Centrioles are essential components of the animal centrosome and play crucial roles in the formation of cilia and flagella. They are cylindrical structures composed of nine triplet microtubules organized around a central cartwheel. Recent studies have identified spindle assembly abnormal protein SAS-6 as a critical component necessary for formation of the cartwheel. However, the molecular details of how the cartwheel participates in centriolar microtubule assembly have not been clearly understood. In this report, we show that the C-terminal tail (residues 470-657) of human SAS-6, HsSAS-6 C, the region that has been shown to extend toward the centriolar wall where the microtubule triplets are organized, nucleated and induced microtubule polymerization in vitro. The N-terminus (residues 1-166) of HsSAS-6, the domain known to be involved in formation of the central hub of the cartwheel, did not, however, exert any effect on microtubule polymerization. HsSAS-6 C bound to the microtubules and localized along the lengths of the microtubules in vitro. Microtubule pull-down and coimmunoprecipitation (Co-IP) experiments with S-phase synchronized HeLa cell lysates showed that the endogenous HsSAS-6 coprecipitated with the microtubules, and it mediated interaction with tubulin. Isothermal calorimetry titration and size exclusion chromatography showed that HsSAS-6 C bound to the αβ-tubulin dimer in vitro. The results demonstrate that HsSAS-6 possesses an intrinsic microtubule assembly promoting activity and further implicate that its outer exposed C-terminal tail may play critical roles in microtubule assembly and stabilizing microtubule attachment with the centriolar cartwheel.
Inspired by the remarkable sophistication and complexity of natural metalloproteins, the field of protein design and engineering has traditionally sought to understand and recapitulate the design principles that underlie the interplay between metals and protein scaffolds. Yet, some recent efforts in the field demonstrate that it is possible to create new metalloproteins with structural, functional and physico-chemical properties that transcend evolutionary boundaries. This essay aims to highlight some of these efforts and draw attention to the ever-expanding scope of bioinorganic chemistry and its new connections to synthetic biology, biotechnology, supramolecular chemistry and materials engineering.
Edited by Karin Musier-Forsyth Serine-arginine (SR) proteins are essential splicing factors that promote numerous steps associated with mRNA processing and whose biological function is tightly regulated through multi-site phosphorylation. In the nucleus, the cdc2-like kinases (CLKs) phosphorylate SR proteins on their intrinsically disordered Arg-Ser (RS) domains, mobilizing them from storage speckles to the splicing machinery. The CLKs have disordered N termini that bind tightly to RS domains, enhancing SR protein phosphorylation. The N termini also promote nuclear localization of CLKs, but their transport mechanism is presently unknown. To explore cytoplasmic-nuclear transitions, several classical nuclear localization sequences in the N terminus of the CLK1 isoform were identified, but their mutation had no effect on subcellular localization. Rather, we found that CLK1 amplifies its presence in the nucleus by forming a stable complex with the SR protein substrate and appropriating its NLS for transport. These findings indicate that, along with their well-established roles in mRNA splicing, SR proteins use disordered protein-protein interactions to carry their kinase regulator from the cytoplasm to the nucleus.
Aggregation of Tau, a natively unfolded protein, is responsible for tauopathies, a class of neurodegenerative disorders. An active peptide sequence containing 20 amino acids is selected from the Tau microtubule binding region, which includes the essential V306-K311 residue, to monitor the structural change that initiates aggregation at very low concentrations. The synthesis of a peptide sequence is accomplished by employing solid-phase protocols. The active domain of Tau possesses an amino functionality on lysine and free thiol on cysteine. The former end is selectively labeled to rhodamine 101 which is further bound to the InP/ZnS quantum dot surface through the thiol linkage. Efficient resonance energy transfer is observed in its unfolded conformation which is confirmed using various steady state fluorescence techniques. The average distance between the quantum dot core and the chromophore is probed by Förster resonance energy transfer (FRET) as 24.5 ± 0.8 Å. Heparin, a negatively charged glycosaminoglycan, is used for inducing aggregation of the active domain of the Tau peptide. Structural changes in the peptide monomer, on addition of heparin, could be monitored at nanomolar concentrations through the inhibition of energy transfer from quantum dots to rhodamine dye.
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