Tolloid cleavage activates latent GDF8 by priming the pro‐complex for dissociation

Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF‐β family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid‐cleaved GDF8 pro‐complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro‐complexes reveals a V‐shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring‐like, cross‐armed conformation of latent TGF‐β1. Surprisingly, Tolloid‐cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen–deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain–growth factor dissociation, increased exchange in regions that correspond in pro‐TGF‐β1 to the α1‐helix, latency lasso, and β1‐strand in the prodomain and to the β6′‐ and β7′‐strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain–growth factor interfaces and primes the growth factor for release from the prodomain.     

Investigation of the dithiocarbamate-induced coupling of allylidene and carbonyl ligands on a tungsten center

Reaction of the allylidene tungsten complex [W(CPhCHCHMe)Br₂(CO)₂(4-picoline)] (1) with the dithiocarbamates MS₂CNR₂ (a: M=Na, R=Et; b: M=Na, R=Me; c: M=Li, R=Ph) in THF at 50 °C affords the vinylketene tungsten complexes [W(S₂CNR₂)₂(OCCPhCHCHMe)(CO)] (2a–c). At lower temperatures, four reaction intermediates (3–6) may be discerned. Spectroscopic studies indicate that these compounds contain η⁴-allyldithiocarbamate ligands which are generated by addition of dithiocarbamate across the metal-carbon double bond of the allylidene-tungsten unit in 1. The structures of [W(S₂CNEt₂)₂(OCCPhCHCHMe)(CO)] (2a) and of one intermediate, [W(η⁴-Et₂NCS₂CPhCHCHMe)(S₂CNEt₂)(CO)₂] (5a) were elucidated by X-ray crystallography.     

Stochastic population dynamics and life-history variation in marine fish species

Abstract We examined whether differences in life-history characteristics can explain interspecific variation in stochastic population dynamics in nine marine fish species living in the Barents Sea system. After observation errors in population estimates were accounted for, temporal variability in natural mortality rate, annual recruitment, and population growth rate was negatively related to generation time. Mean natural mortality rate, annual recruitment, and population growth rate were lower in long-lived species than in short-lived species. Thus, important species-specific characteristics of the population dynamics were related to the species position along the slow-fast continuum of life-history variation. These relationships were further associated with interspecific differences in ecology: species at the fast end were mainly pelagic, with short generation times and high natural mortality, annual recruitment, and population growth rates, and also showed high temporal variability in those demographic traits. In contrast, species at the slow end were long-lived, deepwater species with low rates and reduced temporal variability in the same demographic traits. These interspecific relationships show that the life-history characteristics of a species can predict basic features of interspecific variation in population dynamical characteristics of marine fish, which should have implications for the choice of harvest strategy to facilitate sustainable yields.     

Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding

Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin γ1 (IgG1) antibody. IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs both in vivo and in vitro. For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response. We investigated the impact of differential galactosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the FcγRIIIa receptor. The results showed that methionine oxidation and galactosylation both impact IgG1 conformation, whereas fucosylation appears to have little or no impact to the conformation. FcγRIIIa binding was strongly influenced by both the glycan structure/composition (namely galactose and fucose) and conformational changes that were induced by some of the modifications.The structure of many proteins can be altered by post-translational modifications (1). Although the impact of post-translational modifications (PTMs)¹ on protein structure is more understood for some modifications (e.g. phosphorylation; see Ref. 2), it is less defined for other PTMs and in many cases is protein-dependent. Because there are many important downstream effects of PTMs, including changes in protein localization, protein and cellular diversification, protein functionality, protein stability, protein life cycle, and so forth, understanding how PTMs alter protein structure for as many proteins as possible in a timely manner is a highly desirable goal. Furthermore, in an age where recombinant proteins are being used to treat disease, it becomes ever more important to understand how particular modifications may alter the structure and eventually the function of therapeutic proteins. To realize these goals, methods that permit access to conformational information for modified forms of therapeutic proteins must be developed and refined. In this report, we will illustrate how MS can contribute to structural proteomics by describing our recent work with a recombinant monoclonal antibody (an IgG1), which represents an important class of therapeutic proteins.Many biopharmaceutical companies are pursuing antibody drugs (3). In particular, the IgG1 subclass of antibodies has evolved into a commonly used therapeutic option for the treatment of a wide range of diseases. IgG1s consist of a dimer of identical heavy chains and light chains that fold to form (from N to C terminus) the variable, CL, CH1, CH2, and CH3 domains (as an example, see Ref. 4). Individual domains are structurally stable and are primarily composed of antiparallel β-sheets arranged in an immunoglobulin-like β-sandwich (5). The variable, CL, and CH1 domains are collectively referred to as the Fab (fragment antigen binding) portion of IgG1, which is responsible for recognizing a specific antigen. The CH2 and CH3 domains together are referred to as the Fc (fragment crystallizable) portion, which carries out effector functions such as binding to Fcγ receptors. These effector functions are essential to many therapeutic antibodies, especially when antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity are involved in the mechanisms of action (6).As a biopharmaceutical, IgG1 monoclonal antibodies are critically monitored throughout production (7). In many cases, the impact of structural modifications in these and other formulated versions of biopharmaceuticals are not well understood at a functional level. In the case of IgG1s, with over 1300 amino acid residues and a molecular mass approaching 150 kDa, a large array of PTMs can be incorporated both in vivo (during cellular synthesis) and in vitro (as a result of handling and processing steps that occur during purification, vialing, and storage). Commonly monitored PTMs on IgG1s include methionine oxidation, asparagine and glutamine deamidation, N-terminal acetylation or cyclization, glycation of lysine, and variable glycosylation (8). Some of these modifications affect only a small percentage of the protein product, and their presence may not change overall outcome. Others, however, can have significant impact on the structure, function, and biological activities of a protein that can involve self-association as well as interactions with other proteins (9). The same PTMs can affect different IgG1 molecules in different ways or have no effect(s) at all. Therefore assessing the presence of PTMs, determining the relative level of the modifications, and understanding the structural effects of PTMs are all important during development of protein biopharmaceuticals.Two commonly studied IgG1 modifications are methionine oxidation and glycosylation, each of which has been shown to affect biological function (6, 10). Methionine oxidation has been implicated in protein stability (inducing aggregation), and increased oxidation levels have been shown to provoke an immunogenic response (11–13). Elevated levels of methionine oxidation in an IgG1 were shown to impact neonatal Fc receptor (FcRn) and protein A binding (10). Variable glycosylation (i.e. different levels of sialic acid, galactose, fucose, or high mannose structures) is known to influence thermal stability and effector functions (14–16). Previous studies have shown that removal of fucose from the glycan present on the Fc portion of an IgG1 can greatly enhance Fc binding to FcγRIIIa, but removal of the entire glycan nearly abolishes FcγRIIIa binding (17). As oxidation and changes to the glycan are both common IgG1 modifications, we were interested in determining the conformational effects of oxidation, afucosylation, and galactosylation and correlating any conformational changes that were observed with changes of FcγRIIIa binding activity.Conformational analysis of large proteins like antibodies, however, is not trivial. Traditional biophysical techniques such as circular dichroism, DSC, and fluorescence provide useful information, but these techniques look at the entire protein and provide only a global view (18). NMR and x-ray crystallography can both provide high resolution structural analysis, but each is faced with limitations that often make the study of an intact IgG1 difficult or nearly impossible (19–21). Recently we described how hydrogen/deuterium exchange (H/DX) MS could be used to study the conformation and conformational dynamics of an intact IgG1 with resolution down to stretches of several amino acid residues (22). For the present work, we used H/DX MS to study the impact of galactosylation, oxidation, and afucosylation on the conformation and dynamics of an intact IgG1. We also studied the complex of IgG1 and FcγRIIIa to map the points of interaction and probe any changes in the dynamics of the IgG1 as a result of FcγRIIIa interaction. Finally, we correlated the functional activity of all the proteins that were studied by H/DX MS with the observed conformational disturbance(s). Such correlations are important to connect structure with function and to understand whether a particular PTM is something that may affect the therapeutic value of a recombinant protein.     

