Mass spectrometrical analysis of recombinant human growth hormone (Genotropin(®)) reveals amino acid substitutions in 2% of the expressed protein

BACKGROUND: The structural integrity of recombinant proteins is of critical importance to their application as clinical treatments. Recombinant growth hormone preparations have been examined by several methodologies. In this study recombinant human growth hormone (rhGH; Genotropin(®)), expressed in E. coli K12, was structurally analyzed by two-dimensional gel electrophoresis and MALDI-TOF-TOF, LC-MS and LC-MS/ MS sequencing of the resolved peptides. RESULTS: Electrospray LC-MS analysis revealed one major protein with an average molecular mass of 22126.8 Da and some additional minor components. Electrospray LC-MS/MS evaluation of the enzymatically digested Genotropin(® )sample resulted in the identification of amino acid substitutions at the residues M(14), M(125), and M(170); di-methylation of K(70 )(or exchange to arginine); deamidation of N(149), and N(152), and oxidation of M(140), M(125 )and M(170). Peak area comparison of the modified and parental peptides indicates that these changes were present in ~2% of the recombinant preparation. CONCLUSION: Modifications of the recombinant human growth hormone may lead to structural or conformational changes, modification of antigenicity and development of antibody formation in treated subjects. Amino acid exchanges may be caused by differences between human and E. coli codon usage and/or unknown copy editing mechanisms. While deamidation and oxidation can be assigned to processing events, the mechanism for possible di-methylation of K(70 )remains unclear

N-terminal β-strand underpins biochemical specialization of an ATG8 isoform

Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein-protein interaction resource, obtained using in planta immunoprecipitation (IP) followed by mass spectrometry (MS), to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal β-strand-and, in particular, a single amino acid polymorphism-underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this protein's ATG8-interacting motif (AIM). Additional proteomics experiments indicated that the N-terminal β-strand shapes the broader ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants

ESCO1 and CTCF enable formation of long chromatin loops by protecting cohesinSTAG1 from WAPL.

Eukaryotic genomes are folded into loops. It is thought that these are formed by cohesin complexes via extrusion, either until loop expansion is arrested by CTCF or until cohesin is removed from DNA by WAPL. Although WAPL limits cohesin's chromatin residence time to minutes, it has been reported that some loops exist for hours. How these loops can persist is unknown. We show that during G1-phase, mammalian cells contain acetylated cohesinSTAG1 which binds chromatin for hours, whereas cohesinSTAG2 binds chromatin for minutes. Our results indicate that CTCF and the acetyltransferase ESCO1 protect a subset of cohesinSTAG1 complexes from WAPL, thereby enable formation of long and presumably long-lived loops, and that ESCO1, like CTCF, contributes to boundary formation in chromatin looping. Our data are consistent with a model of nested loop extrusion, in which acetylated cohesinSTAG1 forms stable loops between CTCF sites, demarcating the boundaries of more transient cohesinSTAG2 extrusion activity

Dissociation of Cohesin from Chromosome Arms and Loss of Arm Cohesion during Early Mitosis Depends on Phosphorylation of SA2

Cohesin is a protein complex that is required to hold sister chromatids together. Cleavage of the Scc1 subunit of cohesin by the protease separase releases the complex from chromosomes and thereby enables the separation of sister chromatids in anaphase. In vertebrate cells, the bulk of cohesin dissociates from chromosome arms already during prophase and prometaphase without cleavage of Scc1. Polo-like kinase 1 (Plk1) and Aurora-B are required for this dissociation process, and Plk1 can phosphorylate the cohesin subunits Scc1 and SA2 in vitro, consistent with the possibility that cohesin phosphorylation by Plk1 triggers the dissociation of cohesin from chromosome arms. However, this hypothesis has not been tested yet, and in budding yeast it has been found that phosphorylation of Scc1 by the Polo-like kinase Cdc5 enhances the cleavability of cohesin, but does not lead to separase-independent dissociation of cohesin from chromosomes. To address the functional significance of cohesin phosphorylation in human cells, we have searched for phosphorylation sites on all four subunits of cohesin by mass spectrometry. We have identified numerous mitosis-specific sites on Scc1 and SA2, mutated them, and expressed nonphosphorylatable forms of both proteins stably at physiological levels in human cells. The analysis of these cells lines, in conjunction with biochemical experiments in vitro, indicate that Scc1 phosphorylation is dispensable for cohesin dissociation from chromosomes in early mitosis but enhances the cleavability of Scc1 by separase. In contrast, our data reveal that phosphorylation of SA2 is essential for cohesin dissociation during prophase and prometaphase, but is not required for cohesin cleavage by separase. The similarity of the phenotype obtained after expression of nonphosphorylatable SA2 in human cells to that seen after the depletion of Plk1 suggests that SA2 is the critical target of Plk1 in the cohesin dissociation pathway

