New frontiers in short-chain alkyl-coenzyme M reductases.

Exploring patterns and causes of intraspecific trait variation is crucial for a better understanding of the effects of climate change on plant populations and ecosystems. However, our current understanding of the intraspecific trait variation is mainly based on structural (morphological) traits, and we have limited knowledge on patterns and causes of variation in biochemical traits (e.g. leaf pigments), which are also crucial for plant adaptation. As a result, we also do not know how similar the climatic effects on structural versus biochemical traits are. Using plant traits from 110 genotypes representing 11 Festuca rubra populations grown in 4 different climates, we studied trait covariation among structural traits (linked to fitness, resource use, gas exchange and reproduction) and biochemical traits (linked to photosynthesis, photoprotection and oxidative stress). We also disentangled the relative role of the climate of origin and the climate of cultivation in the structural versus biochemical traits and tested for adaptive plasticity in the traits. We found that (1) biochemical traits did not covary with structural traits and represent independent ‘photoharvesting–photoprotection’ strategy dimension of functional variation; (2) interactive effects of climate of origin and cultivation were more pronounced for biochemical than structural traits. (3) Trait plasticity was affected by the climate of origin (precipitation and temperature as well as their interaction); (4) F. rubra showed both adaptive and maladaptive plasticity, and adaptiveness depended upon trait type, cultivation climate and climate of origin. Overall, our results suggest that structural and biochemical plant traits respond differentially to climate and thus the response of one group of traits cannot be predicted from the other. Responses are also strongly determined by interactions between the climate of origin and cultivation. Thus, more studies on variation in biochemical traits, their correspondence to other traits, and their variation with climate are needed. Read the free Plain Language Summary for this article on the Journal blog.

Recombinant protein expression: Challenges in production and folding related matters

RyR2 Gain-of-Function and Not So Sudden Cardiac Death

Cellular O-linked N-acetylglucosamine (O-GlcNAc) levels are modulated by two enzymes: uridine diphosphate-N-acetyl-D-glucosamine:polypeptidyltransferase (OGT) and O-GlcNAcase (OGA). To quantitatively address the activity of these enzymes on protein substrates, we generated five structurally diverse proteins in both unmodified and O-GlcNAc-modified states. We found a remarkably invariant upper limit for k(cat)/K(m) values for human OGA (hOGA)-catalyzed processing of these modified proteins, which suggests that hOGA processing is driven by the GlcNAc moiety and is independent of the protein. Human OGT (hOGT) activity ranged more widely, by up to 15-fold, suggesting that hOGT is the senior partner in fine tuning protein O-GlcNAc levels. This was supported by the observation that K(m,app) values for UDP-GlcNAc varied considerably (from 1 μM to over 20 μM), depending on the protein substrate, suggesting that some OGT substrates will be nutrient-responsive, whereas others are constitutively modified. The ratios of k(cat)/K(m) values obtained from hOGT and hOGA kinetic studies enable a prediction of the dynamic equilibrium position of O-GlcNAc levels that can be recapitulated in vitro and suggest the relative O-GlcNAc stoichiometries of target proteins in the absence of other factors. We show that changes in the specific activities of hOGT and hOGA measured in vitro on calcium/calmodulin-dependent kinase IV (CaMKIV) and its pseudophosphorylated form can account for previously reported changes in CaMKIV O-GlcNAc levels observed in cells. These studies provide kinetic evidence for the interplay between O-GlcNAc and phosphorylation on proteins and indicate that these effects can be mediated by changes in hOGT and hOGA kinetic activity.

