Determination of two sites of automethylation in bovine erythrocyte protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase

Protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed thePCM fraction [Lindquist and McFadden (1994),J. Protein Chem. 13, 23–30]. The altered aspartyl sites serving as methyl acceptors inPCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [³H]automethylated enzyme that contain a [³H]methyl ester. Methylation was positively identified at positions Asn₁₈₈ and Asp₂₁₇ in the enzyme sequence, a consequence of the spontaneous alteration of these sites tol-isoaspartyl ord-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows thatPCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.Abbreviations PCM protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase - EDTA disodium ethylenediaminetetraacetate - PMSF phenylmethylsulfonyl fluoride - TEA trifluoroacetic acid - HPLC high-pressure liquid chromatography     

Automethylation of protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase, a response to enzyme aging

A question that is central to understanding the mechanisms of aging and cellular deterioration is whether enzymes involved in recognition and metabolism of spontaneously damaged proteins are themselves damaged, either becoming substrates for their own activity; or being unable to act upon themselves, initiating cascades of cellular damage. We show here byin vitro experiments that protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM) from bovine erythrocytes does methylate age-dependent amino acid damage in its own sequence. The subpopulation that is methylated, termed thePCM fraction, appears to be formed through age-dependent deamidation of an asparaginyl site to either anl-isoaspartyl ord-aspartyl site because (a) the stoichiometry of automethylation of purified PCM is less than 1%, a value typical of the substoichiometric methylation of many other aged protein substrates, (b)PCM is slightly more acidic than the bulk of PCM, and (c) the methyl esterified site inPCM has the characteristic base-lability of this type of methyl ester. Also, the methyl group is not incorporated into the enzyme as an active site intermediate because the incorporated methyl group is not chased onto substrate protein. The effect of enzyme dilution on the rate of the automethylation reaction is consistent with methylation occurring between protein molecules, showing that the pool of PCM is autocatalytic even though individual molecules may not be. The automethylation and possible self-repair of the PCM pool has implications for maintaining thein vivo efficiency of methylation-dependent protein repair.     

In vitro production of proteoglycans in the articular-epiphyseal cartilage of growing pigs

The failure of cartilage mineralization in osteochondrotic cartilage may be due to an impaired proteoglycan production. Thein vitro production of proteoglycans was therefore studied in the joint cartilage of growing pigs, aged 9–18 weeks, after incubation of cartilage samples with³⁵S-sulfate. Cartilage was obtained from different areas of the femoral condyles and samples from these areas were further divided into three layers, where the superficial layer contains articular cartilage and the basal layers consist of growth cartilage. There was no significant difference in the overall amount of³⁵S-proteoglycans synthesized in different areas of the condyles. However, the total production of³⁵S-proteoglycans per mg tissue was highest in the basal layer in all areas. This was not due to a larger number of cells; the superficial layer contained more DNA per mg tissue than the basal layer. Gel chromatography on Sepharose CL-2B of the cartilage extracts, which resulted in the separation of large proteoglycans (K _av 0.4) from proteoglycans of small hydrodynamic size (K _av 0.8), showed that the relative amount of large proteoglycans increased with the distance from the articular surface. Again, no difference in the relative amounts of large and small proteoglycans were found when cartilage from different areas were compared. Osteochondrotic cartilage was detected in the pigs aged 12–18 weeks. In areas where osteochondrotic cartilage were present, the total production of³⁵S-proteoglycans was lowered and the relative amount of large proteoglycans was less than that found in the adjoining areas devoid of osteochondrotic lesions. The data available indicate that the higher relative amount of small proteoglycans in the osteochondrotic cartilage was partly caused by degradation of the large proteoglycans (aggrecan).     

Compositional heterogeneity of the Escherichia coli genome: A role for VSP repair?

E. coli genes that contain a high frequency of the tetranucleotide CTAG are also rich in the tetramers CTTG, CCTA, CCAA, TTGG, TAGG, and CAAG (group-I tetramers). Conversely, E. coli genes lacking CTAG are rich in the tetranucleotides CCTG, CCAG, CTGG, and CAGG (group-II tetramers). These two gene samples differ also in codon usage, amino acid composition, frequency of Dcm sites, and contrast vocabularies. Group-I tetramers have in common that they are depleted by very-short-patch repair (VSP), while group-II tetramers are favored by VSP activity. The VSP system repairs G:T mismatches to G:C, thereby increasing the overall G+C content of the genome; for this reason the CTAG-rich sample has a lower G+C content than the CTAG-poor sample. This compositional heterogeneity can be tentatively explained by a low level of VSP activity on the CTAG-rich sample. A negative correlation is found between the frequency of group-I tetramers and the level of gene expression, as measured by the Codon Adaptation Index (CAI). A possible link between the rate of VSP activity and the level of gene expression is considered.Correspondence to: A. Marine     

