specificity or affinity of the individual components alone.

     

Synthesis of potentially bioactive PABA-related <Emphasis Type="Italic">N</Emphasis>-(aminoalkyl)lactamic amino acids and esters via selective S<Subscript>N</Subscript>Ar reactions

Abstract  

Potentially bioactive N-(aminoalkyl)lactamic amino acids and esters were synthesized in satisfactory to good yields by S_NAr reactions of aromatic acids with N-(3-aminopropyl)lactams followed by esterification with tertiary amino alcohols. The addition–elimination S_NAr mechanism was confirmed by NMR and MS measurements.     

Diverse bacterial genomes encode an operon of two genes, one of which is an unusual class-I release factor that potentially recognizes atypical mRNA signals other than normal stop codons

Background  

While all codons that specify amino acids are universally recognized by tRNA molecules, codons signaling termination of translation are recognized by proteins known as class-I release factors (RF). In most eukaryotes and archaea a single RF accomplishes termination at all three stop codons. In most bacteria, there are two RFs with overlapping specificity, RF1 recognizes UA(A/G) and RF2 recognizes U(A/G)A.     

Structure Motivator: A tool for exploring small three-dimensional elements in proteins

Backround

Aspartyl aminopeptidase (DNPEP), with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated.

Results

The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-??-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids.

Conclusions

The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.     

Multi-omics approach to study the growth efficiency and amino acid metabolism in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> at various specific growth rates

Background  

Lactococcus lactis is recognised as a safe (GRAS) microorganism and has hence gained interest in numerous biotechnological approaches. As it is fastidious for several amino acids, optimization of processes which involve this organism requires a thorough understanding of its metabolic regulations during multisubstrate growth.     

Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance.     

<Emphasis Type="Italic">ASAView</Emphasis>: Database and tool for solvent accessibility representation in proteins

Background  

Accessible surface area (ASA) or solvent accessibility of amino acids in a protein has important implications. Knowledge of surface residues helps in locating potential candidates of active sites. Therefore, a method to quickly see the surface residues in a two dimensional model would help to immediately understand the population of amino acid residues on the surface and in the inner core of the proteins.     

Fingerprinting the substrate specificity of M1 and M17 aminopeptidases of human malaria, Plasmodium falciparum

Background

Plasmodium falciparum, the causative agent of human malaria, expresses two aminopeptidases, PfM1AAP and PfM17LAP, critical to generating a free amino acid pool used by the intraerythrocytic stage of the parasite for proteins synthesis, growth and development. These exopeptidases are potential targets for the development of a new class of anti-malaria drugs.

Methodology/Principal Findings

To define the substrate specificity of recombinant forms of these two malaria aminopeptidases we used a new library consisting of 61 fluorogenic substrates derived both from natural and unnatural amino acids. We obtained a detailed substrate fingerprint for recombinant forms of the enzymes revealing that PfM1AAP exhibits a very broad substrate tolerance, capable of efficiently hydrolyzing neutral and basic amino acids, while PfM17LAP has narrower substrate specificity and preferentially cleaves bulky, hydrophobic amino acids. The substrate library was also exploited to profile the activity of the native aminopeptidases in soluble cell lysates of P. falciparum malaria.

Conclusions/Significance

This data showed that PfM1AAP and PfM17LAP are responsible for majority of the aminopeptidase activity in these extracts. These studies provide specific substrate and mechanistic information important for understanding the function of these aminopeptidases and could be exploited in the design of new inhibitors to specifically target these for anti-malaria treatment.     

DNA binding by Corynebacterium glutamicum TetR-type transcription regulator AmtR

Background  

The TetR family member AmtR is the central regulator of nitrogen starvation response in Corynebacterium glutamicum. While the AmtR regulon was physiologically characterized in great detail up to now, mechanistic questions of AmtR binding were not addressed. This study presents a characterization of functionally important amino acids in the DNA binding domain of AmtR and of crucial nucleotides in the AmtR recognition motif.     

Solution structure of the <Emphasis Type="Italic">Legionella pneumophila</Emphasis> Mip-rapamycin complex

Background  

Legionella pneumphila is the causative agent of Legionnaires' disease. A major virulence factor of the pathogen is the homodimeric surface protein Mip. It shows peptidyl-prolyl cis/trans isomerase activty and is a receptor of FK506 and rapamycin, which both inhibit its enzymatic function. Insight into the binding process may be used for the design of novel Mip inhibitors as potential drugs against Legionnaires' disease.     

Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS

Background  

The enzymes responsible for the synthesis of poly-ADP-ribose are named poly-ADP-ribose polymerases (PARP). PARP-2 is a nuclear protein, which regulates a variety of cellular functions that are mainly controlled by protein-protein interactions. A previously described non-conventional bipartite nuclear localization sequence (NLS) lies in the amino-terminal DNA binding domain of PARP-2 between amino acids 1–69; however, this targeting sequence has not been experimentally examined or validated.     

Study on roles of anaplerotic pathways in glutamate overproduction of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> by metabolic flux analysis

Background  

Corynebacterium glutamicum has several anaplerotic pathways (anaplerosis), which are essential for the productions of amino acids, such as lysine and glutamate. It is still not clear how flux changes in anaplerotic pathways happen when glutamate production is induced by triggers, such as biotin depletion and the addition of the detergent material, Tween 40. In this study, we quantitatively analyzed which anaplerotic pathway flux most markedly changes the glutamate overproduction induced by Tween 40 addition.     

The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

Background  

SH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3).