A plant-specific cyclin-dependent kinase is involved in the control of G2/M progression in plants.

Cyclin-dependent kinases (CDKs) control the key transitions in the eukaryotic cell cycle. All the CDKs known to control G(2)/M progression in yeast and animals are distinguished by the characteristic PSTAIRE motif in their cyclin-binding domain and are closely related. Higher plants contain in addition a number of more divergent non-PSTAIRE CDKs with still obscure functions. We show that a plant-specific type of non-PSTAIRE CDKs is involved in the control of the G(2)/M progression. In synchronized tobacco BY-2 cells, the corresponding protein, accumulated in a cell cycle-regulated fashion, peaking at the G(2)/M transition. The associated histone H1 kinase activity reached a maximum in mitosis and required a yet unidentified subunit to be fully active. Down-regulation of the associated kinase activity in transgenic tobacco plants using a dominant-negative mutation delayed G(2)/M transition. These results provide the first evidence that non-PSTAIRE CDKs are involved in the control of the G(2)/M progression in plants.

Regulation of cyclin-dependent kinases in Arabidopsis thaliana.

In plants, different families of cyclin-dependent kinases (CDKs) and cyclins have been identified, indicating that also in plants the progression through the cell cycle is regulated by CDKs. In all eukaryotes, CDKs exert their activity through well-controlled phosphorylations of specific substrates on serine/threonine residues. Such post-translational modifications are universal mechanisms in signal transduction pathways. They allow the organism to differentiate, regulate growth and/or adapt to environmental changes, the latter being crucial for plants because of their sedentary life-style. This adaptation might explain the occurrence of a special CDK type with plant-specific features. This review focuses on the involvement of plant CDKs in different phases of the cell cycle in Arabidopsis thaliana and outlines their regulation by binding to other proteins, and by phosphorylation and dephosphorylation.

The Arabidopsis thaliana PIN1At gene encodes a single-domain phosphorylation-dependent peptidyl prolyl cis/trans isomerase.

A homologue of the human site-specific prolyl cis/trans isomerase PIN1 was identified in Arabidopsis thaliana. The PIN1At gene encodes a protein of 119 amino acids that is 53% identical with the catalytic domain of the human PIN1 parvulin. Steady-state PIN1At mRNA is found in all plant tissues tested. We show by two-dimensional NMR spectroscopy that the PIN1At is a prolyl cis/trans isomerase with specificity for phosphoserine-proline bonds. PIN1At is the first example of an eukaryotic parvulin without N- or C-terminal extensions. The N-terminal WW domain of 40 amino acids, typical of all the phosphorylation-dependent eukaryotic parvulins, is absent. However, triple-resonance NMR experiments showed that PIN1At contained a hydrophobic helix similar to the alpha1 helix observed in PIN1 that could mediate the protein-protein interactions.

Recombinant production of the p10CKS1At protein from Arabidopsis thaliana and 13C and 15N double-isotopic enrichment for NMR studies.

The CKS1At gene product, p10CKS1At from Arabidopsis thaliana, is a member of the cyclin-dependent kinase subunit (CKS) family of small proteins. These proteins bind the cyclin-dependent kinase (CDK)/cyclin complexes and play an essential, but still not precisely known role in cell cycle progression. To solve the structure of p10CKS1At, a protocol was needed to produce the quantity of protein large enough for nuclear magnetic resonance (NMR) spectroscopy. The first attempt to express CKS1At in Escherichia coli under the control of the T7 promoter was not successful. E. coli BL21(DE3) cotransformed with the CKS1At gene and the E. coli argU gene that encoded the arginine acceptor tRNAUCU produced a sufficient amount of p10CKS1At to start the structural study by NMR. Replacement of four rare codons in the CKS1At gene sequence, including a tandem arginine, by highly used codons in E. coli, restored also a high expression of the recombinant protein. Double-isotopic enrichment by 13C and 15N is reported that will facilitate the NMR study. Isotopically labeled p10CKS1At was purified to yield as much as 55 mg from 1 liter of minimal media by a two-step chromatographic procedure. Preliminary results of NMR spectroscopy demonstrate that a full structural analysis using triple-resonance spectra is feasible for the labeled p10CKS1At protein.

The Arabidopsis Cks1At protein binds the cyclin-dependent kinases Cdc2aAt and Cdc2bAt.

