The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach.

The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. Understanding and manipulating protein stability provide crucial information on the molecular determinants that modulate structure and stability, and expand the applications of de novo proteins. Since the (ß/⍺)8-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel, and subjected them to a thorough folding analysis. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design.

In Kluyveromyces lactis a Pair of Paralogous Isozymes Catalyze the First Committed Step of Leucine Biosynthesis in Either the Mitochondria or the Cytosol.

Divergence of paralogous pairs, resulting from gene duplication, plays an important role in the evolution of specialized or novel gene functions. Analysis of selected duplicated pairs has elucidated some of the mechanisms underlying the functional diversification of Saccharomyces cerevisiae (S. cerevisiae) paralogous genes. Similar studies of the orthologous pairs extant in pre-whole genome duplication yeast species, such as Kluyveromyces lactis (K. lactis) remain to be addressed. The genome of K. lactis, an aerobic yeast, includes gene pairs generated by sporadic duplications. The genome of this organism comprises the KlLEU4 and KlLEU4BIS paralogous pair, annotated as putative α-isopropylmalate synthases (α-IPMSs), considered to be the orthologs of the S. cerevisiae ScLEU4/ScLEU9 paralogous genes. The enzymes encoded by the latter two genes are mitochondrially located, differing in their sensitivity to leucine allosteric inhibition resulting in ScLeu4-ScLeu4 and ScLeu4-ScLeu9 sensitive dimers and ScLeu9-ScLeu9 relatively resistant homodimers. Previous work has shown that, in a Scleu4Δ mutant, ScLEU9 expression is increased and assembly of ScLeu9-ScLeu9 leucine resistant homodimers results in loss of feedback regulation of leucine biosynthesis, leading to leucine accumulation and decreased growth rate. Here we report that: (i) K. lactis harbors a sporadic gene duplication, comprising the KlLEU4, syntenic with S. cerevisiae ScLEU4 and ScLEU9, and the non-syntenic KlLEU4BIS, arising from a pre-WGD event. (ii) That both, KlLEU4 and KlLEU4BIS encode leucine sensitive α-IPMSs isozymes, located in the mitochondria (KlLeu4) and the cytosol (KlLeu4BIS), respectively. (iii) That both, KlLEU4 or KlLEU4BIS complement the Scleu4Δ Scleu9Δ leucine auxotrophic phenotype and revert the enhanced ScLEU9 transcription observed in a Scleu4Δ ScLEU9 mutant. The Scleu4Δ ScLEU9 growth mutant phenotype is only fully complemented when transformed with the syntenic KlLEU4 mitochondrial isoform. KlLEU4 and KlLEU4BIS underwent a different diversification pathways than that leading to ScLEU4/ScLEU9. KlLEU4 could be considered as the functional ortholog of ScLEU4, since its encoded isozyme can complement both the Scleu4Δ Scleu9Δ leucine auxotrophy and the Scleu4Δ ScLEU9 complex phenotype.

Zombie bugs? Manipulation of kissing bug behavior by the parasite Trypanosoma cruzi.

The parasite manipulation hypothesis states that the parasite modifies host's behavior thereby increasing the probability that the parasite will pass from an intermediate host to its final host. We used the kissing bugs Triatoma pallidipennis and T. longipennis and two isolates of the Trypanosoma cruzi parasite (Chilpancingo and Morelos) to test these ideas. These insects are intermediate hosts of this parasite, which is the causal agent of Chagas disease. The Chilpancingo isolate is more pathogenic than the Morelos isolate, in the bugs. We expected that infected bugs would be more active and likely at detecting human-like odors. Given the differences in pathogenicity between isolates, we expected the Chilpancingo isolate to induce these effects more strongly and lead to higher parasite number than the Morelos isolate. Finally, infected bugs would gain less mass (a mechanism thought to increase bite rate, and thus transmission) than non-infected bugs. Having determined that both isolate haplotypes belong to the Tc1a group, we found that: (a) young instars of both species were more active and likely to detect human odor when they were infected, regardless of the isolate; (b) there was no difference in parasite abundance depending on isolate; and, (c) infected bugs did not end up with less weight than uninfected bugs. These results suggest that T. cruzi can manipulate the bugs, which implies a higher risk to contract Chagas disease than previously thought.

The cost of being a killer's accomplice: Trypanosoma cruzi impairs the fitness of kissing bugs.