The ability to form full-length intron RNA circles is a general property of nuclear group I introns

In addition to splicing, group I intron RNA is capable of an alternative two-step processing pathway that results in the formation of full-length intron circular RNA. The circularization pathway is initiated by hydrolytic cleavage at the 3′ splice site and followed by a transesterification reaction in which the intron terminal guanosine attacks the 5′ splice site presented in a structure analogous to that of the first step of splicing. The products of the reactions are full-length circular intron and unligated exons. For this reason, the circularization reaction is to the benefit of the intron at the expense of the host. The circularization pathway has distinct structural requirements that differ from those of splicing and appears to be specifically suppressed in vivo. The ability to form full-length circles is found in all types of nuclear group I introns, including those from the Tetrahymena ribosomal DNA. The biological function of the full-length circles is not known, but the fact that the circles contain the entire genetic information of the intron suggests a role in intron mobility.     

The ecology of ruffe,Gymnocephalus cernuus (Pisces: Percidae) introduced to Mildevatn,western Norway

Synopsis Diet, habitat use, diel and seasonal activity and a number of population parameters were studied on ruffe,Gymnocephalus cernuus, introduced to Mildevatn, western Norway. This lake is sited outside the natural range of the ruffe and has a lower fish diversity and a different fish species composition than within its native range. From June through September the ruffe was planktivorous and mainly caught at 4 to 6 m depth in the benthic zone. At other times of year ruffe was feeding on zoobenthos and caught deeper in the benthic zone. Ruffe was mainly day active. Zooplankton feeding during summer is the clearest difference compared to ruffe populations living within its natural range. Presence of large zooplankton organisms available for ruffe is suggested as the main reason for the difference found in food choice. The availability of large zooplankton is probably due to community structure caused by a predator and lack of interspecific competition for zooplankton in the deeper parts of the lake. Piscivorous brown trout.Salmo trutta, restrict the habitat of threespined stickleback,Gasterosteus aculeatus, to the zone of littoral vegetation, allowing high densities of larger zooplankton species likeBythotrephes longimanus to be present in the lake. Brown trout is present only in the upper light and well oxygenated parts of the lake, leaving a refuge for the ruffe, where they can feed on the rich zooplankton community.     

Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases

Bruton’s tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.     

Genetic diversity in a germplasm collection of roseroot (Rhodiola rosea) in Norway studied by AFLP

Rhodiola rosea is widely distributed in Norway, but so far limited knowledge exists on the level of genetic diversity. To initiate a selective breeding program, Amplified Fragment Length Polymorphism (AFLP) analysis was used to estimate genetic diversity within the Norwegian R. rosea germplasm collection. AFLP analysis of 97 R. rosea clones using five primer combinations gave a total of 109 polymorphic bands. We detected high percentage of polymorphic bands (PPB) with a mean of 82.3% among the clones of R. rosea. Each of the 97 R. rosea clones could be unambiguously identified based on these primer combinations. Estimates of genetic similarities were obtained by the Dice coefficient, and a final dendrogram was constructed with the Unweighted Pair Group Method with Arithmetic mean (UPGMA). Genetic similarity based on the AFLP data ranged from 0.440 to 0.950 with a mean of 0.631. This genetic analysis showed that there was no close genetic similarity among clones related to their original growing county. No gender-specific markers were found in the R. rosea clones. Analysis of molecular variance (AMOVA) revealed a significantly greater variation within regions (92.03%) than among regions (7.97%). A low level of genetic differentiation (F_ST = 0.043) was observed, indicating a high level of gene flow, which had a strong influence on the genetic structure at different counties. Our results indicate high gene flow among R. rosea clones that might be a result of seed dispersal rather than cross-pollination. Further world-wide studies are required to compare the level of genetic diversity and more studies in R. rosea detailing the consequences of different patterns of gene flow (pollen spread and dispersal of seeds and clonal plants) will be useful for characterization of roseroot.     

Ha-ras-1 alleles in Norwegian lung cancer patients

Summary We have examined DNA restriction fragment length polymorphisms (RFLP) of the Ha-ras-1 gene in DNA from 118 lung cancer patients and 123 unaffected controls. When DNA samples were digested with MspI/ HpaII restriction endonucleases. Southern blot analysis demonstrated 4 common, 4 intermediate and 7 different rare alleles in the combined population after hybridization to the pGDa1 probe. Six of the rare alleles were unique for the lung cancer group and 1 rare allele for the control group. The frequency of rare alleles in lung cancer patients (10/236) was significantly different (P<0.01) from the control group (1/246). The lung cancer group also had a significantly lower frequency of the common 2.57 kb fragment than the controls (P<0.02). The results thus indicate that Ha-ras genotyping may be of value in lung cancer risk assessment.