Statistical discovery of site inter-dependencies in sub-molecular hierarchical protein structuring

Background

Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families.

Results

The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function.

Conclusions

Our results demonstrate that the method we present here using a k- modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family.

     

Head and shoulder posture affect scapular mechanics and muscle activity in overhead tasks

Forward head and rounded shoulder posture (FHRSP) is theorized to contribute to alterations in scapular kinematics and muscle activity leading to the development of shoulder pain. However, reported differences in scapular kinematics and muscle activity in those with forward head and rounded shoulder posture are confounded by the presence of shoulder pain. Therefore, the purpose of this study was to compare scapular kinematics and muscle activity in individuals free from shoulder pain, with and without FHRSP. Eighty volunteers were classified as having FHRSP or ideal posture. Scapular kinematics were collected concurrently with muscle activity from the upper and lower trapezius as well as the serratus anterior muscles during a loaded flexion and overhead reaching task using an electromagnetic tracking system and surface electromyography. Separate mixed model analyses of variance were used to compare three-dimensional scapular kinematics and muscle activity during the ascending phases of both tasks. Individuals with FHRSP displayed significantly greater scapular internal rotation with less serratus anterior activity, during both tasks as well as greater scapular upward rotation, anterior tilting during the flexion task when compared with the ideal posture group. These results provide support for the clinical hypothesis that FHRSP impacts shoulder mechanics independent of shoulder pain.     

Cell cycle phosphorylation of mitotic exit network (MEN) proteins

Phosphorylation of proteins is an important mechanism used to regulate most cellular processes. Recently, we completed an extensive phosphoproteomic analysis of the core proteins that constitute the Saccharomyces cerevisiae centrosome. Here, we present a study of phosphorylation sites found on the mitotic exit network (MEN) proteins, most of which are associated with the cytoplasmic face of the centrosome. We identified 55 sites on Bfa1, Cdc5, Cdc14 and Cdc15. Eight sites lie in cyclin-dependent kinase motifs (Cdk, S/T-P), and 22 sites are completely conserved within fungi. More than half of the sites were found in centrosomes from mitotic cells, possibly in preparation for their roles in mitotic exit. Finally, we report phosphorylation site information for other important cell cycle and regulatory proteins.Key words: in vivo phosphorylation, yeast centrosome, mitotic exit network (MEN), cell cycle, protein kinase, Cdk (cyclin-dependent kinase)/Cdc28, Plk1 (polo-like kinase)/Cdc5Reversible protein phosphorylation leads to changes in targeting, structure and stability of proteins and is used widely to modulate biochemical reactions in the cell. We are interested in phosphoregulation of centrosome duplication and function in the yeast Saccharomyces cerevisiae. Centrosomes nucleate microtubules and, upon duplication during the cell cycle, form the two poles of the bipolar mitotic spindle used to segregate replicated chromosomes into the two daughter cells. Timing and spatial cues are highly regulated to ensure that elongation of the mitotic spindle and separation of sister chromatids occur prior to progression into late telophase and initiation of mitotic exit. The mitotic exit network (MEN) regulates this timing through a complex signaling cascade activated at the centrosome that triggers the end of mitosis, ultimately through mitotic cyclin-dependent kinase (Cdk) inactivation (reviewed in ref. ¹).The major components of the MEN pathway (Fig. 1) are a Ras-like GTPase (Tem1), an activator (Lte1) with homology to nucleotide exchange factors, a GTPase-activating protein (GAP) complex (Bfa1/Bub2), several protein kinases [Cdc5 (Plk1 in humans), Cdc15 and Dbf2/Mob1] and Cdc14 phosphatase (reviewed in ref. ^2–5). Tem1 initiates the signal for the MEN pathway when switched to a GTP-active state. Prior to activation at anaphase, it is held at the centrosome in an inactive GDP-bound state by an inhibiting GAP complex, Bfa1/Bub2.⁶ The Bfa1/Bub2 complex and the inactive Tem1 are localized at the mother centrosome destined to move into the budded cell upon chromosome segregation, whereas the activator Lte1 is localized at the tip of the budded cell. These separate localizations ensure that Lte1 and Tem1 only interact in late anaphase, when the mitotic spindle elongates.^7,8 Lte1 has been thought to activate Tem1 as a nucleotide exchange factor, although more recent evidence suggests that it may instead affect Bfa1 localization.⁹ In addition, full activation of Tem1 is achieved through Cdc5 phosphorylation of the negative regulator Bfa1 ¹⁰ and potentially through phosphorylation of Lte1. GTP-bound Tem1 is then able to recruit Cdc15 to the centrosome, allowing for Dbf2 activation.³ The final step in the MEN pathway is release of Cdc14 from the nucleolus, which is at least partially due to phosphorylation by Dbf2¹¹ an leads to mitotic cyclin degradation and inactivation of the mitotic kinase.²Open in a separate windowFigure 1Schematic representation of the MEN proteins and pathway. MEN protein localization is shown within a metaphase cell when mitotic exit is inhibited and in a late anaphase cell when mitotic exit is initiated. Primary inhibition and activation events are described below the cells.Recently, we performed a large-scale analysis of phosphorylation sites on the 18 core yeast centrosomal proteins present in enriched centrosomal preparations.¹² In total, we mapped 297 sites on 17 of the 18 proteins and described their cell cycle regulation, levels of conservation and demonstrated defects in centrosome assembly and function resulting from mutating selected sites. MEN proteins were also identified in the centrosome preparations. This was expected, because Nud1, one of the 18 core centrosome components, is known to recruit several MEN proteins to the centrosome¹³ as part of its function in mitotic exit.^14,15 As phosphorylation is essential to several steps in the MEN pathway, beginning with recruitment of Bfa1/Bub2 by phosphorylated Nud1,¹⁵ we were interested in mapping in vivo phosphorylation sites on the MEN proteins associated with centrosomes and identifying when they occur during the cell cycle.We combined centrosome enrichment with mass spectrometry analysis to examine phosphorylation from asynchronously growing cells.¹² Centrosomes were also prepared from cells arrested in G₁ and mitosis¹² to monitor potentially cell cycle-regulated sites. We obtained significant coverage of a number of the MEN proteins, several of which have human homologs (​and33, column 1), of which eight sites lie within Cdk/Cdc28 motifs [S/T(P)], (23 Mob1 and Dbf2 are known phosphoproteins²⁴ for which we observed peptide coverage but no phosphorylation. Surprisingly, we did not detect phosphorylation on Bub2 despite the high peptide coverage; it is possible that the mitotic centrosome preparations (using a Cdc20 depletion protocol) affect the phosphorylation state of Bub2, as Bub2 is required for mitotic exit arrest in cdc20 mutants.²⁵ Additionally, specific phosphorylation sites have not been mapped on Bub2, suggesting that modifications on this protein may be difficult to observe by mass spectrometry. Lte1 does not localize to the centrosome, and we did not recover Lte1 peptides in our preparations. Many phosphorylation events on MEN proteins were observed in mitotic centrosomal preparations, most likely in preparation for their subsequent role in exit from mitosis (

