Unraveling the interplay between PKA inhibition and Cdk1 activation during oocyte meiotic maturation.

Oocytes are arrested in prophase I. In vertebrates, meiotic resumption is triggered by hormonal stimulation that results in cAMP-dependent protein kinase (PKA) downregulation leading to Cdk1 activation. Yet the pathways connecting PKA to Cdk1 remain unclear. Here, we identify molecular events triggered by PKA downregulation occurring upstream of Cdk1 activation. We describe a two-step regulation controlling cyclin B1 and Mos accumulation, which depends on both translation and stabilization. Cyclin B1 accumulation is triggered by PKA inhibition upstream of Cdk1 activation, while its translation requires Cdk1 activity. Conversely, Mos translation initiates in response to the hormone, but the protein accumulates only downstream of Cdk1. Furthermore, two successive translation waves take place, the first controlled by PKA inhibition and the second by Cdk1 activation. Notably, Arpp19, an essential PKA effector, does not regulate the early PKA-dependent events. This study elucidates how PKA downregulation orchestrates multiple pathways that converge toward Cdk1 activation and induce the oocyte G2/M transition.

Ilf3 and NF90 functions in RNA biology.

Double-stranded RNA-binding proteins (DRBPs) are known to regulate many processes of RNA metabolism due, among others, to the presence of double-stranded RNA (dsRNA)-binding motifs (dsRBMs). Among these DRBPs, Interleukin enhancer-binding factor 3 (Ilf3) and Nuclear Factor 90 (NF90) are two ubiquitous proteins generated by mutually exclusive and alternative splicings of the Ilf3 gene. They share common N-terminal and central sequences but display specific C-terminal regions. They present a large heterogeneity generated by several post-transcriptional and post-translational modifications involved in their subcellular localization and biological functions. While Ilf3 and NF90 were first identified as activators of gene expression, they are also implicated in cellular processes unrelated to RNA metabolism such as regulation of the cell cycle or of enzymatic activites. The implication of Ilf3 and NF90 in RNA biology will be discussed with a focus on eukaryote transcription and translation regulation, on viral replication and translation as well as on noncoding RNA field.

Proteomic analysis of interleukin enhancer binding factor 3 (Ilf3) and nuclear factor 90 (NF90) interactome.

Interleukin enhancer binding factor 3 (Ilf3) and Nuclear Factor 90 (NF90) are two ubiquitous proteins generated by alternative splicing from the ILF3 gene that provides each protein with a long and identical N-terminal domain of 701 amino acids and a specific C-terminal domain of 210 and 15 amino acids, respectively. They exhibit a high polymorphism due to their posttranscriptional and posttranslational modifications. Ilf3 and NF90 functions remain unclear although they have been described as RNA binding proteins but have been implicated in a large scale of cellular phenomena depending on the nature of their interacting partners, the composition of their protein complexes and their subcellular localization. In order to better understand the functions of Ilf3 and NF90, we have investigated their protein partners by an affinity chromatography approach. In this report, we have identified six partners of Ilf3 and NF90 that interact with their double-stranded RNA binding motifs: hnRNP A/B, hnRNP A2/B1, hnRNP A3, hnRNP D, hnRNP Q and PSF. These hnRNP are known to be implicated in mRNA stabilization, transport and/or translation regulation whereas PSF is a splicing factor. Furthermore, Ilf3, NF90 and most of their identified partners have been shown to be present in large complexes. Altogether, these data suggest an implication of Ilf3 and NF90 in mRNA metabolism. This work allows to establish a link between Ilf3 and NF90 functions, as RNA binding proteins, and their interacting partners implicated in these functions.

A substrate-based approach to convert SerpinB1 into a specific inhibitor of proteinase 3, the Wegener's granulomatosis autoantigen.

The physiological and pathological functions of proteinase 3 (PR3) are not well understood due to its close similarity to human neutrophil elastase (HNE) and the lack of a specific inhibitor. Based on structural analysis of the active sites of PR3 and HNE, we generated mutants derived from the polyvalent inhibitor SerpinB1 (monocyte/neutrophil elastase inhibitor) that specifically inhibit PR3 and that differ from wt-SerpinB1 by only 3 or 4 residues in the reactive center loop. The rate constant of association between the best SerpinB1 mutant and PR3 is 1.4 × 107 M⁻¹ · s⁻¹, which is ∼100-fold higher than that observed with wt-SerpinB1 and compares with that of α1-protease inhibitor (α1-PI) toward HNE. SerpinB1(S/DAR) is cleaved by HNE, but due to differences in rate, inhibition of PR3 by SerpinB1(S/DAR) is only minimally affected by the presence of HNE even when the latter is in excess. SerpinB1(S/DAR) inhibits soluble PR3 and also membrane-bound PR3 at the surface of activated neutrophils. Moreover, SerpinB1(S/DAR) clears induced PR3 from the surface of activated neutrophils. Overall, these specific inhibitors of PR3 will be valuable for defining biological functions of the protease and may prove useful as therapeutics for PR3-related inflammatory diseases, such as Wegener's granulomatosis.

