Engineering and structural characterization of a linear polyubiquitin-specific antibody

Polyubiquitination is an essential posttranslational modification that plays critical roles in cellular signaling. PolyUb (polyubiquitin) chains are formed by linking the carboxyl-terminus of one Ub (ubiquitin) subunit to either a lysine residue or the amino-terminus of an adjacent Ub. Linkage through the amino-terminus results in linear polyubiquitination that has recently been demonstrated to be a key step in nuclear factor κB activation; however, tools to study linear chains have been lacking. We therefore engineered a linear-linkage-specific antibody that is functional in Western blot, immunoprecipitation, and immunofluorescence applications. A crystal structure of the linear-linkage-specific antibody Fab fragment in complex with linear diubiquitin provides molecular insight into the nature of linear chain specificity. We use the antibody to demonstrate that linear polyUb is up-regulated upon tumor necrosis factor α stimulation of cells, consistent with a critical role in nuclear factor κB signaling. This antibody provides an essential tool for further investigation of the function of linear chains.     

Activation of the proapoptotic death receptor DR5 by oligomeric peptide and antibody agonists

The cell-extrinsic apoptotic pathway triggers programmed cell death in response to certain ligands that bind to cell-surface death receptors. Apoptosis is essential for normal development and homeostasis in metazoans, and furthermore, selective activation of the cell-extrinsic pathway in tumor cells holds considerable promise for cancer therapy. We used phage display to identify peptides and synthetic antibodies that specifically bind to the human proapoptotic death receptor DR5. Despite great differences in overall size and structure, the DR5-binding peptides and antibodies shared a tripeptide motif, which was conserved within a disulfide-constrained loop of the peptides and the third complementarity determining region of the antibody heavy chains. The X-ray crystal structure of an antibody in complex with DR5 revealed that the tripeptide motif is buried at the core of the interface, confirming its central role in antigen recognition. We found that certain peptides and antibodies exhibited potent proapoptotic activity against DR5-expressing SK-MES-1 lung carcinoma cells. These phage-derived ligands may be useful for elucidating DR5 activation at the molecular level and for creating synthetic agonists of proapoptotic death receptors.     

Effect of soman on schedule-controlled behavior and brain acetylcholinesterase in rats

Rats were trained to press a lever under a multiple fixed-ratio 25 fixed-interval 50-second (FR25 FI50-sec) schedule of food reinforcement. Soman, 70-90 micrograms/kg, s.c., suppressed response rates in both components, with a slightly greater effect in the FI schedule. The pattern of responding under the FI schedule, however, was maintained until lever-pressing was nearly completely suppressed. At the highest doses, soman occasionally caused tremors or mild tonic seizures with hindlimb abduction. The suppression of response rate was correlated with inhibition of acetylcholinesterase (AChE) in all brain regions examined: cortex, striatum, hippocampus, hypothalamus and brainstem. Cortical AChE was inhibited to the highest degree, while striatal AChE was most resistant to inhibition by soman.     

Soybean seed protein electrophoresis profiles from 15 Asian countries or regions: Hypotheses on paths of dissemination of soybeans from China

Soybean [Glycine max (L.) Merr.] seed protein extracts from 1,603 accessions obtained from 15 Asian countries or regions (not including Japan) were analyzed for the presence of alleles of 2 proteins. Three alleles of the Kunitz. trypsin inhibitor orSBTI-A ₂ designated asTi ^a,Ti ^b andTi ^c are electrophoretically distinguishable from one another by their different Rf values of 0.79, 0.75 and 0.83, respectively. The Sp₁ seed protein or β-amylase has 2 alleles designatedSp1 ^a andSp ₁ ^b which are electrophoretically distinguishable from one another by their Rf values 0.36 and 0.42. About 94 percent of the soybean accessions had theTi ^a allele. Two accessions from Korea,P.I. 157440 andP.I. 196168, do not have theSBTI-A2 protein(ti). Two accessions, one from Pakistan and the other from Korea, were identified as having theTi ^c allele. Only the Korean and central Indian soybean populations have a high frequency for theTi ^b allele. Within Korea, the soybeans from those districts that lie closest to Japan have a high frequency for theTi ^b allele whereas the soybeans from those districts that lie closest to China have a low frequency for theTi ^b allele. TheTi ^b allele is not present in soybeans from the Philippines, Vietnam, Thailand, Malaysia, Burma, Nepal, Pakistan, and Afghanistan. Only 1 accession each from Taiwan and Indonesia have theTi ^b allele. TheSp ₁ ^a allele is not present in soybeans from Taiwan, Vietnam, Thailand, Malaysia, Indonesia, Burma, Pakistan and Afghanistan. The highest frequency for theSp1 ^a allele occurs in soybean germ plasm from northern India and Nepal. The soybeans from Asia (including Japan) were divided into 3 gene centers— primary, secondary, and tertiary—containing 7 germ plasm pools. Paths of dissemination of the soybean from China to the rest of Asia were developed based upon a combination of electrophoretic data and available historical, agronomic, and biogeographical literature.     

