Acyldepsipeptides (ADEPs) are potential antibiotics that dysregulate the activity of the highly conserved tetradecameric bacterial ClpP protease, leading to bacterial cell death. Here, we identified ADEP analogs that are potent dysregulators of the human mitochondrial ClpP (HsClpP). These ADEPs interact tightly with HsClpP, causing the protease to non-specifically degrade model substrates. Dysregulation of HsClpP activity by ADEP was found to induce cytotoxic effects via activation of the intrinsic, caspase-dependent apoptosis. ADEP-HsClpP co-crystal structure was solved for one of the analogs revealing a highly complementary binding interface formed by two HsClpP neighboring subunits but, unexpectedly, with HsClpP in the compact conformation. Given that HsClpP is highly expressed in multiple cancers and has important roles in cell metastasis, our findings suggest a therapeutic potential for ADEPs in cancer treatment.

The platelet-derived growth factor (PDGF) signaling pathway regulates numerous lineages of mesenchymal cell origin during development and in the adult. The transcriptional targets of this pathway have been shown to be required in several PDGF-dependent processes, but the roles of these targets in specific tissues is just beginning to be identified. In this study, we show that five different PDGF target genes are essential for male and/or female fertility. Mutations in each of these five different genes lead to defects in the steroid-producing cells in the testis and/or ovary and altered hormone production, suggesting that the PDGF pathway controls steroidogenesis through these genes in both sexes. Furthermore, conditional mutations of both PDGF receptors revealed a requirement in steroid-producing cells in multiple organs, including the testis, ovary, and adrenal cortex. Therefore, PDGF signaling may constitute a common mechanism in the control of multiple steroidogenic lineages.[Keywords: PDGF; testis; ovary; steroid hormone; Leydig cell; theca cell] Supplemental material is available at http://www.genesdev.org. Steroidogenesis and the development of the steroid-producing cells in the gonads is a tightly controlled process in both sexes, requiring regulation at many stages of development through endocrine, autocrine, and paracrine mechanisms. Defects in these processes lead to infertility, malformation of the reproductive tracts, and abnormal secondary sexual characteristics. In males, low testosterone production leads to abnormalities in spermatogenesis, undescended testes, ambiguous genitalia, and infertility (Habert et al. 2001). In females, the production of estrogen and progesterone is essential for ovulation and the maintenance of pregnancy (Fisher et al. 1998;Toda et al. 2001). Altered levels of steroid hormones are known to be associated with many of the common types of infertility in both men and women, such as hypogonadism and Polycystic Ovary Syndrome (PCOS), yet many of the mechanisms that control steroidogenic cell development and hormone production are not well understood.In the gonads, steroid hormones are synthesized by specialized endocrine cells: the Leydig cells in the testis (Habert et al. 2001) and theca cells in the ovary (Magoffin 2005). In the testis, Leydig cells have at least two clearly defined waves of development, as fetal and adult Leydig cells. Fetal Leydig cells appear very early after sex determination, by embryonic day 12.5 (E12.5) in the mouse, and testosterone production from these cells is necessary for the masculinization of the fetus. Postnatally, fetal Leydig cells are replaced by adult Leydig cells, which are required for the progression and maintenance of spermatogenesis. In the ovary, the production of steroid hormones requires cooperation between two cell types, the theca and granulosa cells (Magoffin 2005). Theca cells are steroidogenic, as they initiate steroid synthesis by importing cholesterol to the mitochondrial membrane and synthesize steroid hormones de novo. ...

An automated method for constructing libraries for 454 sequencing significantly reduces the cost and time required.

Protein homeostasis is critically important for cell viability. Key to this process is the refolding of misfolded or aggregated proteins by molecular chaperones or, alternatively, their degradation by proteases. In most prokaryotes and in chloroplasts and mitochondria, protein degradation is performed by the caseinolytic protease ClpP, a tetradecamer barrel-like proteolytic complex. Dysregulating ClpP function has shown promise in fighting antibiotic resistance and as a potential therapy for acute myeloid leukemia. Here we use methyl-transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, biochemical assays, and molecular dynamics simulations to characterize the structural dynamics of ClpP from (SaClpP) in wild-type and mutant forms in an effort to discover conformational hotspots that regulate its function. Wild-type SaClpP was found exclusively in the active extended form, with the N-terminal domains of its component protomers in predominantly β-hairpin conformations that are less well-defined than other regions of the protein. A hydrophobic site was identified that, upon mutation, leads to unfolding of the N-terminal domains, loss of SaClpP activity, and formation of a previously unobserved split-ring conformation with a pair of 20-Å-wide pores in the side of the complex. The extended form of the structure and partial activity can be restored via binding of ADEP small-molecule activators. The observed structural plasticity of the N-terminal gates is shown to be a conserved feature through studies of and ClpP, suggesting a potential avenue for the development of molecules to allosterically modulate the function of ClpP.

A comprehensive view of molecular chaperone function in the cell was obtained through a systematic global integrative network approach based on physical (protein-protein) and genetic (gene-gene or epistatic) interaction mapping. This allowed us to decipher interactions involving all core chaperones (67) and cochaperones (15) of Saccharomyces cerevisiae. Our analysis revealed the presence of a large chaperone functional supercomplex, which we named the naturally joined (NAJ) chaperone complex, encompassing Hsp40, Hsp70, Hsp90, AAA+, CCT, and small Hsps. We further found that many chaperones interact with proteins that form foci or condensates under stress conditions. Using an in vitro reconstitution approach, we demonstrate condensate formation for the highly conserved AAA+ ATPases Rvb1 and Rvb2, which are part of the R2TP complex that interacts with Hsp90. This expanded view of the chaperone network in the cell clearly demonstrates the distinction between chaperones having broad versus narrow substrate specificities in protein homeostasis.