Discovering small-molecule modulators of protein-protein interactions is a challenging task because of both the generally noncontiguous, large protein surfaces that form these interfaces and the shortage of high-throughput approaches capable of identifying such rare inhibitors. We describe here a robust and flexible methodology that couples disruption of protein-protein complexes to host cell survival. The feasibility of this approach was demonstrated through monitoring a small-molecule-mediated protein-protein association (FKBP12-rapamycin-FRAP) and two cases of dissociation (homodimeric HIV-1 protease and heterodimeric ribonucleotide reductase). For ribonucleotide reductase, we identified cyclic peptide inhibitors from genetically encoded libraries that dissociated the enzyme subunits. A solid-phase synthetic strategy and peptide ELISAs were developed to characterize these inhibitors, resulting in the discovery of cyclic peptides that operate in an unprecedented manner, thus highlighting the strengths of a functional approach. The ability of this method to process large libraries, coupled with the benefits of a genetic selection, allowed us to identify rare, uniquely active small-molecule modulators of protein-protein interactions at a frequency of less than one in 10 million.M any regulatory processes in living organisms are often a consequence of specific protein-protein contacts, and interference with such interactions provides a means to exert control over cellular events. The de novo discovery of small molecules capable of disrupting such protein-protein complexes has been fraught with challenges, yielding very few inhibitors at a low success rate (1-3). These difficulties suggest that large, functionally diverse libraries might be essential for finding unique molecules that are capable of perturbing the intracellular levels of specific protein-protein interactions. The major challenge in sifting through such vast compound pools is the shortage of functional high-throughput assays for detection of the protein complex dissociation (4).Genetic selection is uniquely capable of identifying individual molecules with desired properties from large libraries by using whole cells as reporters and correlating host growth to a desired functional property. Unlike recently popularized affinity-based selections (5), an intracellular genetic selection can directly assay for effects on enzymatic activity or the modulation of a proteinprotein complex, thus bypassing the inherent limitations of in vitro approaches. Additionally, library members must function within the context of the entire host proteome, requiring positive candidates to have an enhanced level of selectivity for their target. This feature represents an important advantage over traditional screen-based methods in drug discovery by allowing both target affinity and selectivity to be simultaneously optimized. The application of a genetic selection to the identification of small-molecule modulators may yield both potent and selective activities as well as uniq...
SUMMARY Faithful genome transmission during cell division requires precise, coordinated action of DNA metabolic enzymes, including proteins responsible for DNA damage detection and repair. Dynamic phosphorylation plays an important role in controlling repair enzymes during the DNA damage response (DDR). Cdc14 phosphatases oppose cyclin-dependent kinase (Cdk) phosphorylation and have been implicated in the DDR in several model systems. Using new insight into Cdc14 specificity we identified the budding yeast Holliday junction resolvase Yen1 as the first DNA repair target of Cdc14. Cdc14 activation at anaphase triggers nuclear accumulation and enzymatic activation of Yen1, likely to resolve persistent recombinational repair intermediates. Consistent with this, expression of a phosphomimetic Yen1 mutant increased sister chromatid non-disjunction. In contrast, lack of Cdk phosphorylation resulted in constitutive activity and elevated crossover-associated repair. The precise timing of Yen1 activation, governed by core cell cycle regulators, helps coordinate DNA repair with chromosome segregation and safeguards against genome destabilization.
In mammals, sperm-oocyte fusion initiates Ca(2+) oscillations leading to a series of events called oocyte activation, which is the first stage of embryo development. Ca(2+) signaling is elicited by the delivery of an oocyte-activating factor by the sperm. A sperm-specific phospholipase C (PLCZ1) has emerged as the likely candidate to induce oocyte activation. Recently, PAWP, a sperm-born tryptophan domain-binding protein coded by WBP2NL, was proposed to serve the same purpose. Here, we studied two infertile brothers exhibiting normal sperm morphology but complete fertilization failure after intracytoplasmic sperm injection. Whole exomic sequencing evidenced a missense homozygous mutation in PLCZ1, c.1465A>T; p.Ile489Phe, converting Ile 489 into Phe. We showed the mutation is deleterious, leading to the absence of the protein in sperm, mislocalization of the protein when injected in mouse GV and MII oocytes, highly abnormal Ca(2+) transients and early embryonic arrest. Altogether these alterations are consistent with our patients' sperm inability to induce oocyte activation and initiate embryo development. In contrast, no deleterious variants were identified in WBP2NL and PAWP presented normal expression and localization. Overall we demonstrate in humans, the absence of PLCZ1 alone is sufficient to prevent oocyte activation irrespective of the presence of PAWP. Additionally, it is the first mutation located in the C2 domain of PLCZ1, a domain involved in targeting proteins to cell membranes. This opens the door to structure-function studies to identify the conserved amino acids of the C2 domain that regulate the targeting of PLCZ1 and its selectivity for its lipid substrate(s).
Motile cilia and sperm flagella share an extremely conserved microtubule-based cytoskeleton, called the axoneme, which sustains beating and motility of both organelles. Ultra-structural and/or functional defects of this axoneme are well-known to cause primary ciliary dyskinesia (PCD), a disorder characterized by recurrent respiratory tract infections, chronic otitis media, situs inversus, male infertility and in most severe cases, hydrocephalus. Only recently, mutations in genes encoding axonemal proteins with preferential expression in the testis were identified in isolated male infertility; in those cases, individuals displayed severe asthenozoospermia due to Multiple Morphological Abnormalities of the sperm Flagella (MMAF) but not PCD features. In this study, we performed genetic investigation of two siblings presenting MMAF without any respiratory PCD features, and we report the identification of the c.2018T > G (p.Leu673Pro) transversion in AK7, encoding an adenylate kinase, expressed in ciliated tissues and testis. By performing transcript and protein analyses of biological samples from individual carrying the transversion, we demonstrate that this mutation leads to the loss of AK7 protein in sperm cells but not in respiratory ciliated cells, although both cell types carry the mutated transcript and no tissue-specific isoforms were detected. This work therefore, supports the notion that proteins shared by both cilia and sperm flagella may have specific properties and/or function in each organelle, in line with the differences in their mode of assembly and organization. Overall, this work identifies a novel genetic cause of asthenozoospermia due to MMAF and suggests that in humans, more deleterious mutations of AK7 might induce PCD.
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