Recombinant expression in the yeast Saccharomyces cerevisiae offers an alternative approach to developing large-scale production systems for class II bacteriocins from lactic acid bacteria, such as enterocin A, mundticin ST4SA and plantaricin 423. An important consideration for bacteriocin activity is disulphide bond formation: mature mundticin ST4SA has one, and plantaricin 423 and enterocin A each have two disulphide bonds. The native bacteriocin operon typically includes accessory proteins that facilitate disulphide bond formation, but this gene is absent in the enterocin A operon. In this study, the recombinant expression of a codon-optimised gene for enterocin A in S. cerevisiae, was compared to that for a codon-optimised plantaricin 423 and mundticin ST4SA, previously successfully expressed in S. cerevisiae. Shake flasks delivered more than twofold higher peptide EntA_Opt levels than PlaX_Opt and MunX_Opt, with even higher EntA_Opt expression levels in batch fermentations. However, the bacteriocin activity of plantaricin 423 was considerably lower than that of enterocin A and mundticin ST4SA. It is postulated that this could be a result of incorrect disulphide bond conformation due to the absence of the plantaricin accessory protein, PlaC, in S. cerevisiae. Nano-LC-MS/MS analysis showed various post-translational modifications for the peptides, with a greater proportion of EntA_Opt peptides with the correct disulphide bond conformation than for PlaX_Opt. This study demonstrated that S. cerevisiae is a promising host to produce recombinant class IIa bacteriocins, particularly enterocin A. However, the co-expression of accessory proteins should be investigated to improve the activity of recombinant plantaricin 423.

The bacterial species "Candidatus Alkanivorans nitratireducens" was recently demonstrated to mediate nitrate-dependent anaerobic oxidation of short-chain gaseous alkanes (SCGAs). In previous bioreactor enrichment studies, the species appeared to reduce nitrate in two phases, switching from denitrification to dissimilatory nitrate reduction to ammonium (DNRA) in response to nitrite accumulation. The regulation of this switch or the nature of potential syntrophic partnerships with other microorganisms remains unclear. Here, we describe anaerobic multispecies cultures of bacteria that couple the oxidation of propane and butane to nitrate reduction and the oxidation of ammonium (anammox). Batch tests with 15N-isotope labelling and multi-omic analyses collectively supported a syntrophic partnership between "Ca. A. nitratireducens" and anammox bacteria, with the former species mediating nitrate-driven oxidation of SCGAs, supplying the latter with nitrite for the oxidation of ammonium. The elimination of nitrite accumulation by the anammox substantially increased SCGA and nitrate consumption rates, whereas it suppressed DNRA. Removing ammonium supply led to its eventual production, the accumulation of nitrite, and the upregulation of DNRA gene expression for the abundant "Ca. A. nitratireducens". Increasing the supply of SCGA had a similar effect in promoting DNRA. Our results suggest that "Ca. A. nitratireducens" switches to DNRA to alleviate oxidative stress caused by nitrite accumulation, giving further insight into adaptability and ecology of this microorganism. Our findings also have important implications for the understanding of the fate of nitrogen and SCGAs in anaerobic environments.

Generally, spermatogenesis and sperm function involve widespread posttranslational modification of regulatory proteins in many different species. Nematode spermatogenesis has been studied in detail, mostly by genetic/molecular genetic techniques in the free-living Caenorhabditis elegans and by biochemistry/cell biology in the pig parasite Ascaris suum. Like other nematodes, both of these species produce sperm that use a form of amoeboid motility termed crawling, and many aspects of spermatogenesis are likely to be similar in both species. Consequently, work in these two nematode species has been largely complementary. Work in C. elegans has identified a number of spermatogenesis-defective genes and, so far, 12 encode enzymes that are implicated as catalysts of posttranslational protein modification. Crawling motility involves extension of a single pseudopod and this process is powered by a unique cytoskeleton composed of Major Sperm Protein (MSP) and accessory proteins, instead of the more widely observed actin. In Ascaris, pseudopod extension and crawling motility can be reconstituted in vitro, and biochemical studies have begun to reveal how posttranslational protein modifications, including phosphorylation, dephosphorylation and proteolysis, participate in these processes.

Obtaining diffraction quality crystals is frequently an iterative process which traditionally has involved screening large numbers of crystallization conditions. Due to advances in high-throughput gene engineering, recombinant expression, and purification, the protein of interest has now become one of the many variables routinely investigated during crystallization trials. As such, construct design is a critical step in the path toward successful crystallization. In this chapter will we address construct design strategies frequently employed to improve the solution and crystallization behavior of proteins. Topics covered include choosing a recombinant expression system and reducing disorder through truncations and surface mutagenesis. Also covered are strategies to reduce heterogeneity from posttranslational modifications, impurities, and aggregation.