In Arabidopsis, two cyclin-dependent kinases (CDK), Cdc2aAt and Cdc2bAt, have been described. Here, we have used the yeast two-hybrid system to identify Arabidopsis proteins interacting with Cdc2aAt. Three different clones were isolated, one of which encodes a Suc1/Cks1 homologue. The functionality of the Arabidopsis Suc1/Cks1 homologue, designed Cks1At, was demonstrated by its ability to rescue the temperature-sensitive cdc2-L7 strain of fission yeast at low and intermediate expression levels. In contrast, high cks1At expression levels inhibited cell division in both mutant and wild-type yeast strains. Cks1At binds both Cdc2aAt and Cdc2bAt in vivo and in vitro. Furthermore, we demonstrate that the fission yeast Suc1 binds Cdc2aAt but only weakly Cdc2bAt, whereas the human CksHs1 associated exclusively with Cdc2aAt.

Acute transcriptional response of the honeybee peptide-antibiotics gene repertoire and required post-translational conversion of the precursor structures.

The cell-free immune repertoire of honeybees (Apis mellifera) consists of four polypeptides that are induced by bacterial infection and, through complementarity, provide broad-spectrum antibacterial defense. apidaecin is overproduced by a combination of low threshold transcriptional activation and a unique, genetically encoded amplification mechanism. In contrast, sizable experimental infections are required for induction of the normally silent hymenoptaecin, abaecin, and bee defensin genes; even so, bee defensin transcription is minimal and delayed, and only minute quantities of corresponding peptide are produced. The specific, temporal organization of the multi-component immune response in bees has therefore likely been selected to cope with infection of prevalent, plant-associated Gram-negative bacteria. Post-translational processing and modifications are substantially different for each of the four antibacterial peptides. While no similarities were observed among precursor structures of the various bee peptides, surprisingly, the signal sequences of abaecin (bee) and drosocin (Drosophila) shared unmistakable homology, possibly indicating common ancestral secretion/processing mechanisms. Finally, we report that bee defensin contains a typical disulfide-rich structure (40 amino acids) but also a unique, amphipathic, putatively amidated carboxyl-terminal tail (10 amino acids). We speculate that this structure is a "co-drug," assembled by fusing "disulfide-rich" and "alpha-helical" class peptide antibiotics, a novel concept in naturally occurring antibacterials.

Biodiversity of apidaecin-type peptide antibiotics. Prospects of manipulating the antibacterial spectrum and combating acquired resistance.

Insects have a unique repertoire of peptide antibiotics but, to date, prospects of clinical applications are not clear. Apidaecin, a small peptide isolated from honeybees, inhibits viability of Gram-negative bacteria; lethal activity is near immediate, independent of a conventional "lytic" mechanism, and involves stereoselective recognition of target molecules. Here we report structural analysis of 14 naturally occurring apidaecin-type peptides and the existence of evolutionarily conserved ("constant") regions. By detailed analysis of activities against clinically relevant bacteria, we demonstrate that the diversity of the intervening ("variable") regions confers specificity to the antibacterial spectrum of each homolog. As a result, apidaecin-homolog-based antibiograms (using 16 peptides) differ markedly between bacterial strains, contrasting the most between Yersinia enterocolitica and Campylobacter jejuni. Furthermore, in at least one instance, acquired resistance to apidaecin could be negated by minor substitutions in the variable regions. The delineation in a short peptide of constant and variable regions, responsible for, respectively, general antibacterial capacity and specificity of the antibacterial spectrum, is unprecedented. Taken together, we provide evidence that antibacterial spectra of apidaecin-type peptides can be manipulated, and that, in some cases, resistance can be countered and perhaps prevented. The current findings will guide rational design of second generation peptide antibiotics for clinical trials.

Apidaecin-type peptide antibiotics function through a non-poreforming mechanism involving stereospecificity.

Insect resistance to bacterial infections is dependent on the production of specialized defense peptides. We report here that lethal activities of apidaecin, a small peptide from honeybees, cannot possibly be the result of a conventional 'lytic' mechanism. Evidence includes the complete lack of membrane permeabilization, at concentrations that exceed lethal doses by four orders of magnitude, and undiminished sensitivity of apidaecin-resistant mutants to 'poreforming' peptides. In addition, the D-enantiomer of apidaecin is completely devoid of antibacterial activities. We propose therefore, that the antagonistic effects of apidaecin involve stereoselective recognition of a chiral cellular target, establishing this peptide as functionally unique among insect antibacterials. Identification of the apidaecin target may provide the scientific basis for rational drug design.