Relatively little is known about the fitness effects and life history trade-offs in medically important parasites and their insect vectors. One such case is the triatomine bugs and the parasite Trypanosoma cruzi, the key actors in Chagas disease. Previous studies have revealed some costs but have not simultaneously examined traits related to development, reproduction, and survival or their possible trade-offs. In addition, these studies have not compared the effects of genetically different T. cruzi strains that differ in their weakening effects in their vertebrate hosts. We compared the body size of the bugs after infection, the number of eggs laid, hatching/non-hatching rate, hatching success, survival, and the resulting number of parasites in Meccus (Triatoma) pallidipennis bugs that were experimentally infected with two strains of T. cruzi (Chilpancingo [CH], the most debilitating in vertebrates; and Morelos [MO], the least debilitating) (both belonging to TcI group). Our results showed that infection affects size (MO < CH; MO and CH = control), number of eggs laid (MO and CH < control) hatching/non-hatching rate (MO < control < CH), hatching success (control < MO, CH = control = MO), and survival (Chilpancingo < Morelos < control). In addition, the CH strain produced more parasites than the MO strain. These results suggest that (a) infection costs depend on the parasite's origin, (b) the more debilitating effects of the CH strain are due to its increased proliferation in the host, and (c) differences in pathogenicity among T. cruzi strains can be maintained through their different effects on hosts' life history traits. Probably, the vectorial capacity mediated by a more aggressive strain could be reduced due to its costs on the triatomine, leading to a lower risk of vertebrate and invertebrate infection in natural populations.

Saccharomyces cerevisiae Differential Functionalization of Presumed ScALT1 and ScALT2 Alanine Transaminases Has Been Driven by Diversification of Pyridoxal Phosphate Interactions.

Saccharomyces cerevisiae arose from an interspecies hybridization (allopolyploidiza-tion), followed by Whole Genome Duplication. Diversification analysis of ScAlt1/ScAlt2 indicated that while ScAlt1 is an alanine transaminase, ScAlt2 lost this activity, constituting an example in which one of the members of the gene pair lacks the apparent ancestral physiological role. This paper analyzes structural organization and pyridoxal phosphate (PLP) binding properties of ScAlt1 and ScAlt2 indicating functional diversification could have determined loss of ScAlt2 alanine transaminase activity and thus its role in alanine metabolism. It was found that ScAlt1 and ScAlt2 are dimeric enzymes harboring 67% identity and intact conservation of the catalytic residues, with very similar structures. However, tertiary structure analysis indicated that ScAlt2 has a more open conformation than that of ScAlt1 so that under physiological conditions, while PLP interaction with ScAlt1 allows the formation of two tautomeric PLP isomers (enolimine and ketoenamine) ScAlt2 preferentially forms the ketoenamine PLP tautomer, indicating a modified polarity of the active sites which affect the interaction of PLP with these proteins, that could result in lack of alanine transaminase activity in ScAlt2. The fact that ScAlt2 forms a catalytically active Schiff base with PLP and its position in an independent clade in "sensu strictu" yeasts suggests this protein has a yet undiscovered physiological function.

Activation of the Hog1 MAPK by the Ssk2/Ssk22 MAP3Ks, in the absence of the osmosensors, is not sufficient to trigger osmostress adaptation in Saccharomyces cerevisiae.

Yeast cells respond to hyperosmotic stress by activating the high-osmolarity glycerol (HOG) pathway, which consists of two branches, Hkr1/Msb2-Sho1 and Sln1, which trigger phosphorylation and nuclear internalization of the Hog1 mitogen-activated protein kinase. In the nucleus, Hog1 regulates gene transcription and cell cycle progression, which allows the cell to respond and adapt to hyperosmotic conditions. This study demonstrates that the uncoupling of the known sensors of both branches of the pathway at the level of Ssk1 and Ste11 impairs cell growth in hyperosmotic medium. However, under these conditions, Hog1 was still phosphorylated and internalized into the nucleus, suggesting the existence of an alternative Hog1 activation mechanism. In the ssk1ste11 mutant, phosphorylated Hog1 failed to associate with chromatin and to activate transcription of canonical hyperosmolarity-responsive genes. Accordingly, Hog1 also failed to induce glycerol production at the levels of a wild-type strain. Inactivation of the Ptp2 phosphatase moderately rescued growth impairment of the ssk1ste11 mutant under hyperosmotic conditions, indicating that downregulation of the HOG pathway only partially explains the phenotypes displayed by the ssk1ste11 mutant. Cell cycle defects were also observed in response to stress when Hog1 was phosphorylated in the ssk1ste11 mutant. Taken together, these observations indicate that Hog1 phosphorylation by noncanonical upstream mechanisms is not sufficient to trigger a protective response to hyperosmotic stress.