Structured modelling of animal cells

Recent advances in computer technology have promoted the design and use of detailed, computer-based models for biological systems. For many non-biological systems, the complexity of such simulations may be considered inappropriate and unwieldy, but in biological systems, and more specifically in animal cell culture, this level of complexity simply mimics what is only beginning to be understood about metabolic processs. With this in mind, we contend that complex, structured models are vital tools in the investigation of fundamental biological processes. An example of such a simulation, which describes the commercial production of therapeutic proteins by animal cell cultures, is considered.     

Retained folates in the rat.

The retention of radioactivity after doses of 14C- and 3H-labelled folic acid is described. Radioactivity was retained in liver, kidney and gut of rats for some time after administration of the dose. The retained radioactivity could not be displaced by large doses of unlabelled folic acid or unlabelled 5-methyltetrahydrofolate. 14C- and 3H-labbelled folates showed similar chromatographic behaviour onion-exchange chromatography to 5-methyltetrahydrofolate, and on ion-exchange and gel-permeation chromatography to synthetic pteroylhepta-gamma-glutamate.     

Structural insights into anaphase-promoting complex function and mechanism

The anaphase-promoting complex or cyclosome (APC/C) controls sister chromatid segregation and the exit from mitosis by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. This unusually large E3 RING-cullin ubiquitin ligase is assembled from 13 different proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D box and KEN box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. Recent structural and biochemical studies of the APC/C are beginning to reveal an understanding of the roles of individual APC/C subunits and co-activators and how they mutually interact to mediate APC/C functions. This review focuses on the findings showing how information on the structural organization of the APC/C provides insights into the role of co-activators and core APC/C subunits in mediating substrate recognition. Mechanisms of regulating and modulating substrate recognition are discussed in the context of controlling the binding of the co-activator to the APC/C, and the accessibility and conformation of the co-activator when bound to the APC/C.     

Topological characteristics of helical repeat proteins.

The recent elucidation of protein structures based upon repeating amino acid motifs, including the armadillo motif, the HEAT motif and tetratricopeptide repeats, reveals that they belong to the class of helical repeat proteins. These proteins share the common property of being assembled from tandem repeats of an alpha-helical structural unit, creating extended superhelical structures that are ideally suited to create a protein recognition interface.     

Genetic dissection of Al tolerance QTLs in the maize genome by high density SNP scan

Background

Aluminum (Al) toxicity is an important limitation to food security in tropical and subtropical regions. High Al saturation on acid soils limits root development, reducing water and nutrient uptake. In addition to naturally occurring acid soils, agricultural practices may decrease soil pH, leading to yield losses due to Al toxicity. Elucidating the genetic and molecular mechanisms underlying maize Al tolerance is expected to accelerate the development of Al-tolerant cultivars.

Results

Five genomic regions were significantly associated with Al tolerance, using 54,455 SNP markers in a recombinant inbred line population derived from Cateto Al237. Candidate genes co-localized with Al tolerance QTLs were further investigated. Near-isogenic lines (NILs) developed for ZmMATE2 were as Al-sensitive as the recurrent line, indicating that this candidate gene was not responsible for the Al tolerance QTL on chromosome 5, qALT5. However, ZmNrat1, a maize homolog to OsNrat1, which encodes an Al³⁺ specific transporter previously implicated in rice Al tolerance, was mapped at ~40 Mbp from qALT5. We demonstrate for the first time that ZmNrat1 is preferentially expressed in maize root tips and is up-regulated by Al, similarly to OsNrat1 in rice, suggesting a role of this gene in maize Al tolerance. The strongest-effect QTL was mapped on chromosome 6 (qALT6), within a 0.5 Mbp region where three copies of the Al tolerance gene, ZmMATE1, were found in tandem configuration. qALT6 was shown to increase Al tolerance in maize; the qALT6-NILs carrying three copies of ZmMATE1 exhibited a two-fold increase in Al tolerance, and higher expression of ZmMATE1 compared to the Al sensitive recurrent parent. Interestingly, a new source of Al tolerance via ZmMATE1 was identified in a Brazilian elite line that showed high expression of ZmMATE1 but carries a single copy of ZmMATE1.

