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Results for “enzyme evolution”

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PubMed2005

Structural and functional diversity generated by alternative mRNA splicing.

Alternative mRNA splicing is becoming increasingly recognized as an important mechanism for the generation of structural and functional diversity in proteins. Recent estimations predict that approximately 50% of all eukaryotic proteins can be alternatively spliced. Several lines of evidence suggest that alternative mRNA splicing results in small changes in protein structure and is likely to fine-tune the function and specificity of the affected protein. However, knowledge of how alternative splicing regulates cellular processes on the molecular level is still limited. It is only recently that structures of alternatively spliced proteins have been solved. These studies have shown that alternative splicing affects the structure not only in the vicinity of the splice site but also at long distance.

Alternative Splicing↗

PubMed2006

Polygalacturonase inhibiting proteins: players in plant innate immunity?

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular leucine-rich repeat (LRR) proteins that recognize and inhibit fungal polygalacturonases (PGs). The PG-PGIP interaction favours the accumulation of elicitor-active oligogalacturonides and causes the activation of defence responses. Small gene families encode PGIP isoforms that differ in affinity and specificity for PGs secreted by different pathogens. The consensus motif within the LRR structure of PGIPs is the same as that of the extracellular receptors of the plant innate immune system. Structural and functional evidence suggest that PGIPs are versatile proteins involved in innate immunity and that they are capable of recognizing different surface motifs of functionally related but structurally variable PGs.

Biological Evolution↗

PubMed2003

The MAPK cascade in equally cleaving spiralian embryos.

Spiralian development is shared by several protostome phyla and characterized by regularities in early cleavage, fate map, and larva. Experimental evidence from multiple spiralian species implicates cells in the D quadrant lineage as the organizer of future axial development of the embryo. However, the mechanisms by which the D quadrant is specified differ between species with equal and unequal spiral cleavage. Equally cleaving mollusc embryos establish the D quadrant via cell-cell interactions between the micromeres and macromeres at the 24- to 36-cell stage. In unequally cleaving embryos, the D quadrant is established at the 4-cell stage via asymmetries in the first 2 cell divisions. We have begun to explore the molecular mechanisms of D quadrant patterning in spiralians. Previously, we showed that, in the unequally cleaving embryo of the mollusc Ilyanassa obsoleta, the MAPK pathway is activated and functionally required in 3D and also in the micromeres known to require a signal from 3D. Here, we examine the role of MAPK signaling in 4 spiralians with equal cleavage. In 3 equally cleaving molluscs, the chiton Chaetopleura, the limpet Tectura, and the snail Lymnaea, the MAPK pathway is activated in the 3D cell but not in the overlying micromeres. In the equally cleaving embryo of the polychaete annelid Hydroides, MAPK activation was not detected in the 3D macromere but was observed in one of its daughter cells, 4d. In addition, inhibiting Tectura MAPK activation disrupts differentiation of 3D and cells induced by it, supporting a functional role for MAPK in axis specification in equally cleaving spiralians. Thus, MAPK signaling may have a conserved role in the D quadrant organizer cell 3D in molluscs. However, there have been at least 2 evolutionary changes in the activation of the MAPK pathway during spiralian evolution. MAPK function in the Ilyanassa micromeres is a recent cooption and, since the divergence of annelids and molluscs, there has been a shift in onset of MAPK activation between 3D and 4d. We propose that this latter shift correlates with a change in the timing of specification of the secondary embryonic axis.

Animals↗

PubMed2005

dUbc9 negatively regulates the Toll-NF-kappa B pathways in larval hematopoiesis and drosomycin activation in Drosophila.

Highly conserved during evolution, the enzyme Ubc9 activates the small ubiquitin-like modifier (SUMO) prior to its covalent ligation to target proteins. We have used mutations in the Drosophila Ubc9 (dUbc9) gene to understand Ubc9 functions in vivo. Loss-of-function mutations in dUbc9 cause strong mitotic defects in larval hematopoietic tissues, an increase in the number of hematopoietic precursors in the lymph gland and of mature blood cells in circulation, and an increase in the proportion of cyclin-B-positive cells. Some blood cells are polyploid and multinucleate, exhibiting signs of genomic instability. We also observe an overabundance of highly differentiated blood cells (lamellocytes), normally not found in healthy larvae. Lamellocytes in mutants are either free in circulation or recruited to form tumorous masses. Hematopoietic defects of dUbc9 mutants are strongly suppressed in the absence of the Rel/NF-kappaB-family transcription factors Dorsal and Dif or in the presence of a non-signaling allele of Cactus, the IkappaB protein in Drosophila. In the larval fat body, dUbc9 negatively regulates the expression of the antifungal peptide gene drosomycin, which is constitutively expressed in dUbc9 mutants in the absence of immune challenge. dUbc9-mediated drosomycin expression requires Dorsal and Dif. Together, our results support a role for dUbc9 in the negative regulation of the Drosophila NF-kappaB signaling pathways in larval hematopoiesis and humoral immunity.

Animals↗

PubMed1998

Evolution of angiotensin-converting enzyme inhibition in hypertension, heart failure, and vascular protection.