Laccases catalyse the oxidation of a range of organic substrates coupled to the reduction of molecular oxygen to water. They are members of the ubiquitous blue multi-copper oxidase family. These enzymes are implicated in a wide variety of biological activities. Most of the laccases studied thus far are of fungal origin. Large variety of potential substrates has raised interest in the use of laccases in several industrial applications, such as pulp delignification, textile dye bleaching, effluent detoxification, biopolymer modification and bioremediation. Cloning of the laccase genes followed by heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (different substrate specificities and improved stabilities) for industrial applications. Heterologous expression of Pleurotus ostreatus laccases POXC and POXA1b in two yeasts and a first approach of directed evolution experiments are reported. The yeasts of choice were Saccharomyces cerevisiae, proven to be success-full in recombinant laccase expression and directed-evolution experiments, and Kluyveromyces lactis, a non-conventional yeast offering significant advantages, such as high-level secretion of non-hyperglycosylated recombinant proteins. Expression vectors were set up cloning the cDNAs under the control of different promoters. Furthermore, the laccase leader peptides (poxc and poxa1b), as well as the yeast derived signal peptides (S. cerevisiae invertase and K. lactis killer toxin), were alternatively used to direct the secretion of active laccase into the culture medium. The laccase signals proved to be more effective to drive the secretion of recombinant proteins in both hosts. Levels of laccase secreted activity were markedly different: rPOXA1b transformants always gave much higher activity than rPOXC transformants, and production of both laccases in S. cerevisiae was significantly lower than that in K. lactis. Recombinant laccases from K. lactis were purified to electrophoretic homogeneity and characterized. rPOXA1b specific activity was similar to that of the native protein, whilst rPOXC specific activity was much lower than that of the native POXC. Mass spectrometry analyses of the recombinant proteins allowed to verify their primary structures and to identify post-translational modifications. Our data confirm that K. lactis has a lower tendency, respect to S. cerevisiae, to hyperglycosylate recombinant proteins. The S. cerevisiae laccase expression systems were further used to set off directed evolution experiments. Mutated cDNAs libraries with different mutation rate were created, and homologous recombination experiments were performed, giving rise to libraries of mutated laccase secreting yeasts. Moreover a screening procedure to isolate clones exhibiting desired property was realized. As a result, this work allowed obtaining the heterologous expression of two P. ostreatus laccases in yeasts, and their purification and characterisation. Moreover, this research work broadened the potentiality of the developed expression system addressing enzymes to such large markets and different industrial application such as pulp and textile bleaching, and enzymatic remediation of waste streams. A new laccase host (K. lactis) has been built on, and its promising performances will lead to further investigate its utilization for further structure-activities studies, as well as for directed evolution. Results obtained demonstrate the potential of the recombinant expression for the study of potential industrial interest.

The short-chain gaseous alkanes (ethane, propane, and butane; SCGAs) are important components of natural gas, yet their fate in environmental systems is poorly understood. Microbially mediated anaerobic oxidation of SCGAs coupled to nitrate reduction has been demonstrated for propane, but is yet to be shown for ethane or butane-despite being energetically feasible. Here we report two independent bacterial enrichments performing anaerobic ethane and butane oxidation, respectively, coupled to nitrate reduction to dinitrogen gas and ammonium. Isotopic 13C- and 15N-labelling experiments, mass and electron balance tests, and metabolite and meta-omics analyses collectively reveal that the recently described propane-oxidizing "Candidatus Alkanivorans nitratireducens" was also responsible for nitrate-dependent anaerobic oxidation of the SCGAs in both these enrichments. The complete genome of this species encodes alkylsuccinate synthase genes for the activation of ethane/butane via fumarate addition. Further substrate range tests confirm that "Ca. A. nitratireducens" is metabolically versatile, being able to degrade ethane, propane, and butane under anoxic conditions. Moreover, our study proves nitrate as an additional electron sink for ethane and butane in anaerobic environments, and for the first time demonstrates the use of the fumarate addition pathway in anaerobic ethane oxidation. These findings contribute to our understanding of microbial metabolism of SCGAs in anaerobic environments.

