Obtaining the high-resolution structures of proteins and their complexes is a crucial aspect of understanding the mechanisms of life. Experimental structure determination methods are time-consuming, expensive and cannot keep pace with the growing number of protein sequences available through genomic DNA sequencing. Thus, the ability to accurately predict the structure of proteins from their sequence is a holy grail of structural and computational biology that would remove a bottleneck in our efforts to understand as well as rationally engineer living systems. Recent advances in protein structure prediction, in particular the breakthrough with the AI-based tool AlphaFold2 (AF2), hold promise for achieving this goal, but the practical utility of AF2 remains to be explored. Focusing on proteins with essential roles in centrosome and centriole biogenesis, we demonstrate the quality and usability of the AF2 prediction models and we show that they can provide important insights into the modular organization of two key players in this process, CEP192 and CEP44. Furthermore, we used the AF2 algorithm to elucidate and then experimentally validate previously unknown prime features in the structure of TTBK2 bound to CEP164, as well as the Chibby1-FAM92A complex for which no structural information was available to date. These findings have important implications in understanding the regulation and function of these complexes. Finally, we also discuss some practical limitations of AF2 and anticipate the implications for future research approaches in the centriole/centrosome field.

Highlights d CCDC61 is a paralog of SAS6, XRCC4, XLF, and PAXX d CCDC61 can form protofilaments with a 3-fold screw axis in vitro d CCDC61 binds to microtubules mainly via its coiled-coil domain d Microtubule binding of CCDC61 is important for its function in Chlamydomonas

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Highlights d CEP164-NTD contains a TTBK2-binding WW domain inserted into an a-helical bundle d CEP164-Q11P and R93W mutants cause ciliopathies by disrupting complex formation d CEP164-NTD binding does not stimulate TTBK2 activity but impairs its EB1 engagement d Disordered regions in TTBK2 and CEP164 might facilitate substrate access by TTBK2

Selenophosphate synthetase (SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the activation of selenide with adenosine 5 0 -triphosphate (ATP) to generate selenophosphate, the essential selenium donor for selenocysteine synthesis. Recombinant full-length Leishmania major SPS (LmSPS2) was recalcitrant to crystallization. Therefore, a limited proteolysis technique was used and a stable N-terminal truncated construct (ÁN-LmSPS2) yielded suitable crystals. The Trypanosoma brucei SPS orthologue (TbSPS2) was crystallized by the microbatch method using paraffin oil. X-ray diffraction data were collected to resolutions of 1.9 Å for ÁN-LmSPS2 and 3.4 Å for TbSPS2.

Eukaryotes from the Excavata superphylum have been used as models to study the evolution of cellular molecular processes. Strikingly, human parasites of the Trypanosomatidae family ( T . brucei , T . cruzi and L . major ) conserve the complex machinery responsible for selenocysteine biosynthesis and incorporation in selenoproteins (SELENOK/SelK, SELENOT/SelT and SELENOTryp/SelTryp), although these proteins do not seem to be essential for parasite viability under laboratory controlled conditions. Selenophosphate synthetase (SEPHS/SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the formation of selenophosphate, the biological selenium donor for selenocysteine synthesis. We solved the crystal structure of the L . major selenophosphate synthetase and confirmed that its dimeric organization is functionally important throughout the domains of life. We also demonstrated its interaction with selenocysteine lyase (SCLY) and showed that it is not present in other stable assemblies involved in the selenocysteine pathway, namely the phosphoseryl-tRNA Sec kinase (PSTK)-Sec-tRNA Sec synthase (SEPSECS) complex and the tRNA Sec -specific elongation factor (eEFSec) complex. Endoplasmic reticulum stress with dithiothreitol (DTT) or tunicamycin upon selenophosphate synthetase ablation in procyclic T . brucei cells led to a growth defect. On the other hand, only DTT presented a negative effect in bloodstream T . brucei expressing selenophosphate synthetase-RNAi. Furthermore, selenoprotein T (SELENOT) was dispensable for both forms of the parasite. Together, our data suggest a role for the T . brucei selenophosphate synthetase in the regulation of the parasite’s ER stress response.

NEK6 is a central kinase in developing castration-resistant prostate cancer (CRPC). However, the pathways regulated by NEK6 in CRPC are still unclear. Cancer cells have high reactive oxygen species (ROS) levels and easily adapt to this circumstance and avoid cell death by increasing antioxidant defenses. We knocked out the NEK6 gene and evaluated the redox state and DNA damage response in DU-145 cells. The knockout of NEK6 decreases the clonogenic capacity, proliferation, cell viability, and mitochondrial activity. Targeting the NEK6 gene increases the level of intracellular ROS; decreases the expression of antioxidant defenses (SOD1, SOD2, and PRDX3); increases JNK phosphorylation, a stress-responsive kinase; and increases DNA damage markers (p-ATM and γH2AX). The exogenous overexpression of NEK6 also increases the expression of these same antioxidant defenses and decreases γH2AX. The depletion of NEK6 also induces cell death by apoptosis and reduces the antiapoptotic Bcl-2 protein. NEK6-lacking cells have more sensitivity to cisplatin. Additionally, NEK6 regulates the nuclear localization of NF-κB2, suggesting NEK6 may regulate NF-κB2 activity. Therefore, NEK6 alters the redox balance, regulates the expression of antioxidant proteins and DNA damage, and its absence induces the death of DU-145 cells. NEK6 inhibition may be a new strategy for CRPC therapy.