Conclusions

High ZmMATE1 expression, controlled either by three copies of the target gene or by an unknown molecular mechanism, is responsible for Al tolerance mediated by qALT6. As Al tolerant alleles at qALT6 are rare in maize, marker-assisted introgression of this QTL is an important strategy to improve maize adaptation to acid soils worldwide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-153) contains supplementary material, which is available to authorized users.     

Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect on the in vitro biological activity of IL-2. In addition soluble IL-2 receptor alpha and beta polypeptides do not compete with heparin for the binding of IL-2. IL-2 bound by heparin is still recognized by two IL-2 specific monoclonal antibodies, 3H9 and H2- 8, whose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin. Human IL-2 analogs with single amino acid substitutions at positions Lys43, Thr51, and Gln126 analogs no longer bind to heparin. By contrast the Arg38Ala analog retains heparin full heparin binding activity. These experimental findings together with molecular modeling studies suggest two putative heparin binding sites on human IL-2, one involving four basic residues, Lys48, Lys49, Lys54, and His55, and the other being a discontinuous site comprising Lys43, Lys64, Arg81, and Arg83. Neither of these two clusters is completely conserved in murine IL-2. Overall our data suggest that the binding of human IL-2 to heparin and heparan sulfate does not interfere with IL-2/IL-2 receptor interactions. Therefore, binding to glycosaminoglycan may be a mechanism for retaining the cytokine in an active form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.      

Differential oxidation of protein-tyrosine phosphatases

Oxidation is emerging as an important regulatory mechanism of protein-tyrosine phosphatases (PTPs). Here we report that PTPs are differentially oxidized, and we provide evidence for the underlying mechanism. The membrane-proximal RPTPalpha-D1 was catalytically active but not readily oxidized as assessed by immunoprobing with an antibody that recognized oxidized catalytic site cysteines in PTPs (oxPTPs). In contrast, the membrane-distal RPTPalpha-D2, a poor PTP, was readily oxidized. Oxidized catalytic site cysteines in PTP immunoprobing and mass spectrometry demonstrated that mutation of two residues in the Tyr(P) loop and the WPD loop that reverse catalytic activity of RPTPalpha-D1 and RPTPalpha-D2 also reversed oxidizability, suggesting that oxidizability and catalytic activity are coupled. However, catalytically active PTP1B and LAR-D1 were readily oxidized. Oxidizability was strongly dependent on pH, indicating that the microenvironment of the catalytic cysteine has an important role. Crystal structures of PTP domains demonstrated that the orientation of the absolutely conserved PTP loop arginine correlates with oxidizability of PTPs, and consistently, RPTPmu-D1, with a similar conformation as RPTPalpha-D1, was not readily oxidized. In conclusion, PTPs are differentially oxidized at physiological pH and H(2)O(2) concentrations, and the PTP loop arginine is an important determinant for susceptibility to oxidation.     

Crystal structure of the M-fragment of alpha-catenin: implications for modulation of cell adhesion.

The cytoskeletal protein alpha-catenin, which shares structural similarity with vinculin, is required for cadherin-mediated cell adhesion, and functions to modulate cell adhesive strength and to link the cadherins to the actin-based cytoskeleton. Here we describe the crystal structure of a region of alpha-catenin (residues 377-633) termed the M-fragment. The M-fragment is composed of a tandem repeat of two antiparallel four-helix bundles of virtually identical architectures that are related in structure to the dimerization domain of alpha-catenin and the tail region of vinculin. These results suggest that alpha-catenin is composed of repeating antiparallel helical domains. The region of alpha-catenin previously defined as an adhesion modulation domain corresponds to the C-terminal four-helix bundle of the M-fragment, and in the crystal lattice these domains exist as dimers. Evidence for dimerization of the M-fragment of alpha-catenin in solution was detected by chemical cross-linking experiments. The tendency of the adhesion modulation domain to form dimers may explain its biological activity of promoting cell-cell adhesiveness by inducing lateral dimerization of the associated cadherin molecule.