Functional and chemical characterization of Hymenoptaecin, an antibacterial polypeptide that is infection-inducible in the honeybee (Apis mellifera).

As part of our ongoing search for novel antimicrobial agents and their use in singular or combined drug therapy, we have isolated a series of polypeptides from the lymph fluid of honeybees. These polypeptides are synthesized de novo, following experimental infection of the insect with live Escherichia coli cells, and confer a broad-spectrum antibacterial defense to the host. We have dissected this humoral "immune" system into its constituent components. In addition to the previously characterized apidaecins and abaecin, we also isolated a member of the defensin family of peptide antibiotics and, now, a novel 93-amino acid long, cationic polypeptide, termed hymenoptaecin. Detailed analysis established the complete chemical structure, including a 2-pyrrolidone-5-carboxylic acid at the N terminus, and indicated major differences with all known antibacterial polypeptides. Under physiological conditions, it inhibits viability of Gram-negative and Gram-positive bacteria, including several human pathogens. Lethal effects against E. coli are secondary to sequential permeabilization of outer and inner membrane. In combination, the six-constituent "peptide antibiotics" of bee lymph provide wide-spectrum antibacterial protection in vitro by virtue of complementarity rather than synergism.

Apidaecin multipeptide precursor structure: a putative mechanism for amplification of the insect antibacterial response.

Apidaecins are the most prominent components of the honeybee humoral defense against microbial invasion. Our analysis of cDNA clones indicated that up to 12 of these short peptides (2 kDa) can be generated by processing of single precursor proteins; different isoforms are hereby linked in one promolecule. Assembly of the multipeptide precursors and the putative three-step maturation are strongly reminiscent of yeast alpha-mating factor. Bioactive apidaecins are flanked by the two 'processing' sequences, EAEPEAEP (or variants) and RR; joined together, they form a single unit that is repeated numerous times. The number of such repeats is variable and was reflected in the observed diversity of transcript lengths. Each such transcript is likely to be encoded by a different gene, forming a tight gene cluster. While transcriptional activation upon bacterial challenge is not exceptionally fast, the multigene and multipeptide precursor nature of the apidaecin genetic information allows for amplification of the response, resulting in a real overproduction of peptide antibiotic. Enhanced efficiency of the 'immune' response to bacterial infection through such a mechanism is, to our knowledge, unique among insects.

Isolation and characterization of abaecin, a major antibacterial response peptide in the honeybee (Apis mellifera).

Honeybee (Apis mellifera) are frequently exposed to and likely to be infected by plant-associated bacteria. We mimicked this process by injecting bees with live bacteria and isolated five induced antibacterial substances by comparative liquid chromatographic mapping of the hemolymph. Three of these antibiotics belong to a unique family of small (18 amino acids) peptides: the apidaecins [Casteels et al. (1989) EMBO J. 8, 2387-2391]. We have now characterized a fourth bee immune response peptide. The complete sequence was established by Edman degradation of the peptide and fragments thereof. It is 34 amino acids long and contains 10 proline residues. The amino-terminal half is related to the apidaecins; similar proline motifs are also present in the amino-terminal quarter of the much longer fly diptericins. The newly identified peptide's broad spectrum, lower specific activities against Gram-negative plant pathogens and its inability to inhibit bacterial growth at medium ionic strength are different from the apidaecins. Moreover, the highest observed specific activity was against an apidaecin-resistant Xanthomonas strain. In contrast to the immediate action of apidaecins, bactericidal activity is delayed. We propose the name 'abaecin' for this new antibacterial response peptide.

Apidaecins: antibacterial peptides from honeybees.

Although insects lack the basic entities of the vertebrate immune system, such as lymphocytes and immunoglobulins, they have developed alternative defence mechanisms against infections. Different types of peptide factors, exhibiting bactericidal activity, have been detected in some insect species. These humoral factors are induced upon infection. The present report describes the discovery of the apidaecins, isolated from lymph fluid of the honeybee (Apis mellifera). The apidaecins represent a new family of inducible peptide antibiotics with the following basic structure: GNNRP(V/I)YIPQPRPPHPR(L/I). These heat-stable, non-helical peptides are active against a wide range of plant-associated bacteria and some human pathogens, through a bacteriostatic rather than a lytic process. Chemically synthesized apidaecins display the same bactericidal activity as their natural counterparts. While only active antibacterial peptides are detectable in adult honeybee lymph, bee larvae contain considerable amounts of inactive precursor molecules.