A Mathematical Model of Force Generation by Flexible Kinetochore-Microtubule Attachments

Important mechanical events during mitosis are facilitated by the generation of force by chromosomal kinetochore sites that attach to dynamic microtubule tips. Several theoretical models have been proposed for how these sites generate force, and molecular diffusion of kinetochore components has been proposed as a key component that facilitates kinetochore function. However, these models do not explicitly take into account the recently observed flexibility of kinetochore components and variations in microtubule shape under load. In this paper, we develop a mathematical model for kinetochore-microtubule connections that directly incorporates these two important components, namely, flexible kinetochore binder elements, and the effects of tension load on the shape of shortening microtubule tips. We compare our results with existing biased diffusion models and explore the role of protein flexibility inforce generation at the kinetochore-microtubule junctions. Our model results suggest that kinetochore component flexibility and microtubule shape variation under load significantly diminish the need for high diffusivity (or weak specific binding) of kinetochore components; optimal kinetochore binder stiffness regimes are predicted by our model. Based on our model results, we suggest that the underlying principles of biased diffusion paradigm need to be reinterpreted.     

An activity-dependent assay for ricin and related RNA N-glycosidases based on electrochemiluminescence

Synthetic biotinylated RNA substrates were cleaved by the combined actions of ricin holotoxin and a chemical agent, N,N'-dimethylethylenediamine. The annealing of the product with a ruthenylated oligodeoxynucleotide resulted in the capture of ruthenium chelate onto magnetic beads, enabling the electrochemiluminescence (ECL)-based detection of RNA N-glycosidase activities of toxins. ECL immunoassays and the activity assay exhibited similar limits of detection just below signals with 0.1 ng/ml of ricin; the ECL response was linear as the ricin concentration increased by two orders of magnitude. Activities were detected with other adenine-specific RNA N-glycosidases, including Ricinus communis agglutinin (RCA), saporin, and abrin II. The substrate that provided the greatest sensitivity was composed of a four-residue loop, GdAGA, in a hairpin structure. When the 2'-deoxyadenosine (dA) was substituted with adenosine (A), 2'-deoxyinosine, or 2'-deoxyuridine, toxin-dependent signals were abolished. Placing the GdAGA motif in a six-residue loop or replacing it with GdAdGA or GdAAA resulted in measurable activities and signal patterns that were reproducible for a given toxin. Data indicated that saporin and abrin II shared one pattern, while ricin and RCA shared a distinct pattern. A monoclonal antibody that enhanced the activities of ricin, RCA, and abrin II to different extents, thus improving the diagnostic potential of the assay, was identified .     

