Computational identification and phylogenetic analysis of the oil-body structural proteins,oleosin and caleosin,in castor bean and flax

Oil bodies (OBs) are the intracellular particles derived from oilseeds. These OBs store lipids as a carbon resource, and have been exploited for a variety of industrial applications including biofuels. Oleosin and caleosin are the common OB structural proteins which are enabling biotechnological enhancement of oil content and OB-based pharmaceutical formations via stabilizing OBs. Although the draft whole genome sequence information for Ricinus communis L. (castor bean) and Linum usitatissimum L. (flax), important oil seed plants, is available in public database, OB-structural proteins in these plants are poorly indentified. Therefore, in this study, we performed a comprehensive bioinformatic analysis including analysis of the genome sequence, conserved domains and phylogenetic relationships to identify OB structural proteins in castor bean and flax genomes. Using comprehensive analysis, we have identified 6 and 15 OB-structural proteins from castor bean and flax, respectively. A complete overview of this gene family in castor bean and flax is presented, including the gene structures, phylogeny and conserved motifs, resulting in the presence of central hydrophobic regions with proline knot motif, providing an evolutionary proof that this central hydrophobic region had evolved from duplications in the primitive eukaryotes. In addition, expression analysis of L-oleosin and caleosin genes using quantitative real-time PCR demonstrated that seed contained their maximum expression, except that RcCLO-1 expressed maximum in cotyledon. Thus, our comparative genomics analysis of oleosin and caleosin genes and their putatively encoded proteins in two non-model plant species provides insights into the prospective usage of gene resources for improving OB-stability.     

A role for caleosin in degradation of oil-body storage lipid during seed germination

Caleosin is a Ca(2+)-binding oil-body surface protein. To assess its role in the degradation of oil-bodies, two independent insertion mutants lacking caleosin were studied. Both mutants demonstrated significant delay of breakdown of the 20:1 storage lipid at 48 and 60 h of germination. Additionally, although germination rates for seeds were not affected by the mutations, mutant seedlings grew more slowly than wild type when measured at 48 h of germination, a defect that was corrected with continued growth for 72 and 96 h in the light. After 48 h of germination, wild-type central vacuoles had smooth contours and demonstrated internalization of oil bodies and of membrane containing alpha- and delta-tonoplast intrinsic proteins (TIPs), markers for protein storage vacuoles. In contrast, mutant central vacuoles had distorted limiting membranes displaying domains with clumps of the two TIPs, and they contained fewer oil bodies. Thus, during germination caleosin plays a role in the degradation of storage lipid in oil bodies. Its role involves both the normal modification of storage vacuole membrane and the interaction of oil bodies with vacuoles. The results indicate that interaction of oil bodies with vacuoles is one mechanism that contributes to the degradation of storage lipid.     

Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil-body formation in seeds

Two genomic clones, encoding isoforms A and B of the 24 kDa soybean oleosin and containing 5 kbp and 1 kbp, respectively, of promoter sequence, were inserted separately into rapeseed plants. T₂ seeds from five independent transgenic lines, three expressing isoform A and two expressing isoform B, each containing one or two copies of the transgene, were analysed in detail. In all five lines, the soybean transgenes exhibited the same patterns of mRNA and protein accumulation as the resident rapeseed oleosins, i.e. their expression was absolutely seed-specific and peaked at the mid-late stages of cotyledon development. The 24 kDa soybean oleosin was targeted to and stably integrated into oil bodies, despite the absence of a soybean partner isoform. The soybean protein accumulated in young embryos mainly as a 23 kDa polypeptide, whereas a 24 kDa protein predominated later in development. The ratio of rapeseed:soybean oleosin in the transgenic plants was about 5:1 to 6:1, as determined by SDS-PAGE and densitometry. Accumulation of these relatively high levels of soybean oleosin protein did not affect the amount of endogenous rapeseed oleosin. Immunoblotting studies showed that about 95% of the recombinant soybean 24 kDa oleosin (and the endogenous 19 kDa rapeseed oleosin) was targeted to oil bodies, with the remainder associated with the microsomal fraction. Sucrose density-gradient centrifugation showed that the oleosins were associated with a membrane fraction of buoyant density 1.10–1.14 g ml^?1, which partially overlapped with several endoplasmic reticulum (ER) markers. Unlike oleosins associated with oil bodies, none of the membrane-associated oleosins could be immunoprecipitated in the presence of protein A-Sepharose, indicating a possible conformational difference between the two pools of oleosin. Complementary electron microscopy-immunocytochemical studies of transgenic rapeseed revealed that all oil bodies examined could be labelled with both the soybean or rapeseed anti-oleosin antibodies, indicating that each oil body contained a mixed population of soybean and rapeseed oleosins. A small but significant proportion of both soybean and rapeseed oleosins was located on ER membranes in the vicinity of oil bodies, but none were detected on the bulk ER cisternae. This is the first report of apparent targeting of oleosins via ER to oil bodies in vivo and of possible associated conformational/ processing changes in the protein. Although oil-body formation per se can occur independently of oleosins, it is proposed that the relative net amounts of oleosin and oil accumulated during the course of seed development are a major determinant of oil-body size in desiccation-tolerant seeds.     

Structures of N-termini of helices in proteins.

We have surveyed 393 N-termini of alpha-helices and 156 N-termini of 3(10)-helices in 85 high resolution, non-homologous protein crystal structures for N-cap side-chain rotamer preferences, hydrogen bonding patterns, and solvent accessibilities. We find very strong rotamer preferences that are unique to N-cap sites. The following rules are generally observed for N-capping in alpha-helices: Thr and Ser N-cap side chains adopt the gauche - rotamer, hydrogen bond to the N3 NH and have psi restricted to 164 +/- 8 degrees. Asp and Asn N-cap side chains either adopt the gauche - rotamer and hydrogen bond to the N3 NH with psi = 172 +/- 10 degrees, or adopt the trans rotamer and hydrogen bond to both the N2 and N3 NH groups with psi = 1-7 +/- 19 degrees. With all other N-caps, the side chain is found in the gauche + rotamer so that the side chain does not interact unfavorably with the N-terminus by blocking solvation and psi is unrestricted. An i, i + 3 hydrogen bond from N3 NH to the N-cap backbone C = O in more likely to form at the N-terminus when an unfavorable N-cap is present. In the 3(10)-helix Asn and Asp remain favorable N-caps as they can hydrogen bond to the N2 NH while in the trans rotamer; in contrast, Ser and Thr are disfavored as their preferred hydrogen bonding partner (N3 NH) is inaccessible. This suggests that Ser is the optimum choice of N-cap when alpha-helix formation is to be encouraged while 3(10)-helix formation discouraged. The strong energetic and structural preferences found for N-caps, which differ greatly from positions within helix interiors, suggest that N-caps should be treated explicitly in any consideration of helical structure in peptides or proteins.     

Self-organization and regulation of intrinsically disordered proteins with folded N-termini

How do mostly disordered proteins coordinate the specific assembly of very large signal transduction protein complexes? A newly emerging hypothesis may provide some clues towards a molecular mechanism.     

Differential,temporal and spatial expression of genes involved in storage oil and oleosin accumulation in developing rapeseed embryos: implications for the role of oleosins and the mechanisms of oil-body formation