Over 100 years of scientific investigation has established that angiotensin-converting enzyme (ACE) plays an important role in both the renin-angiotensin system and the kinin-kallekrein system. ACE inhibitors--which stem the production of angiotensin II, a potent vasoconstrictor--have proved to be useful agents in the management of hypertension, in the prevention and treatment of heart failure, and in the improvement of endothelial function. Generally, ACE inhibitors are as efficacious as beta blockers and thiazide diuretics in reducing blood pressure and also induce regression of left ventricular hypertrophy. Many trials-including the Cooperative North Scandinavian Enalapril Survival Study (CONSENSUS), the Veteran's Administration Cooperative Vasodilator-Heart Failure Trial (V-HeFT I), the Studies of Left Ventricular Dysfunction (SOLVD), and the Survival and Ventricular Enlargement (SAVE) trial--have demonstrated the ability of ACE inhibitors to reduce mortality within a wide range of heart failure, from asymptomatic left ventricular dysfunction to severe heart failure. The SAVE trial specifically evaluated the effects on post-myocardial infarction mortality and remodeling and found that ACE inhibitors were effective in reducing both. Further studies are assessing the potential additional antiatherosclerotic aspects of ACE inhibitors. Initial research indicates a reversal of endothelial dysfunction in atherosclerotic animals, and subsequent clinical trials, including the Trial on Reversing ENdothelial Dysfunction (TREND), support the likelihood of a similar effect in humans. Beneficial effects in hypertension or heart failure may also be gained with other interruptors of the renin-angiotensin system, such as angiotensin receptor blockers. Results from studies assessing these receptor blockers will bring greater understanding to the mechanism of action of ACE inhibitors.

Angiotensin Receptor Antagonists↗

PubMed2002

Enzyme function less conserved than anticipated.

The level of sequence similarity that implies similarity in protein structure is well established. Recently, many groups proposed thresholds for similarity in sequence implying similarity in enzymatic function. All previous results suggest the strong conservation of enzymatic function above levels of 50% pairwise sequence identity. Here, I argue that all groups substantially overestimated the conservation of enzyme function because their data sets were either too biased, or too small. An unbiased analysis suggested that less than 30% of the pair fragments above 50% sequence identity have entirely identical EC numbers. Another surprising finding was that even BLAST E-values below 10(-50) did not suffice to automatically transfer enzyme function without errors. As expected, most misclassifications originated from similarities in relatively short regions and/or from transferring annotations for different domains. Both problems cannot be corrected easily by adjusting the thresholds for automatic transfer of genome annotations. A score relating sequence identity to alignment length (distance from HSSP-threshold) outperformed statistical BLAST scores for high sequence similarity. In particular, the distance score allowed error-free transfer of enzyme function for the 10% most similar enzyme pairs. The results illustrated how difficult it is to assess the conservation of protein function and to guarantee error-free genome annotations, in general: sets with millions of pair comparisons might not suffice to arrive at statistically significant conclusions. In practice, the revised detailed estimates for the sequence conservation of enzyme function may provide important benchmarks for everyday sequence analysis and for more cautious automatic genome annotations.

Amino Acid Sequence↗

PubMed2002

One fold with many functions: the evolutionary relationships between TIM barrel families based on their sequences, structures and functions.

The eightfold (betaalpha) barrel structure, first observed in triose-phosphate isomerase, occurs ubiquitously in nature. It is nearly always an enzyme and most often involved in molecular or energy metabolism within the cell. In this review we bring together data on the sequence, structure and function of the proteins known to adopt this fold. We highlight the sequence and functional diversity in the 21 homologous superfamilies, which include 76 different sequence families. In many structures, the barrels are "mixed and matched" with other domains generating additional variety. Global and local structure-based alignments are used to explore the distribution of the associated functional residues on this common structural scaffold. Many of the substrates/co-factors include a phosphate moiety, which is usually but not always bound towards the C-terminal end of the sequence. Some, but not all, of these structures, exhibit a structurally conserved "phosphate binding motif". In contrast metal-ligating residues and catalytic residues are distributed along the sequence. However, we also found striking structural superposition of some of these residues. Lastly we consider the possible evolutionary relationships between these proteins, whose sequences are so diverse that even the most powerful approaches find few relationships, yet whose active sites all cluster at one end of the barrel. This extreme example of the "one fold-many functions" paradigm illustrates the difficulty of assigning function through a structural genomics approach for some folds.

Aldose-Ketose Isomerases↗

PubMed2002

Directed evolution of barnase stability using proteolytic selection.

We report the construction of a phage-displayed repertoire of mutants of the ribonuclease barnase from Bacillus amyloliquefaciens. The construction was guided by the natural variability between two closely related ribonucleases, barnase and binase from Bacillus intermedius. This repertoire was selected using a proteolytic selection method, allowing sorting of the library according to the resistance of the mutants toward proteolysis. Susceptibility toward proteolysis has been correlated with flexibility and unfolding, and is thus expected to yield mutants with increased thermal stability. Enrichment of barnase mutants with specific combinations of amino acid residues at four of the randomised positions was observed. Three of these enriched amino acid residues are present in neither barnase nor binase. For some of the mutations, the improvement in proteolytic stability does not lead to a pronounced improvement in thermodynamic stability, indicating that the factors governing the proteolytic stability in some cases may be different from those governing the thermodynamic stability, e.g. propensity to local unfolding.The results obtained add important knowledge to a novel use of phage display technology for selection of thermodynamically stable proteins. Only by carefully establishing the parameters that can be adjusted, and recognising the influence this will have on the outcome of selection, will it be possible to realise the powerful technique of proteolytic selection.

Amino Acid Sequence↗

PubMed1976