Anaerobic microorganisms are thought to play a critical role in regulating the flux of short-chain gaseous alkanes (SCGAs; including ethane, propane and butane) from terrestrial and aquatic ecosystems to the atmosphere. Sulfate has been confirmed to act as electron acceptor supporting microbial anaerobic oxidation of SCGAs, yet several other energetically more favourable acceptors co-exist with these gases in anaerobic environments. Here, we show that a bioreactor seeded with biomass from a wastewater treatment facility can perform anaerobic propane oxidation coupled to nitrate reduction to dinitrogen gas and ammonium. The bioreactor was operated for more than 1000 days, and we used 13C- and 15N-labelling experiments, metagenomic, metatranscriptomic, metaproteomic and metabolite analyses to characterize the microbial community and the metabolic processes. The data collectively suggest that a species representing a novel order within the bacterial class Symbiobacteriia is responsible for the observed nitrate-dependent propane oxidation. The closed genome of this organism, which we designate as ‘Candidatus Alkanivorans nitratireducens’, encodes pathways for oxidation of propane to CO2 via fumarate addition, and for nitrate reduction, with all the key genes expressed during nitrate-dependent propane oxidation. Our results suggest that nitrate is a relevant electron sink for SCGA oxidation in anaerobic environments, constituting a new microbially-mediated link between the carbon and nitrogen cycles.

The mechanisms by which human immunodeficiency virus (HIV) circumvents and coopts cellular machinery to replicate and persist in cells are not fully understood. HIV accessory proteins play key roles in the HIV life cycle by altering host pathways that are often dependent on post-translational modifications (PTMs). Thus, the identification of HIV accessory protein host targets and their PTM status is critical to fully understand how HIV invades, avoids detection and replicates to spread infection. To date, a comprehensive characterization of HIV accessory protein host targets and modulation of their PTM status does not exist. The significant gap in knowledge regarding the identity and PTMs of HIV host targets is due, in part, to technological limitations. Here, we applied current mass spectrometry techniques to define mechanisms of viral protein action by identifying host proteins whose abundance is affected by the accessory protein Vpr and the corresponding modulation of down-stream signaling pathways, specifically those regulated by phosphorylation. By utilizing a novel, inducible HIV-1 CD4+ T-cell model system expressing either the wild type or a vpr-negative viral genome, we overcame challenges associated with synchronization and infection-levels present in other models. We report identification and abundance dynamics of over 7000 proteins and 28,000 phospho-peptides. Consistent with Vpr's ability to impair cell-cycle progression, we observed Vpr-mediated modulation of spindle and centromere proteins, as well as Aurora kinase A and cyclin-dependent kinase 4 (CDK4). Unexpectedly, we observed evidence of Vpr-mediated modulation of the activity of serine/arginine-rich protein-specific kinases (SRPKs), suggesting a possible role for Vpr in the regulation of RNA splicing. This study presents a new experimental system and provides a data-resource that lays the foundation for validating host proteins and phosphorylation-pathways affected by HIV-1 and its accessory protein Vpr.