The temporal and spatial expression of oleosin and ₉-stearoyl-ACP desaturase genes and their products has been examined in developing embryos of rapeseed, Brassica napus L. var. Topas. Expression of oleosin and stearate desaturase genes was measured by in situ hybridisation at five different stages of development ranging from the torpedo stage to a mature-desiccating embryo. The temporal pattern of gene expression varied dramatically between the two classes of gene. Stearate desaturase gene expression was relatively high, even at the torpedo stage, whereas oleosin gene expression was barely detectable at this stage. By the stage of maximum embryo fresh weight, stearate desaturase gene expression had declined considerably while oleosin gene expression was at its height.In contrast to their differential temporal expression, the in situ labelling of both classes of embryo-specific gene showed similar, relatively uniform patterns of spatial expression throughout the embryo sections. Immunogold labelling of ultra-thin sections from radicle tissue with anti-oleosin antibodies showed similar patterns to sections from cotyledon tissue. However, whereas at least three oleosin isoforms were detectable on western blots of homogenates from cotyledons, only one isoform was found in radicles. This suggests that some of the oleosin isoforms may be expressed differentially in the various types of embryo tissue. The differential timing of stearate desaturase and oleosin gene expression was mirrored by similar differences in the timing of the accumulation of their ultimate products, i.e. storage oil and oleosin proteins. Oil-body fractions prepared from young (2.5 mg) embryos contained very little oleosin protein, as examined by SDS-PAGE and western blotting, whereas identically prepared fractions from dry seeds contained over 10% (w/w) oleosin. Dehydration of oil bodies from young embryos resulted in their breakdown and coalescence into large clumps of oil which could not be re-emulsified, even after rehydration. In contrast, the oleosin-rich oil bodies from mature embryos were stable to dehydration and subsequent rehydration. It is suggested that, in developing rapeseed embryos, the accumulation of storage oil and oleosins is not concomitant but that the eventual deposition of oleosins onto the surfaces of storage oil bodies is essential for their stability during seed desiccation.Abbreviations ABA abscisic acid - ACP acyl carrier protein - GLC gas-liquid chromatography - PBS phosphate-buffered saline     

Structural homology discloses a bifunctional structural motif at the N-termini of G alpha proteins

In a family of proteins, often the three-dimensional structure has been experimentally determined only for one member or a few members of the family. Homology modeling can be used to model the structures of all other members of the family and thus allow comparison of these structures. This approach was applied to heterotrimeric G proteins that require anchorage to the plasma membrane to properly interact with membrane-bound receptors and downstream effectors. Lipid modification by palmitoylation is a fundamental contributor to this localization, but the signals leading to this modification are still unknown. In this work, homology models of all the different human G(alpha) paralogs were generated using automated homology modeling, and the electrostatic potential of these proteins was calculated and visualized. This approach identifies a basic, positively charged, structural motif in the N-termini of heterotrimeric G proteins, which is not readily discernible from sequence alone. The basic motif is much reduced in those G(alpha) subunits that also undergo myristoylation, suggesting that the basic patches and myristoylation play overlapping roles. These motifs can affect both membrane affinity and orientation and determine the palmitoylation of G(alpha) subunits in cooperation with the G(betagamma) subunits, as has been corroborated by previous experimental studies. Furthermore, other palmitoylated proteins such as GAP-43 and RGS proteins share this alpha-helical basic motif in their N-terminus. It therefore appears that this structural motif is more widely applicable as a membrane-targeting and palmitoylation-determining signal. The work presented here highlights the possibilities available for experimentalists to discover structural motifs that are not readily observed by analysis of the linear sequence.     

Membrane perturbation effects of peptides derived from the N-termini of unprocessed prion proteins

Peptides derived from the unprocessed N-termini of mouse and bovine prion proteins (mPrPp and bPrPp, respectively), comprising hydrophobic signal sequences followed by charged domains (KKRPKP), function as cell-penetrating peptides (CPPs) with live cells, concomitantly causing toxicity. Using steady-state fluorescence techniques, including calcein leakage and polarization of a membrane probe (diphenylhexatriene, DPH), as well as circular dichroism, we studied the membrane interactions of the peptides with large unilamellar phospholipid vesicles (LUVs), generally with a 30% negative surface charged density, comparing the effects with those of the CPP penetratin (pAntp) and the pore-forming peptide melittin. The prion peptides caused significant calcein leakage from LUVs concomitant with increased membrane ordering. Fluorescence correlation spectroscopy (FCS) studies of either rhodamine-entrapping (REVs) or rhodamine-labeled (RLVs) vesicles, showed that addition of the prion peptides resulted in significant release of rhodamine from the REVs without affecting the overall integrity of the RLVs. The membrane leakage effects due to the peptides had the following order of potency: melittin > mPrPp > bPrPp > pAntp. The membrane perturbation effects of the N-terminal prion peptides suggest that they form transient pores (similar to melittin) causing toxicity in parallel with their cellular trafficking.     