An easy and reliable method for establishment and maintenance of leaf and root cell cultures ofArabidopsis thaliana

Cell suspension cultures are useful for a wide range of biochemical and physiological studies, yet their production can be technically demanding and often unreliable. Here we describe a protocol for producing Arabidopsis cell suspension cultures that is reliable and easy to use.     

Molecular characterization of malarial parasites in captive passerine birds

Seven of 28 passerine birds that died in captivity were positive for malarial parasites by polymerase chain reaction targeting the mitochondrial cytochrome b (cytB) and apicoplast ribosomal RNA (rRNA) genes. Each bird was infected with a single parasite lineage having a unique genotype. Apicoplast rRNA sequences were present both in Haemoproteus spp. and Plasmodium spp. and had typically high adenosine + thymidine content. Phylogenies for cytB and apicoplast rRNA sequences were largely congruent and supported previous studies that suggest that Plasmodium-Haemoproteus spp. underwent synchronous speciation with their avian hosts, interrupted by sporadic episodes of host switching. Apicoplast phylogeny further indicated that Haemoproteus spp. are ancestral to Plasmodium spp. All the 7 infected passerine birds had histologic lesions of malaria, and malarial parasites may have contributed to the death of at least 4 animals. These findings provide new genetic data on passerine hematozoa, including initial sequences of apicoplast DNA, and emphasize the relevance of parasite prevalence, evolutionary relationships, and host switching to modern management and husbandry practices of captive birds.     

Changing concepts in plant hormone action

Summary A plant hormone is not, in the classic animal sense, a chemical synthesized in one organ, transported to a second organ to exert a chemical action to control a physiological event. Any phytohormone can be synthesized everywhere and can influence different growth and development processes at different places. The concept of physiological activity under hormonal control cannot be dissociated from changes in concentrations at the site of action, from spatial differences and changes in the tissue's sensitivity to the compound, from its transport and its metabolism, from balances and interactions with the other phytohormones, or in their metabolic relationships, and in their signaling pathways as well. Secondary messengers are also involved. Hormonal involvement in physiological processes can appear through several distinct manifestations (as environmental sensors, homeostatic regulators and spatio-temporal synchronizers, resource allocators, biotime adjusters, etc.), dependent on or integrated with the primary biochemical pathways. The time has also passed for the hypothesized ‘specific’ developmental hormones, rhizocaline, canlocaline, and florigen: root, stem, and flower formation result from a sequential control of specific events at the right places through a coordinated control by electrical signals, the known phytohormones and nonspecific molecules of primary and secondary metabolism, and involve both cytoplasmic and apoplastic compartments. These contemporary views are examined in this review.     

Isoaspartate in ribosomal protein S11 of Escherichia coli

Isoaspartyl sites, in which an aspartic acid residue is linked to its C-flanking neighbor via its beta-carboxyl side chain, are generally assumed to be an abnormal modification arising as proteins age. The enzyme protein L-isoaspartate methyltransferase (PIMT), present in many bacteria, plants, and animals, catalyzes the conversion of isoaspartate to normal alpha-linked aspartyl bonds and is thought to serve an important repair function in cells. Having introduced a plasmid into Escherichia coli that allows high-level expression of rat PIMT, we explored the possibility that the rat enzyme reduces isoaspartate levels in E. coli proteins, a result predicted by the repair hypothesis. The present study demonstrates that this is indeed the case; E. coli cells expressing rat PIMT had significantly lower isoaspartate levels than control cells, especially in stationary phase. Moreover, the distribution of isoaspartate-containing proteins in E. coli differed dramatically between logarithmic- and stationary-phase cultures. In stationary-phase cells, a number of proteins in the molecular mass range of 66 to 14 kDa contained isoaspartate, whereas in logarithmic-phase cells, nearly all of the detectable isoaspartate resided in a single 14-kDa protein which we identified as ribosomal protein S11. The near stoichiometric levels of isoaspartate in S11, estimated at 0.5 mol of isoaspartate per mol of S11, suggests that this unusual modification may be important for S11 function.