Sequential primed kinases create a damage-responsive phosphodegron on Eco1.

Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation, which triggers ubiquitination by SCF(Cdc4). We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7-Dbf4 and the GSK-3 homolog Mck1. These kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing establishment of cohesion after S phase. This elaborate regulatory system, involving three independent kinases and stringent substrate selection by a ubiquitin ligase, enables robust control of cohesion establishment during normal growth and after stress

Dataset supporting the proteomic differences found between excretion/secretion products from two isolates of Fasciola hepatica newly excysted juveniles (NEJ) derived from different snail hosts

Here we present the proteomic profile datasets of two Fasciola hepatica NEJ isolates derived from different snail hosts: Lymnaea viatrix and Pseudosuccinea columella. The data used in the analysis are related to the article ‘A proteomic comparison of excretion/secretion products in Fasciola hepatica newly excysted juveniles (NEJ) derived from Lymnaea viatrix or Pseudosuccinea columella’ (Di Maggio et al., 2019)

Across intra-mammalian stages of the liver fluke Fasciola hepatica: a proteomic study

Fasciola hepatica is the agent of fasciolosis, a foodborne zoonosis that affects livestock production and human health. Although flukicidal drugs are available, re-infection and expanding resistance to triclabendazole demand new control strategies. Understanding the molecular mechanisms underlying the complex interaction with the mammalian host could provide relevant clues, aiding the search for novel targets in diagnosis and control of fasciolosis. Parasite survival in the mammalian host is mediated by parasite compounds released during infection, known as excretory/secretory (E/S) products. E/S products are thought to protect parasites from host responses, allowing them to survive for a long period in the vertebrate host. This work provides in-depth proteomic analysis of F. hepatica intra-mammalian stages, and represents the largest number of proteins identified to date for this species. Functional classification revealed the presence of proteins involved in different biological processes, many of which represent original findings for this organism and are important for parasite survival within the host. These results could lead to a better comprehension of host-parasite relationships, and contribute to the development of drugs or vaccines against this parasite

Saliva from nymph and adult females of Haemaphysalis longicornis: a proteomic study

BACKGROUND: Haemaphysalis longicornis is a major vector of Theileria spp., Anaplasma phagocytophilum, Babesia spp. and Coxiella burnetti in East Asian countries. All life stages of ixodid ticks have a destructive pool-feeding style in which they create a pool-feeding site by lacerating host tissue and secreting a variety of biologically active compounds that allows the tick to evade host responses, enabling the uptake of a blood meal. The identification and functional characterization of tick saliva proteins can be useful to elucidate the molecular mechanisms involved in tick development and to conceive new anti-tick control methods. METHODS: H. longicornis tick saliva was collected from fully engorged nymphs and fully engorged adults induced by dopamine or pilocarpine, respectively. Saliva was digested with trypsin for LC-MS/MS sequencing and peptides were searched against tick and rabbit sequences. RESULTS: A total of 275 proteins were identified, of which 135 were tick and 100 were rabbit proteins. Of the tick proteins, 30 proteins were identified exclusively in fully engorged nymph saliva, 74 in fully engorged adult females, and 31 were detected in both stages. The identified tick proteins include heme/iron metabolism-related proteins, oxidation/detoxification proteins, enzymes, proteinase inhibitors, tick-specific protein families, and cytoskeletal proteins. Proteins involved in signal transduction, transport and metabolism of carbohydrate, energy, nucleotide, amino acids and lipids were also detected. Of the rabbit proteins, 13 were present in nymph saliva, 48 in adult saliva, and 30 were present in both. The host proteins include immunoglobulins, complement system proteins, antimicrobial proteins, serum albumin, peroxiredoxin, serotransferrin, apolipoprotein, hemopexin, proteinase inhibitors, and hemoglobin/red blood cells-related products. CONCLUSIONS: This study allows the identification of H. longicornis saliva proteins. In spontaneously detached tick saliva various proteins were identified, although results obtained with saliva of fully engorged ticks need to be carefully interpreted. However, it is interesting to note that proteins identified in this study were also described in other tick saliva proteomes using partially engorged tick saliva, including hemelipoprotein, proteases, protease inhibitors, proteins related to structural functions, transporter activity, metabolic processes, and others. In conclusion, these data can provide a deeper understanding to the biology of H. longicornis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-015-0918-y) contains supplementary material, which is available to authorized users

Sequential primed kinases create a damage-responsive phosphodegron on Eco1.

Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation, which triggers ubiquitination by SCF(Cdc4). We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7-Dbf4 and the GSK-3 homolog Mck1. These kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing establishment of cohesion after S phase. This elaborate regulatory system, involving three independent kinases and stringent substrate selection by a ubiquitin ligase, enables robust control of cohesion establishment during normal growth and after stress

EThcD and 213 nm UVPD for Top-Down Analysis of Bovine Seminal Plasma Proteoforms on Electrophoretic and Chromatographic Time Frames

Seminal plasma is a critical and complex fluid that carries sperm to eggs to initiate the fertilization process. Here, we present a top-down mass spectrometry (TDMS) strategy for identifying proteins and posttranslational modifications (PTMs) in bovine seminal plasma. In this study, proteins were separated using sheathless capillary zone electrophoresis (CZE)-MS and reversed-phase liquid chromatography (LC)-MS, and then fragmented using electron-transfer/higher-energy collisional dissociation (EThcD) and 213 nm ultraviolet photodissociation (213 nm UVPD) to provide more comprehensive information about the proteomic landscape of this biological fluid. Four hundred and seventeen proteoforms were identified by sheathless CZE-MS, and one hundred and seventy-two species were unique to this method. LC-MS identified 3090 proteoforms, including 1707 unique species. All identifications were within ±10 ppm (mass error) and with a P-Score ≤1 × 10-04. Pooling results (triplicate measurements) from sheathless CZE-MS and LC-MS resulted in the identification of 1433 proteoforms (EThcD) and 2151 proteoforms (213 nm UVPD) with 612 species unique for EThcD and 1021 for 213 nm UVPD. The average sequence coverage was found to be higher for EThcD (28%) than for 213 nm UVPD (23%). The use of sheathless CZE-MS and LC-MS with EThcD and 213 nm UVPD provided complementary protein profiling and proteoform data that were more comprehensive than those of either method alone

Mechanism-Based Post-Translational Modification and Inactivation in Terpene Synthases

Terpenes are ubiquitous natural chemicals with diverse biological functions spanning all three domains of life. In specialized metabolism, the active sites of terpene synthases (TPSs) evolve in shape and reactivity to direct the biosynthesis of a myriad of chemotypes for organismal fitness. As most terpene biosynthesis mechanistically involves highly reactive carbocationic intermediates, the protein surfaces catalyzing these cascade reactions possess reactive regions possibly prone to premature carbocation capture and potentially enzyme inactivation. Here, we show using proteomic and X-ray crystallographic analyses that cationic intermediates undergo capture by conserved active site residues leading to inhibitory self-alkylation. Moreover, the level of cation-mediated inactivation increases with mutation of the active site, upon changes in the size and structure of isoprenoid diphosphate substrates, and alongside increases in reaction temperatures. TPSs that individually synthesize multiple products are less prone to self-alkylation then TPSs possessing relatively high product specificity. In total, the results presented suggest that mechanism-based alkylation represents an overlooked mechanistic pressure during the evolution of cation-derived terpene biosynthesis