UGUS, a reporter for use with destabilizing N-termini.

Due to constraints in vector construction, reporter polypeptides often carry N-terminal sequences of extraneous origin. Since protein half-life can be influenced by small determinants in the N-terminus, such foreign sequences can destabilize proteins and compromise results of reporter-based studies. We provide a real-life example of this problem (destabilizing sequences derived from a ribosomal protein) and show that it can be solved with the ubiquitin fusion technique, in which ubiquitin sequences are placed upstream of the reporter, in our case beta-glucuronidase. Post-translational processing by characterized pathways removes the ubiquitin together with destabilizing sequences, generating a stable reporter whose N-terminus is constant.     

Chemical properties of the N-termini of human haemoglobin.

The chemical properties, namely pK and reactivity, of the N-termini of oxyhaemoglobin and deoxyhaemoglobin toward acetic anhydride and 1-fluoro-2,4-dinitrobenzene (Dnp-F) were determined by the competitive-labelling approach [Kaplan, Stevenson & Hartley, (1971) Biochem. J. 124, 289-229; Duggleby & Kaplan (1975) Biochemistry 14, 5168-5175]. At physiological pH and temperature, the valine-1 alpha and valine-1-beta amino groups had unusually low pK values, but showed only minimal changes in their pK values on deoxygenation. Between pH 7.5 and pH 8.0 a deviation was observed in the pH-reactivity profiles and the apparent pK values became markedly pH-dependent. It was found that Dnp-F, but not acetic anhydride, had an abnormally high reactivity toward the N-termini. It is concluded that the valine-1 alpha and valine-1 beta N-termini make little or no contribution to the alkaline Bohr effect at physiological pH values. The high reactivity toward Dnp-F is attributed to an interaction or binding near the N-terminal region, and the discontinuity in the pH-reactivity profile at moderate alkaline pH values to a conformational change which alters the environment of these groups.     

Vibrational analysis of peptides, polypeptides, and proteins. XXX. Normal mode analyses of gamma-turns

The normal modes have been calculated for three kinds of low energy gamma-turn structures resulting from recent conformational energy calculations by Némethy. Frequencies have been computed for a gamma-turn, a mirror-related gamma-turn, and an inverse gamma-turn of CH3-CO-(L-Ala)n-NH-CH3, with n = 3 and n = 5, and for certain 14C and 15N derivatives of the n = 3 molecule. Correlations are evident between amide frequencies and gamma-turn structures, and it is found that only amide I modes of peptide groups in the turn are relatively insensitive to the lengths of attached chains.     

Synthesis of the major oil-body membrane protein in developing rapeseed (Brassica napus) embryos. Integration with storage-lipid and storage-protein synthesis and implications for the mechanism of oil-body formation.

