SUMMARY The oocyte-to-embryo transition marks the onset of development. The initial phase of this profound change from the differentiated oocyte to the totipotent embryo occurs in the absence of both transcription and mRNA degradation. Here we combine global polysome profiling, ribosome-footprint profiling, and quantitative mass spectrometry in a comprehensive approach to delineate the translational and proteomic changes at this important transition in Drosophila. Our results show that PNG kinase is a critical regulator of the extensive changes in the translatome, acting uniquely at this developmental window. Analysis of the proteome in png mutants provided insights into the contributions of translation to changes in protein levels, revealing a compensatory dynamic between translation and protein turnover during proteome remodeling at the return to totipotency. The proteome changes additionally suggested new regulators of meiosis and early embryogenesis, including the conserved H3K4 demethylase LID, which we demonstrated is required during this period despite transcriptional inactivity.
The onset of development is marked by two major, posttranscriptionally controlled, events: oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, we previously described proteome remodeling during Drosophila egg activation. Here, we describe our quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey, to our knowledge, of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. We show that Muskelin, found to be up-regulated during oocyte maturation, is required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. We find down-regulation of this class of proteins to be mediated partially by APC/C CORT , a meiosis-specific form of the E3 ligase anaphase promoting complex/ cyclosome (APC/C).he change from oocyte to embryo marks the onset of development. Oocyte maturation is a prerequisite for the oocyte-toembryo transition. In most animals, oocytes undergo a prolonged arrest in prophase I to permit oocyte growth, differentiation, and stockpiling of maternal components. This arrest is released at oocyte maturation, commonly in response to hormonal cues. The nuclear envelope then breaks down, followed by assembly of the meiotic spindle. A secondary meiotic arrest ensues, occurring at metaphase II in most vertebrates and metaphase I in insects (1). The oocyte-to-embryo transition initiates with egg activation, which causes the release of the secondary arrest and the completion of meiosis. In many organisms, egg activation requires fertilization, but egg activation is independent of fertilization in Drosophila (1). In all cases, the onset of embryogenesis requires sperm entry, fusion of the male and female pronuclei, and the start of the mitotic embryonic divisions.How is this complex transition from differentiated oocyte into totipotent embryo regulated? Both oocyte maturation and egg activation occur in a transcriptionally silent context (2-4). These events are thus posttranscriptionally controlled, and translational regulation has been shown to play a crucial role. For example, resumption of meiosis in Xenopus depends on translational activation of cyclin B and mos mRNA by CPEB-mediated polyad...
The important role of histone posttranslational modifications, particularly methylation and acetylation, in Plasmodium falciparum gene regulation has been established. However, the role of histone phosphorylation remains understudied. Here, we investigate histone phosphorylation utilizing liquid chromatography and tandem mass spectrometry to analyze histones extracted from asexual blood stages using two improved protocols to enhance preservation of PTMs. Enrichment for phosphopeptides lead to the detection of 14 histone phospho-modifications in P. falciparum. The majority of phosphorylation sites were observed at the N-terminal regions of various histones and were frequently observed adjacent to acetylated lysines. We also report the identification of one novel member of the P. falciparum histone phosphosite binding protein repertoire, Pf14-3-3I. Recombinant Pf14-3-3I protein bound to purified parasite histones. In silico structural analysis of Pf14-3-3 proteins revealed that residues responsible for binding to histone H3 S10ph and/or S28ph are conserved at the primary and the tertiary structure levels. Using a battery of H3 specific phosphopeptides, we demonstrate that Pf14-3-3I preferentially binds to H3S28ph over H3S10ph, independent of modification of neighbouring residues like H3S10phK14ac and H3S28phS32ph. Our data provide key insight into histone phosphorylation sites. The identification of a second member of the histone modification reading machinery suggests a widespread use of histone phosphorylation in the control of various nuclear processes in malaria parasites.
Vaccinia H1-related (VHR) phosphatase is a dual specificity phosphatase that is required for cell-cycle progression and plays a role in cell growth of certain cancers. Therefore, it represents a potential drug target. VHR is structurally and biochemically well characterized, yet its regulatory principles are still poorly understood. Understanding its regulation is important, not only to comprehend VHR's biological mechanisms and roles but also to determine its potential and druggability as a target in cancer. Here, we investigated the functional role of the unique "variable insert" region in VHR by selectively introducing the photo-cross-linkable amino acid para-benzoylphenylalanine (pBPA) using the amber suppression method. This approach led to the discovery of VHR dimerization, which was further confirmed using traditional chemical cross-linkers. Phe68 in VHR was discovered as a residue involved in the dimerization. We demonstrate that VHR can dimerize inside cells, and that VHR catalytic activity is reduced upon dimerization. Our results suggest that dimerization could occlude the active site of VHR, thereby blocking its accessibility to substrates. These findings indicate that the previously unknown transient self-association of VHR acts as a means for the negative regulation of its catalytic activity.
To facilitate subcutaneous dosing, biotherapeutics need to exhibit properties that enable high-concentration formulation and long-term stability in the formulation buffer. For antibody–drug conjugates (ADCs), the introduction of drug-linkers can lead to increased hydrophobicity and higher levels of aggregation, which are both detrimental to the properties required for subcutaneous dosing. Herein we show how the physicochemical properties of ADCs could be controlled through the drug-linker chemistry in combination with prodrug chemistry of the payload, and how optimization of these combinations could afford ADCs with significantly improved solution stability. Key to achieving this optimization is the use of an accelerated stress test performed in a minimal formulation buffer.
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