The fine structure of chickpea (Cicer arietinum L.) chromosomes as revealed by pachytene analysis

A standard pachytene karyotype of chickpea (Cicer arietinum L.) is presented for the first time. Individual pachytene chromosomes were identified and described in detail. An idiogram was prepared on the basis of chromosome length, arm ratio, and distribution of heterochromatin and euchromatin. Chickpea pachytene chromosomes belong to the differentiated type with darker staining heterochromatin proximal to and lighter staining euchromatin distal to the centromeres. Chromosomes were numbered from 1 to 8 following a descending order of length. The total length of the chromosome complement at pachytene was 335.33 , and chromosome size ranged from 58.05 to 30.53 .     

Cytological characterization of transgenic soybean

 Some of the transgenic soybean [Glycine max (L.) Merr.] plants produced by bombarding embryogenic suspension cultures with DNA-coated particles exhibit morphological aberrations, including stunted plant growth, leathery dark green leaves and partialto-total seed sterility. In general, cultures from two Asgrow soybean lines (A2242, A2872) that were maintained for 8 months or longer produced primary transformants with reduced fertility. Cytological examination (mitotic pro-metaphase to metaphase chromosomes) of cells of suspension cultures, of roots from germinating somatic embryos, and of plants (R₀ and R₁) derived from A2242, revealed, besides diploidy (2n=40), various chromosomal aberrations such as deletions, duplications, trisomics and tetraploidy. Diploid transgenic plants with a normal karyotype from A2242 generally exhibited good fertility. No chromosomal abnormalities were observed in A2872-derived plants. However, plants regenerated from relatively old cultures of A2872 (more than 1 year in culture) showed a range of phenotypic abnormalities although they all contained 2n=40 chromosomes. These results indicate that soybean genotypes differ in their susceptibility to chromosomal instability induced by tissue culture. Therefore, chromosome analysis of cell cultures and the plants derived from them can help eliminate chromosomally and genetically abnormal material from gene-transfer experiments. Received: 6 June 1997/Accepted: 9 October 1997     

Ubiquitin chain editing revealed by polyubiquitin linkage-specific antibodies

Posttranslational modification of proteins with polyubiquitin occurs in diverse signaling pathways and is tightly regulated to ensure cellular homeostasis. Studies employing ubiquitin mutants suggest that the fate of polyubiquitinated proteins is determined by which lysine within ubiquitin is linked to the C terminus of an adjacent ubiquitin. We have developed linkage-specific antibodies that recognize polyubiquitin chains joined through lysine 63 (K63) or 48 (K48). A cocrystal structure of an anti-K63 linkage Fab bound to K63-linked diubiquitin provides insight into the molecular basis for specificity. We use these antibodies to demonstrate that RIP1, which is essential for tumor necrosis factor-induced NF-kappaB activation, and IRAK1, which participates in signaling by interleukin-1beta and Toll-like receptors, both undergo polyubiquitin editing in stimulated cells. Both kinase adaptors initially acquire K63-linked polyubiquitin, while at later times K48-linked polyubiquitin targets them for proteasomal degradation. Polyubiquitin editing may therefore be a general mechanism for attenuating innate immune signaling.     

PatternLab for proteomics: a tool for differential shotgun proteomics

Background  

A goal of proteomics is to distinguish between states of a biological system by identifying protein expression differences. Liu et al. demonstrated a method to perform semi-relative protein quantitation in shotgun proteomics data by correlating the number of tandem mass spectra obtained for each protein, or "spectral count", with its abundance in a mixture; however, two issues have remained open: how to normalize spectral counting data and how to efficiently pinpoint differences between profiles. Moreover, Chen et al. recently showed how to increase the number of identified proteins in shotgun proteomics by analyzing samples with different MS-compatible detergents while performing proteolytic digestion. The latter introduced new challenges as seen from the data analysis perspective, since replicate readings are not acquired.     

Attenuating lymphocyte activity: the crystal structure of the BTLA-HVEM complex

Five CD28-like proteins exert positive or negative effects on immune cells. Only four of these five receptors interact with members of the B7 family. The exception is BTLA (B and T lymphocyte attenuator), which instead interacts with the tumor necrosis factor receptor superfamily member HVEM (herpes virus entry mediator). To better understand this interaction, we determined the 2.8-A crystal structure of the BTLA-HVEM complex. This structure shows that BTLA binds the N-terminal cysteine-rich domain of HVEM and employs a unique binding surface compared with other CD28-like receptors. Moreover, the structure shows that BTLA recognizes the same surface on HVEM as gD (herpes virus glycoprotein D) and utilizes a similar binding motif. Light scattering analysis demonstrates that the extracellular domain of BTLA is monomeric and that BTLA and HVEM form a 1:1 complex. Alanine-scanning mutagenesis of HVEM was used to further define critical binding residues. Finally, BTLA adopts an immunoglobulin I-set fold. Despite structural similarities to other CD28-like members, BTLA represents a unique co-receptor.