The synthesis of the major protein and lipid storage reserves during embryogenesis in oilseed rape (Brassica napus L., cv. Mikado) has been examined by biochemical, immunological and immunocytochemical techniques. The mature seeds contained about 45% (w/w) storage oil and 25% (w/w) protein. There were three major seed protein components, i.e. about 40-50% total protein was cruciferin, 20% was napin and 20% was a 18 kDa hydrophobic polypeptide associated with the proteinaceous membrane surrounding the storage oil bodies. Embryogenesis was divided into four overlapping stages with regard to the synthesis of these storage components: (1) for the first 3 weeks after flowering, little, if any, synthesis of storage components was observed; (2) storage-oil synthesis began at about week 3, and maximal rates were from weeks 4 to 7; (3) synthesis of the soluble storage proteins cruciferin and napin started at week 6 and rates were maximal between weeks 8 and 11; (4) the final stage was the synthesis of the 19 kDa oil-body polypeptide, which started at weeks 8-10 and was at a maximal rate between weeks 10 and 12. The synthesis of the 19 kDa oil-body protein therefore occurred independently of the synthesis of the soluble seed storage proteins. This former synthesis did not occur until shortly before the insertion of the 19 kDa polypeptide into the oil-body membrane. No evidence was found, either from sucrose-density-gradient-centrifugation experiments or from immunogold-labelling studies, for its prior accumulation in the endoplasmic reticulum. Conventional and immunogold-electron-microscopic studies showed that oil bodies were synthesized in the early to middle stages of seed development without a strongly electron-dense membrane. Such a membrane was only found at later stages of seed development, concomitantly with the synthesis of the 19 kDa protein. It is proposed that, in rapeseed embryos, oil bodies are initially formed with no proteinaceous membrane. Such a membrane is formed later in development after insertion by ribosomes of the hydrophobic 19 kDa polypeptide directly into the oil bodies.     

Immunohistochemical analyses of cell cycle-related proteins, apoptosis, and proliferation in pituitary adenomas.

To analyze the cell cycle regulatory mechanisms in the growth of pituitary adenomas, we investigated immunohistochemically the expression of the cell cycle-related proteins cyclin A and p27 in 48 pituitary adenomas. The frequency of apoptosis and the proliferative potential were also examined. The percentage of apoptotic cells was evaluated by immunohistochemical analysis using the anti-single-strand DNA antibody. The proliferative potential was assessed using the anti-Ki-67 antibody. The mean cyclin A labeling index (LI) for the non-recurrent group was 1.03% and for the recurrent group 2.31%. A positive linear correlation between cyclin A LI and Ki-67 LI was found. The mean p27 LI for the non-recurrent group was 67.4% and for the recurrent group 47.0%. There were significant differences in cyclin A LI and p27 LI between the non-recurrent group and the recurrent group. The mean apoptotic rate for the non-recurrent group was 0.87% and for the recurrent group 1.05%. There was no significant difference. Multivariate regression analysis revealed that high cyclin A LI and high Ki-67 LI were significant factors for shorter progression-free survival. The results suggest that the cyclin A LI is a useful prognostic factor in pituitary adenomas. (J Histochem Cytochem 49:1193-1194, 2001)     

The N-terminal domains of SOCS proteins: a conserved region in the disordered N-termini of SOCS4 and 5

Suppressors of cytokine signaling (SOCS) proteins function as negative regulators of cytokine signaling and are involved in fine tuning the immune response. The structure and role of the SH2 domains and C‐terminal SOCS box motifs of the SOCS proteins are well characterized, but the long N‐terminal domains of SOCS4–7 remain poorly understood. Here, we present bioinformatic analyses of the N‐terminal domains of the mammalian SOCS proteins, which indicate that these domains of SOCS4, 5, 6, and 7 are largely disordered. We have also identified a conserved region of about 70 residues in the N‐terminal domains of SOCS4 and 5 that is predicted to be more ordered than the surrounding sequence. The conservation of this region can be traced as far back as lower vertebrates. As conserved regions with increased structural propensity that are located within long disordered regions often contain molecular recognition motifs, we expressed the N‐terminal conserved region of mouse SOCS4 for further analysis. This region, mSOCS4_86–155, has been characterized by circular dichroism and nuclear magnetic resonance spectroscopy, both of which indicate that it is predominantly unstructured in aqueous solution, although it becomes helical in the presence of trifluoroethanol. The high degree of sequence conservation of this region across different species and between SOCS4 and SOCS5 nonetheless implies that it has an important functional role, and presumably this region adopts a more ordered conformation in complex with its partners. The recombinant protein will be a valuable tool in identifying these partners and defining the structures of these complexes. Proteins 2011. © 2012 Wiley Periodicals, Inc.     

