Interkingdom Complementation Reveals Structural Conservation and Functional Divergence of 14-3-3 Proteins

The 14-3-3s are small acidic cytosolic proteins that interact with multiple clients and participate in essential cellular functions in all eukaryotes. Available structural and functional information about 14-3-3s is largely derived from higher eukaryotes, which contain multiple members of this protein family suggesting functional specialization. The exceptional sequence conservation among 14-3-3 family members from diverse species suggests a common ancestor for 14-3-3s, proposed to have been similar to modern 14-3-3ε isoforms. Structural features of the sole family member from the protozoan Giardia duodenalis (g14-3-3), are consistent with this hypothesis, but whether g14-3-3 is functionally homologous to the epsilon isoforms is unknown. We use inter-kingdom reciprocal functional complementation and biochemical methods to determine whether g14-3-3 is structurally and functionally homologous with members of the two 14-3-3 conservation groups of the metazoan Drosophila melanogaster. Our results indicate that although g14-3-3 is structurally homologous to D14-3-3ε, functionally it diverges presenting characteristics of other 14-3-3s. Given the basal position of Giardia in eukaryotic evolution, this finding is consistent with the hypothesis that 14-3-3ε isoforms are ancestral to other family members.     

Dimerization Is Essential for 14-3-3�� Stability and Function in Vivo

Members of the conserved 14-3-3 protein family spontaneously self-assemble as homo- and heterodimers via conserved sequences in the first four (αA-αD) of the nine helices that comprise them. Dimeric 14-3-3s bind conserved motifs in diverse protein targets involved in multiple essential cellular processes including signaling, intracellular trafficking, cell cycle regulation, and modulation of enzymatic activities. However, recent mostly in vitro evidence has emerged, suggesting functional and regulatory roles for monomeric 14-3-3s. We capitalized on the simplicity of the 14-3-3 family in Drosophila to investigate in vivo 14-3-3ζ monomer properties and functionality. We report that dimerization is essential for the stability and function of 14-3-3ζ in neurons. Moreover, we reveal the contribution of conserved amino acids in helices A and D to homo- and heterodimerization and their functional consequences on the viability of animals devoid of endogenous 14-3-3ζ. Finally, we present evidence suggesting endogenous homeostatic adjustment of the levels of the second family member in Drosophila, D14-3-3ϵ, to transgenic monomeric and dimerization-competent 14-3-3ζ.     

The cyclic AMP system andDrosophila learning

Molecular and Cellular Biochemistry - The cyclic AMP (cAMP) system plays a critical role in olfactory learning in the fruit fly,Drosophila melanogaster, as evidenced by the following: [1] The dunce...     

Cell type-specific processing of human Tau proteins in Drosophila

Accumulation of hyperphosphorylated Tau is associated with a number of neurodegenerative diseases collectively known as tauopathies. Differences in clinical and cognitive profiles among them suggest differential sensitivity of neuronal populations to Tau levels, phosphorylation and mutations. We used tissue specific expression of wild type and mutant human tau transgenes to demonstrate differential phosphorylation and stability in a cell type-specific manner, which includes different neuronal types and does not correlate with the level of accumulated protein. Rather, they likely reflect the spatial distribution or regulation of Tau-targeting kinases and phosphatases.     

The receptor tyrosine kinase Alk controls neurofibromin functions in Drosophila growth and learning

Anaplastic Lymphoma Kinase (Alk) is a Receptor Tyrosine Kinase (RTK) activated in several cancers, but with largely unknown physiological functions. We report two unexpected roles for the Drosophila ortholog dAlk, in body size determination and associative learning. Remarkably, reducing neuronal dAlk activity increased body size and enhanced associative learning, suggesting that its activation is inhibitory in both processes. Consistently, dAlk activation reduced body size and caused learning deficits resembling phenotypes of null mutations in dNf1, the Ras GTPase Activating Protein-encoding conserved ortholog of the Neurofibromatosis type 1 (NF1) disease gene. We show that dAlk and dNf1 co-localize extensively and interact functionally in the nervous system. Importantly, genetic or pharmacological inhibition of dAlk rescued the reduced body size, adult learning deficits, and Extracellular-Regulated-Kinase (ERK) overactivation dNf1 mutant phenotypes. These results identify dAlk as an upstream activator of dNf1-regulated Ras signaling responsible for several dNf1 defects, and they implicate human Alk as a potential therapeutic target in NF1.     

The Power and Richness of Modelling Tauopathies in <Emphasis Type="Italic">Drosophila</Emphasis>

Tauopathies are a group of neurodegenerative disorders characterised by altered levels of phosphorylation or mutations in the neuronal microtubule protein Tau. The heterogeneous pathology of tauopathies suggests differential susceptibility of different neuronal types to wild-type and mutant Tau. The genetic power and facility of the Drosophila model has been instrumental in exploring the molecular aetiologies of tauopathies, identifying additional proteins likely contributing to neuronal dysfunction and toxicity and novel Tau phosphorylations mediating them. Importantly, recent results indicate tissue- and temporal-specific effects on dysfunction and toxicity coupled with differential effects of distinct Tau isoforms within them. Therefore, they reveal an unexpected richness of the Drosophila model that, coupled with its molecular genetic power, will likely play a significant role in our understanding of multiple tauopathies potentially leading to their differential treatment.     

The pH-dependent stability of wild-type and mutant transthyretin oligomers

A reduction in pH is known to induce the disassociation of the tetrameric form of transthyretin and favor the formation of amyloid fibers. Using continuum electrostatic techniques, we calculate the titration curves and the stability of dimer and tetramer formation of transthyretin as a function of pH. We find that the tetramer and the dimer become less stable than the monomer as the pH is lowered. The free energy difference is 13.8 kcal/mol for dimer formation and 27 kcal/mol for tetramer formation, from the monomers, when the pH is lowered from 7 to 3.9. Similar behavior is observed for both the wild-type and the mutant protein. Certain residues (namely Glu-72, His-88, His-90, Glu-92, and Tyr-116), play an important role in the binding process, as seen by the considerable pK(1/2) change of these residues upon dimer formation.