Inspired by vitamin A for anti-ageing: Searching for plant-derived functional retinoid analogues.

Background: Cosmetic treatments that inspire one's appearance to resemble their younger portrait often utilize ingredients that confer acute effects, particularly hydration by creating hydrophobic barriers or transient elevation of barrier water content. But superior therapies successfully promote morphogenesis of the dermal-epidermal junction, inspiring extracellular matrix (ECM) formation. This can be achieved by agonism of the very well-known retinoid nuclear receptors using the endogenous ligand all-trans retinoic acid (tRA), tRA precursors or plant-based functional analogues, with reduced side effects. Aims Materials and Methods: While there are already many promising cosmetic ingredients available from the world's flora, higher potency is favoured, so increasing known candidates is a worth undertaking. Functional analogues of retinoic acid can be identified by culturing fibroblasts with lipophilic candidates from the plant kingdom and assessing gene-arrays. Modern approaches to validating these findings include the coculturing of fibroblasts with keratinocytes as a measure to predict the potential effects of crosstalk. Results and Discussion: In this regard, the most promising plant-derived candidates are of terpene or meroterpene origin, including derivatives of squalene and phytol. Surprisingly pimaric or abietic acids and labdane diterpenes are also noteworthy agonists of the retinoic acid receptor, stimulating collagen expression in dermal fibroblasts. Conclusion: There are numerous derivatives of these terpenes available from the world's flora and research conducted thus far encourages further screening of these chemical candidates.

Enhanced retinoid response by a combination of the vitamin A ester retinyl propionate with niacinamide and a flavonoid containing Ceratonia siliqua extract in retinoid responsive in vitro models.

OBJECTIVES: Retinoids have been used for decades as efficacious topical agents to treat photoaged skin. The purpose of our present research is to evaluate whether the activity of the vitamin A ester retinyl propionate (RP) can be enhanced by niacinamide (Nam) and a flavonoid containing Ceratonia siliqua (CS) fruit extract in retinoid responsive in vitro models. METHODS: Retinyl propionate was tested alone and in combination with Nam and CS in an RARα reporter cell line for promoter activation and compared to trans-retinoic acid (tRA) activation. These treatments were also tested in keratinocytes for gene expression profiling by qPCR using a panel of 40 retinoid responsive genes. RESULTS: tRA or RP elicited RARα reporter activation in a dose-dependent manner. The combination of 0.5 µM or 2 µM RP with 10 mM Nam had a 56% and 95% signal increase compared to RP, respectively. The addition of 1% CS to 0.5 µM or 2 µM RP with 10 mM Nam elicited a further increase of 114% and 156%, respectively, over RP and Nam combinations. All retinoids elicited an increase in expression of 40 retinoid sensitive genes over control levels. Of the 40 genes, 27 were enhanced by either 0.1 µM RP or 0.5 µM RP with 10 mM Nam and 1% CS. Nam or CS had very modest activity in both models. CONCLUSION: The combination of RP with Nam and CS showed a higher retinoid response than RP in two separate retinoid responsive in vitro models. We hypothesize Nam and CS enhances RP activity by modulating metabolism to tRA via increasing NAD+ pools and inhibiting reduction of retinal (RAL) back to retinol, respectively. The findings provide evidence that this combination may have enhanced efficacy for treating the appearance of photoaged skin.

OBJECTIFS: Les rétinoïdes sont utilisés depuis des décennies comme agents topiques efficaces pour traiter la peau photo-âgée. Le but de notre recherche actuelle est d'évaluer si l'activité du propionate rétinyl ester de vitamine A (RP) peut être augmentée par le niacinamide (Nam) et un flavonoïde contenant un extrait de fruit de Ceratonia Siliqua (CS) dans les modèles in vitro sensibles aux rétinoïdes. MÉTHODES: RP a été testé seul et en combinaison avec Nam et CS dans une ligne de cellule rapporteur de RARα pour l'activation du promoteur et par rapport à l'activation de l'acide transrétinoïque(tRA). Ces traitements ont également été testés dans les kératinocytes pour le profilage d'expression génique par qPCR à l'aide d'un panel de 40 gènes rétinoïdes sensibles. RÉSULTATS: tRA ou RP ont provoqué l'activation du promoteur RARα d'une manière dépendante de la dose. La combinaison de 0,5 µM ou 2 µM de RP avec 10 mM de Nam a permis une augmentation respectivement de 56% et 95% du signal par rapport à RP. L'ajout de 1 % de CS à 0,5 µM ou 2 µM de RP avec 10 mM de Nam a permis une nouvelle augmentation de 114 % et 156 %, respectivement, qu'avec la combinaison RP et Nam. Tous les rétinoïdes ont provoqué une augmentation de l'expression de 40 gènes sensibles aux rétinoïdes sur les niveaux de contrôle. Sur les 40 gènes, 27 ont été améliorés soit par 0,1 µM de RP ou 0.5 µM de RP avec 10 mM de Nam et 1% de CS. Nam ou CS avaient une activité très modeste dans les deux modèles. CONCLUSION: La combinaison de RP avec Nam et CS a montré une réponse rétinoïde plus élevée que RP dans deux modèles in vitro séparés sensibles aux rétinoïdes. Nous émettons l'hypothèse que Nam et CS améliorent l'activité RP en modulant le métabolisme de tRA par l'augmentation des groupement NAD+ et en inhibant la réduction du rétinal (RAL) en rétinol, respectivement. Les résultats fournissent la preuve que cette combinaison peut améliorer l'efficacité du traitement de l'aspect de la peau photo-âgée.