A matter of timing: System requirements for repair and their temporal dimensions

Research into repair within the circular economy (CE) typically focuses on technical aspects of design, policy, and markets, and often assumes simplified conditions for the user/owner and the product system to explain the barriers to scaling repair activities. However, factors occurring at pre-use stages of the product's life cycle can significantly influence whether, and to what extent, repair is viable or possible, that is, warranty duration, after-sale service provision, and access to necessities. The passing of time can directly and indirectly affect the ability, difficulty, and thus, the likelihood of repair activities being performed at each stage of the product's life cycle. Drawing from the literature and applying inductive systems-thinking tools, we propose a framework for considering the “System of Repairability.” We delineate how the passing of time (temporal dimensions) affects one's ‘‘ability to repair,’’ as a product progresses through different life cycle phases (i.e., breakdown vs. repair vs. disposal), and the point(s) at which the repair is considered or attempted (i.e., year of usage). By integrating life cycle and temporal (time-based) dimensions into a broad System of Repairability framework, we clarify relevant interconnections, iterations, sequences, and timing of decision points, stakeholders, and necessary conditions to facilitate an outcome of successful repair at the individual level, and thus intervention strategies for scaling repair within CE. We discuss how a policy mix can address the life cycle of products and the repair system more holistically. We conclude with a future outlook on how temporal dimensions can inform policy strategies and future research.     

High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors

Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3–8.3-fold and 19–22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.     

Human Interindividual Variability in Cancer Risks—Technical and Management Challenges

Existing risk assessment procedures for carcinogens are intended to be “conservative” in the uncertainty dimension—giving estimates that are expected to be higher than true risks for typical people. However, these procedures do not consider the likely variability in susceptibility among individual people. This paper updates previous estimates of the likely extent of this variability for metabolically activated, genetically-acting carcinogens based on recent information on human interindividual variability in metabolic activation, detoxification, and DNA repair. The resulting expected skewness of cancer risk distributions is estimated using Monte Carlo simulations of both variability and uncertainty.

Some risk management implications are:

1. When evaluating the fairness of a particular risk distribution, managers need to gain familiarity with a three-dimensional characterization—X level of risk, for the Yth percentile individual (addressing variability) with Z degree of confidence (addressing uncertainty).

2. To the extent that variability distributions are skewed (e.g., with a long tail extending to high values) population mean risks will tend to exceed risks for median individuals. Together with the skewness in uncertainty distributions, this implies that “expected value” estimates of aggregate population risks—the estimates of interest for cost benefit analyses—are likely to be closer to traditional upper confidence limit risk estimates than has often been assumed in the past.

Characterization of a 160 kD Photosystem II reaction center complex isolated from photoinhibited Dunaliella salina thylakoids

Photoinhibition in the green alga Dunaliella salina is accompanied by the formation of inactive Photosystem II reaction centers. In SDS-PAGE analysis, the latter appear as 160 kD complexes. These complexes are structurally stable, enough to withstand re-electrophoresis of excised gel slices from the 160 kD region. Western blot analyses with specific polyclonal antibodies raised against the D1 or D2 reaction center proteins provided evidence for the presence of both of these polypeptides in the re-electrophoresed 160 kD complex. Incubation of excised gel slices from the 160 kD region, under aerobic conditions at 4°C for a prolonged period of time, caused a break-up of the 160 kD complex into a 52 kD D1-containing and 80 and 26 kD D2-containing pieces. Western blot analysis with polyclonal antibodies raised against the apoproteins of CPI (reaction center proteins of PS I) did not show cross-reaction either with the 160 kD complex or with the 52, 80 and 26 kD pieces. The results show the presence of both D1 and D2 in the 160 kD complex and strengthen the notion of a higher molecular weight D1- and D2-containing complex that forms upon disassembly of photodamaged PS II units.Abbreviations Chl chlorophyll - PS II Photosystem II - D1 the 32 kD reaction center protein of PS II, encoded by the chloroplast psbA gene - D2 the 34 kD reaction center protein of PS II, encoded by the chloroplast psbD gene - CPI the 82 and 83 kD reaction center proteins of PS I, encoded by the chloroplast psaA and psaB genes - HL high light - LL low light This publication is dedicated to the memory of the late Professor Daniel Arnon, whom the first author will fondly remember for his many accounts of past scientific discovery and debate.     

Characterization of a Chlamydomonas reinhardtii gene encoding a protein of the DNA photolyase/blue light photoreceptor family

The organization and nucleotide sequence of a gene from Chlamydomonas reinhardtii encoding a member of the DNA photolyase/blue light photoreceptor protein family is reported. A region of over 7 kb encompassing the gene was sequenced. Northern analysis detected a single 4.2 kb mRNA. The gene consists of eight exons and seven introns, and encodes a predicted protein of 867 amino acids. The first 500 amino acids exhibit significant homology with previously sequenced DNA photolyases, showing the closest relationship to mustard (Sinapis alba) photolyase (43% identity). An even higher identity, 49%, is obtained when the Chlamydomonas gene product is compared to the putative blue-light photoreceptor (HY4) from Arabidopsis thaliana. Both the Chlamydomonas and the Arabidopsis proteins differ from the well characterized DNA photolyases in that they contain a carboxyl terminal extension of 367 and 181 amino acids, respectively. However, there is very little homology between the carboxyl terminal domains of the two proteins. A previously isolated Chlamydomonas mutant, phrl, which is deficient in DNA photolyase activity, especially in the nucleus, was shown by RFLP analysis not to be linked to the gene we have isolated. We propose this gene encodes a candidate Chlamydomonas blue light photoreceptor.