Processed N-termini of mature proteins in higher eukaryotes and their major contribution to dynamic proteomics

N-terminal-ubiquitinylation (NTU) is a newly discovered protein degradation pathway initiated by ubiquitin-tagging of the N-terminal alpha-amino group. We have used data from recent genomic studies, especially those on humans, to up-date and re-interpret biochemical data to identify the sequence features associated with NTU. We compared a mini-proteome for which experimental protein sequence is available with large-scale genomic data. We conclude that N-alpha-acetylation involves less than 30%, and not the widely assumed 90%, of the proteins encoded by any higher eukaryote genome, greatly increasing thereby the number of possible targets for NTU-mediated degradation. Next, straightforward rules linking the first N-terminal residues of any nascent polypeptides to the nature of their processed N-termini are established and dedicated prediction tool is made available at . We provide strong arguments indicating that the nature of the processed N-terminus is a major determinant factor of the half-life of the protein. We finally reveal that one third of the nuclear-encoded proteins starting with an unprocessed and unblocked methionine are at least one order of magnitude less stable than is average in higher eukaryotes. This appears to be the first common feature of proteins undergoing N-terminal ubiquitinylation. Hence, a pool of about 3000 proteins in each proteome could be unstable per se and tagged for rapid degradation via NTU.     

Sequence determinants of the capping box, a stabilizing motif at the N-termini of alpha-helices.

The capping box, a recurrent hydrogen bonded motif at the N-termini of alpha-helices, caps 2 of the initial 4 backbone amide hydrogen donors of the helix (Harper ET, Rose GD, 1993, Biochemistry 32:7605-7609). In detail, the side chain of the first helical residue forms a hydrogen bond with the backbone of the fourth helical residue and, reciprocally, the side chain of the fourth residue forms a hydrogen bond with the backbone of the first residue. We now enlarge the earlier definition of this motif to include an accompanying hydrophobic interaction between residues that bracket the capping box sequence on either side. The expanded box motif--in which 2 hydrogen bonds and a hydrophobic interaction are localized within 6 consecutive residues--resembles a glycine-based capping motif found at helix C-termini (Aurora R, Srinivasan R, Rose GD, 1994, Science 264:1126-1130).     

The domains required to direct core proteins of hepatitis C virus and GB virus-B to lipid droplets share common features with plant oleosin proteins.

In mammalian tissue culture cells, the core protein of hepatitis C virus (HCV) is located at the surface of lipid droplets, which are cytoplasmic structures that store lipid. The critical amino acid sequences necessary for this localization are in a region of core protein that is absent in flavi- and pestiviruses, which are related to HCV. From our sequence comparisons, this region in HCV core was present in the corresponding protein of GBV-B, another virus whose genomic sequence has significant similarity to HCV. Expression of the putative GBV-B core protein revealed that it also was directed to lipid droplets. By extending the comparisons to cellular proteins, there were amino acid sequence similarities between the domains for lipid droplet association in HCV core and plant oleosin proteins. To determine whether these similarities were related functionally, an oleosin encoded by the Brassica napus bniii gene was expressed in different mammalian cell lines, where it retained the capacity to bind to lipid droplets. Analysis of deletion mutants indicated that the critical region within the protein required for this localization was the same for both plant and mammalian cells. A common feature in the viral and plant sequences was a motif containing proline residues. Mutagenesis of these residues in HCV core and plant oleosin abolished lipid droplet association. Finally, the domain within HCV core required for binding to lipid droplets could substitute for the equivalent domain in oleosin, further indicating the functional relatedness between the viral and plant sequences. These studies identify common features in disparate proteins that are required for lipid droplet localization.