High-specific-activity probes and a high-resolution in-gel photo cross-linking assay for protein-DNA complexes.

Photochemical cross-linking has been widely employed to identify proteins interacting with specific sites on DNA. Identification of bound proteins usually relies on transfer of a radiolabel from the DNA to the protein by cross-linking. We set out to fine-map a small viral replication preinitiation complex composed of two protein dimers bound to DNA, the bovine papillomavirus E1E2-ori complex. Here we describe a simple method for generating high-specific-activity probes with a phenyl-azide photoactivatible cross-linking group positioned immediately adjacent to a labeled nucleotide. The method is based on the selective destruction of one 5'-phosphorylated strand of a polymerase chain reaction product with lambda exonuclease and reconstitution of the probe with a phosphorothioate-substituted oligonucleotide, an [alpha-(32)P]dNTP, and thermophilic enzymes. We also developed a high-resolution in-gel cross-linking assay to probe defined protein-DNA complexes. With these methods we have obtained structural information for the papillomavirus E1E2-ori preinitiation complex that would otherwise have been hard to obtain. These approaches should be widely applicable to the study of protein-DNA complexes.

Role of ATP hydrolysis in the DNA translocase activity of the bovine papillomavirus (BPV-1) E1 helicase.

The E1 protein of bovine papillomavirus type-1 is the viral replication initiator protein and replicative helicase. Here we show that the C-terminal approximately 300 amino acids of E1, that share homology with members of helicase superfamily 3 (SF3), can act as an autonomous helicase. E1 is monomeric in the absence of ATP but assembles into hexamers in the presence of ATP, single-stranded DNA (ssDNA) or both. A 16 base sequence is the minimum for efficient hexamerization, although the complex protects approximately 30 bases from nuclease digestion, supporting the notion that the DNA is bound within the protein complex. In the absence of ATP, or in the presence of ADP or the non-hydrolysable ATP analogue AMP-PNP, the interaction with short ssDNA oligonucleotides is exceptionally tight (T(1/2) > 6 h). However, in the presence of ATP, the interaction with DNA is destabilized (T(1/2) approximately 60 s). These results suggest that during the ATP hydrolysis cycle an internal DNA-binding site oscillates from a high to a low-affinity state, while protein-protein interactions switch from low to high affinity. This reciprocal change in protein-protein and protein-DNA affinities could be part of a mechanism for tethering the protein to its substrate while unidirectional movement along DNA proceeds.

Common determinants in DNA melting and helicase-catalysed DNA unwinding by papillomavirus replication protein E1.

E1 and T-antigen of the tumour viruses bovine papillomavirus (BPV-1) and Simian virus 40 (SV40) are the initiator proteins that recognize and melt their respective origins of replication in the initial phase of DNA replication. These proteins then assemble into processive hexameric helicases upon the single-stranded DNA that they create. In T-antigen, a characteristic loop and hairpin structure (the pre-sensor 1beta hairpin, PS1betaH) project into a central cavity generated by protein hexamerization. This channel undergoes large ATP-dependent conformational changes, and the loop/PS1betaH is proposed to form a DNA binding site critical for helicase activity. Here, we show that conserved residues in BPV E1 that probably form a similar loop/hairpin structure are required for helicase activity and also origin (ori) DNA melting. We propose that DNA melting requires the cooperation of the E1 helicase domain (E1HD) and the origin binding domain (OBD) tethered to DNA. One possible mechanism is that with the DNA locked in the loop/PS1betaH DNA binding site, ATP-dependent conformational changes draw the DNA inwards in a twisting motion to promote unwinding.

Epididymal cell secretory activities and the role of proteins in boar sperm maturation.