Inter-Homolog Crossing-Over and Synapsis in Arabidopsis Meiosis Are Dependent on the Chromosome Axis Protein AtASY3

In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR), we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC) formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation

Ionizing radiation, higher plants, and radioprotection: From acute high doses to chronic low doses

© 2018 Caplin and Willey. Understanding the effects of ionizing radiation (IR) on plants is important for environmental protection, for agriculture and horticulture, and for space science but plants have significant biological differences to the animals from which much relevant knowledge is derived. The effects of IR on plants are understood best at acute high doses because there have been; (a) controlled experiments in the field using point sources, (b) field studies in the immediate aftermath of nuclear accidents, and (c) controlled laboratory experiments. A compilation of studies of the effects of IR on plants reveals that although there are numerous field studies of the effects of chronic low doses on plants, there are few controlled experiments that used chronic low doses. Using the Bradford-Hill criteria widely used in epidemiological studies we suggest that a new phase of chronic low-level radiation research on plants is desirable if its effects are to be properly elucidated. We emphasize the plant biological contexts that should direct such research. We review previously reported effects from the molecular to community level and, using a plant stress biology context, discuss a variety of acute high-and chronic low-dose data against Derived Consideration Reference Levels (DCRLs) used for environmental protection. We suggest that chronic low-level IR can sometimes have effects at the molecular and cytogenetic level at DCRL dose rates (and perhaps below) but that there are unlikely to be environmentally significant effects at higher levels of biological organization. We conclude that, although current data meets only some of the Bradford-Hill criteria, current DCRLs for plants are very likely to be appropriate at biological scales relevant to environmental protection (and for which they were intended) but that research designed with an appropriate biological context and with more of the Bradford-Hill criteria in mind would strengthen this assertion. We note that the effects of IR have been investigated on only a small proportion of plant species and that research with a wider range of species might improve not only the understanding of the biological effects of radiation but also that of the response of plants to environmental stress

The Optimal Design of Trade Policy Flexibility in the WTO

This paper is a contribution to the literature on rational design of trade agreements. The World Trade Organization (WTO) is an incomplete contract among sovereign states. Incomplete contracts contain gaps. Ex post, contractual gaps may leave gains from trade unrealized; they may create 'regret' in signatories once unanticipated contingencies or sudden protectionist backlashes have occurred. Trade policy flexibility mechanisms, such as the 'safeguards clause' under Art. XIX GATT, are geared towards seizing ex post regret by allowing parties affected by a protectionist shock to partially and temporarily withdraw from previously made trade liberalization concessions - given that they compensate the victim(s) of such backtracking behavior. This paper examines the somewhat understudied issue of optimal trade policy flexibility design in the WTO: In particular, we analyze whether ex post escape should be organized by means of a unilateral opt-out clause (a 'liability rule' of escape), or a bilateral renegotiation provision (a 'property rule' of escape). Modeling the WTO as a fully non-contingent tariff liberalization contract with contingencies (or 'states of nature') asymmetrically revealed, we find that a liability rule backed by expectation remedies payable to the affected victim Pareto-dominates both a renegotiation clause, as well as any other remedy arrangement connected to a liability rule. Only the remedial design of liability-cum-expectation damages yields the desirable incentives to liberalize ex ante, and to default ex post and therewith is able to replicate the outcomes of the hypothetical contracting ideal of the complete contingent contract