Gibberellic acid induces photosynthetic tissues with non-Kranz anatomy and C4-like biochemical traits in terrestrial-form plants of Eleocharis vivipara. This suggests that the structural and biochemical traits are independently regulated. The amphibious leafless sedge, Eleocharis vivipara Link, develops culms (photosynthetic organs) with C4-like traits and Kranz anatomy under terrestrial conditions, and C3 traits and non-Kranz anatomy under submerged conditions. The conversion from C3 mode to C4-like mode in E. vivipara is reportedly mediated by abscisic acid. Here, we investigated the effects of gibberellic acid (GA) on the differentiation of anatomical and photosynthetic traits because GA is involved in heterophylly in aquatic plants. When 100µM GA was sprayed on terrestrial plants, the newly developed culms had non-Kranz anatomy in the basal part and Kranz-like anatomy in the upper part. In the basal part, the mesophyll cells were well developed, whereas the Kranz (bundle sheath) cells were reduced and contained few chloroplasts and mitochondria. Stomatal frequency was lower in the basal part than in the upper part. Nevertheless, these tissues had abundant accumulation and high activities of C4 photosynthetic enzymes and had C4-like δ13C values, as seen in the culms of the terrestrial form. When submerged plants were grown under water containing GA-biosynthesis inhibitors (uniconazole or paclobutrazol), the new culms had Kranz anatomy. The culms developed under paclobutrazol had the C3 pattern of cellular accumulation of photosynthetic enzymes. These data suggest that GA induces production of photosynthetic tissues with non-Kranz anatomy in terrestrial plants of E. vivipara, without concomitant expression of C3 biochemical traits. The data also suggest that the differentiation of C4 structural and biochemical traits is regulated independently.

Post-translational modifications (PTMs) refer to the covalent modifications of polypeptides after they are synthesized, adding temporal and spatial regulation to modulate protein functions. Being obligate intracellular parasites, viruses rely on the protein synthesis machinery of host cells to support replication, and not surprisingly, many viral proteins are subjected to PTMs. Coronavirus (CoV) is a group of enveloped RNA viruses causing diseases in both human and animals. Many CoV proteins are modified by PTMs, including glycosylation and palmitoylation of the spike and envelope protein, N- or O-linked glycosylation of the membrane protein, phosphorylation and ADP-ribosylation of the nucleocapsid protein, and other PTMs on nonstructural and accessory proteins. In this review, we summarize the current knowledge on PTMs of CoV proteins, with an emphasis on their impact on viral replication and pathogenesis. The ability of some CoV proteins to interfere with PTMs of host proteins will also be discussed.

Methyl‐coenzyme M reductase (MCR) is the key rate‐determining enzyme of methanogenesis as well as the anaerobic oxidation of methane, the essential energy metabolisms of methanogenic archaea and anaerobic methanotrophs (ANME), respectively. MCR is a dimer of heterotrimers with a 2α, 2β, 2γ configuration, and requires the nickel tetrapyrrole prosthetic group, coenzyme F430. The requirement of a unique cofactor, various unusual post translational modifications, and many remaining questions surrounding assembly and activation of MCR has so far largely limited in vitro experiments to native enzymes. To allow further investigation into the catalytic properties and mechanistic aspects of different MCRs, as well as facilitate the development of optimized biocatalytic systems to convert methane to more usable liquid fuels and other valuable compounds, we are developing methods for the heterologous expression of recombinant MCRs in Methanococcus maripaludis, a model methanogen with robust genetic tools.In methanogens, MCR is encoded in the highly conserved MCR gene cluster mcrBDCGA, which encodes two accessory proteins (McrD and McrC) in addition to the MCR‐encoding genes (McrA, McrB, and McrG). The accessory proteins are proposed to be involved in the assembly and activation of MCR. Interestingly, most ANME lack one or more accessory proteins in their MCR gene clusters. We have created a series of MCR expression constructs containing the MCR operons from several ANME organisms as well as several methanogens, with and without accessory protein(s). All constructs contain a his‐tag on the C‐terminus of McrA which allows the purification and determination of proper assembly. We have successfully purified a recombinant ANME‐2d MCR that is assembled and binds F430, whereas in the case of the ANME‐1 MCR, we can only recover the his‐tagged McrA. Since the ANME‐2d MCR operon contains McrD and the ANME‐1 operon does not, this result supports the importance of McrD for proper assembly in vivo. Combined with other results from the expression of recombinant methanogenic MCRs, our results indicate that MCR accessory proteins are organism specific. Current work is focused on demonstrating the role of McrD through in vitro binding studies as well as further elucidating the importance of mcrDand mcrC for recombinant expression of diverse MCRs.