PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin.

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics.

Glucocorticoid resistance conferring mutation in the C-terminus of GR alters the receptor conformational dynamics.

The glucocorticoid receptor is a key regulator of essential physiological processes, which under the control of the Hsp90 chaperone machinery, binds to steroid hormones and steroid-like molecules and in a rather complicated and elusive response, regulates a set of glucocorticoid responsive genes. We here examine a human glucocorticoid receptor variant, harboring a point mutation in the last C-terminal residues, L773P, that was associated to Primary Generalized Glucocorticoid Resistance, a condition originating from decreased affinity to hormone, impairing one or multiple aspects of GR action. Using in vitro and in silico methods, we assign the conformational consequences of this mutation to particular GR elements and report on the altered receptor properties regarding its binding to dexamethasone, a NCOA-2 coactivator-derived peptide, DNA, and importantly, its interaction with the chaperone machinery of Hsp90.

Glucocorticoid receptor complexes form cooperatively with the Hsp90 co-chaperones Pp5 and FKBPs.

The function of steroid receptors in the cell depends on the chaperone machinery of Hsp90, as Hsp90 primes steroid receptors for hormone binding and transcriptional activation. Several conserved proteins are known to additionally participate in receptor chaperone assemblies, but the regulation of the process is not understood in detail. Also, it is unknown to what extent the contribution of these cofactors is conserved in other eukaryotes. We here examine the reconstituted C. elegans and human chaperone assemblies. We find that the nematode phosphatase PPH-5 and the prolyl isomerase FKB-6 facilitate the formation of glucocorticoid receptor (GR) complexes with Hsp90. Within these complexes, Hsp90 can perform its closing reaction more efficiently. By combining chemical crosslinking and mass spectrometry, we define contact sites within these assemblies. Compared to the nematode Hsp90 system, the human system shows less cooperative client interaction and a stricter requirement for the co-chaperone p23 to complete the closing reaction of GR·Hsp90·Pp5/Fkbp51/Fkbp52 complexes. In both systems, hormone binding to GR is accelerated by Hsp90 alone and in the presence of its cofactors. Our results show that cooperative complex formation and hormone binding patterns are, in many aspects, conserved between the nematode and human systems.

Variants of Escherichia coli Subtilase Cytotoxin Subunits Show Differences in Complex Formation In Vitro.

The subtilase cytotoxin (SubAB) of Shiga toxin-producing Escherichia coli (STEC) is a member of the AB5 toxin family. In the current study, we analyzed the formation of active homo- and hetero-complexes of SubAB variants in vitro to characterize the mode of assembly of the subunits. Recombinant SubA1-His, SubB1-His, SubA2-2-His, and SubB2-2-His subunits, and His-tag-free SubA2-2 were separately expressed, purified, and biochemically characterized by circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), and analytical ultracentrifugation (aUC). To confirm their biological activity, cytotoxicity assays were performed with HeLa cells. The formation of AB5 complexes was investigated with aUC and isothermal titration calorimetry (ITC). Binding of SubAB2-2-His to HeLa cells was characterized with flow cytometry (FACS). Cytotoxicity experiments revealed that the analyzed recombinant subtilase subunits were biochemically functional and capable of intoxicating HeLa cells. Inhibition of cytotoxicity by Brefeldin A demonstrated that the cleavage is specific. All His-tagged subunits, as well as the non-tagged SubA2-2 subunit, showed the expected secondary structural compositions and oligomerization. Whereas SubAB1-His complexes could be reconstituted in solution, and revealed a Kd value of 3.9 ± 0.8 µmol/L in the lower micromolar range, only transient interactions were observed for the subunits of SubAB2-2-His in solution, which did not result in any binding constant when analyzed with ITC. Additional studies on the binding characteristics of SubAB2-2-His on HeLa cells revealed that the formation of transient complexes improved binding to the target cells. Conclusively, we hypothesize that SubAB variants exhibit different characteristics in their binding behavior to their target cells.