The final stages of sperm differentiation occur outside the gonad, in the epididymal tubule. These last maturation steps, essential to the quality of spermatozoa, are not under the genomic control of the germ cells. A series of sequential interactions with the epididymal fluid, mostly specific proteins present in the lumen of different regions, are believed to induce the final steps of sperm maturation. In order to provide the luminal changes required for this maturation to occur, the epithelium may resort to two basic mechanisms: absorption and secretion. Far from being a uniform channel, the epididymal duct is a canal with highly specialized regional differentiation of its epithelial ultrastructure and its secretory and absorptive functions. This review focuses on the ultrastructural characteristic of the epithelial cells, their specific secretory activity according to the epididymal regions and their eventual role in sperm maturation of the boar. The chronology of the changes that occur in and on the sperm and in the surrounding environment are described. Relationships between the highly regionalized epididymal activities, sperm characteristics linked to their survival and fertility potential are also presented in this review.

Train A, an RNase A-like protein without RNase activity, is secreted and reabsorbed by the same epididymal cells under testicular control.

Most of the proteins secreted in the epididymis are produced by the proximal region, and several of them are secreted in abundance. Many of these major proteins have now been identified, including a new epididymis-specific RNase A-like Train A protein, which has been recently described in several mammals. This protein is expressed and secreted exclusively in the initial part of the epididymis. RNase A activity was analyzed in the fluids from the testis and from different epididymal regions, but in no case was the Train A protein found to have RNase A activity. The protein was present only in the luminal fluid of the epididymal region that secreted it. Using an in vitro/in vivo microperfusion technique and immunogold electron microscopy labeling, we demonstrated that the epithelium that secreted it specifically reabsorbed the protein that was present in the lumen of the tubule. Thus, the presence of Train A protein in epididymal fluid was the result of a steady state between secretion and absorption. The transcription and translation of Train A mRNA were simultaneous and actively regulated by testicular factors. The function of this protein is unknown, but it does not seem to interact directly with sperm. As for other members of the RNase family (e.g., angiogenin), its biological activity might be expressed after its cellular reabsorption. This new compound might therefore participate in an unknown function in the epithelial cells of this first part of the epididymis by an autocrine pathway.

Identification of a member of a new RNase a family specifically secreted by epididymal caput epithelium.

In this study, we purified the first member of a new ribonuclease (RNase) A family from fluid of the proximal caput of the boar epididymis. This protein, named "Train A," is the most abundant compound secreted in the anterior part of the boar epididymis. After 2D electrophoresis, it is characterized by more than 10 isoforms ranging in size from 26 to 33 kDa and pI from 5 to 8.5. Several tryptic peptides were N-terminal sequenced, and an antiserum against one of these peptides was obtained. The protein was immunolocalized in the epididymal epithelium of the proximal caput, especially in the Golgi zone and the apical cytoplasm of the principal cells. In the lumen, spermatozoa were negative but droplets of reaction product were observed within the lumen. Full lengths of Train A cDNA were obtained from a lambdagt11 boar caput epididymis library and sequenced. The deduced protein is composed of 213 amino acids, including a 23-amino acid peptide signal and a potential N-glycosylation site. The mRNA of this protein has been retrieved and partially sequenced in the bull, horse, and ram, and homologous cDNA is found in databanks for the rat, mouse, and human. All the sequences are highly conserved between species. This protein and its mRNA are male-specific and exclusively expressed in the proximal caput of the epididymis, the only site where they have been found. Train A presents an RNase A family motif in its sequence. The RNase A family is a group of several short proteins (20-14 kDa) with greater and lesser degrees of ribonucleolytic activity and with supposed different roles in vivo. However, the presence of a long-conserved N-terminal specific sequence and the absence of RNase catalytic site for Train A indicate that Train A protein is a member of a new family of RNase A.

Prostaglandin D2 synthase secreted in the caput epididymidis displays spatial and temporal delay between messenger RNA and protein expression during postnatal development.

Highly regionalized protein secretory activity is established progressively throughout the epididymal tubule during postnatal development. Prostaglandin D(2) synthase (PGDS) is a major protein in the ovine epididymis, the secretion of which is restricted to the proximal part of the epididymis. We investigated the mRNA and protein expression of PGDS during ontogenesis. PGDS mRNA was present in the testis and epididymis in the 50-day-old sheep fetus and persisted until 4 mo of age (2 mo before puberty) without mRNA translation in the epididymis. At 4.5 mo, mRNA was present in all of the epididymis, but translation occurred in only the proximal caput. The secreted protein present in the lumen from the caput to the cauda had the same molecular mass and isoelectric point (pI) characteristics as the testicular form. At 5.5 mo, both mRNA and protein expression were restricted to the proximal caput. The protein secreted accumulated in the cauda but was not processed after secretion as it is in the adult animal; no changes in molecular mass or pI were observed. Our results show that for at least one gene (PGDS), transcriptional and posttranscriptional regulation during postnatal development is linked to the presence of unidentified translation factors present in testicular fluid.