Optimized low pH formulation of niacinamide enhances induction of autophagy marker ATG5 gene expression and protein levels in human epidermal keratinocytes.

BACKGROUND: Macromolecules in skin cells are damaged when exposed to environmental stressors, leading to disrupted cellular function and homeostasis. While epidermal turnover can eliminate some of this damage, autophagy can rapidly remove these defective components. Niacinamide (Nam) is known to induce autophagy and optimizing formulations to maximize this response could provide improved homeostasis in stressed skin. OBJECTIVE: To determine (i) whether Nam can induce autophagy related 5 (ATG5), an autophagy marker, in human keratinocytes and (ii) whether optimized low pH Nam formulations can enhance the response in 3D skin models. METHODS: Human keratinocytes treated with Nam were evaluated for autophagosome accumulation and induction of ATG5 by gene expression, immunoblotting and immune-fluorescence microscopy. 3D skin equivalents were topically treated with Nam formulations at pH 5.8 and 3.8. Gene expression profiling and immunoblot analysis of ATG5 were performed. RESULTS: Nam treatment of keratinocytes led to an accumulation of autophagosomes with a maximal signal at 48 h. Gene expression of ATG5 was induced by Nam, and immunoblots stained for ATG5 showed a significant increase after 6 h of treatment. Gene expression profiling of 3D epidermal skin equivalents treated with Nam at pH 3.8 showed stronger induction of autophagy-related genes, including ATG5, compared with pH 5.8 formulas. Enrichment for gene ontology terms on autophagy showed an increased linkage with Nam formulas at pH 3.8. CONCLUSIONS: We found that Nam induces autophagosome accumulation and ATG5 levels in keratinocytes. We also discovered that a Nam formulation at pH 3.8 can further increase levels of ATG5 in 3D skin models when compared to Nam at pH 5.8. These data support that Nam can induce autophagy in keratinocytes and formulations at pH 3.8 can enhance the impact. We hypothesize that optimized formulations at pH 3.8 can improve skin ageing appearance via autophagy induction.

Topical niacinamide does not stimulate hair growth based on the existing body of evidence.

Niacinamide has been suggested to impact hair biology via stimulation of VEGF synthesis. Testing in an in vitro VEGF synthesis assay, it was found that niacinamide cannot stimulate VEGF synthesis across a broad dose-response range.

Nicotinamide preferentially protects glycolysis in dermal fibroblasts under oxidative stress conditions.

BACKGROUND: Daily exposure of human skin to environmental insults such as solar radiation, pollution and smoke can lead to an elevation of oxidative stress, causing premature acceleration of skin ageing. Oxidative stress is known to disrupt cellular metabolism, which negatively impacts the skin's functionality at the cellular and tissue level. OBJECTIVES: To examine the changes in cellular metabolism due to oxidative stress. METHODS: Glycolysis and oxidative phosphorylation rates in human dermal fibroblasts were monitored in real time under controlled nonlethal oxidative stress conditions. Hydrogen peroxide was utilized as a surrogate stressor because numerous environmental stressors as well as intrinsic ageing trigger its production. RESULTS: Hydrogen peroxide ranging between 0.5 and 3 mmol L(-1) caused a significant decrease in glycolytic and oxidative phosphorylation rates along with cellular ATP levels. Nicotinamide (NAM) was found to protect dose dependently as well as restore glycolytic rates concurrent with restoring ATP to control levels. NAM had an effective dose-response range between 0.1 and 1.0 mmol L(-1) , with maximal effects attained at 0.5 mmol L(-1) . Relative to oxidative phosphorylation, NAM was able to provide a diminished level of protection. FK866, a known NAM phosphoribosyltransferase inhibitor, was found to inhibit the protective effects of NAM significantly, suggesting part of the NAM mechanism of action involves nicotinamide adenine dinucleotide (NAD(+) ) synthesis. CONCLUSIONS: These results support previous findings that NAM protects cellular metabolism from oxidative stress by preferentially affecting glycolysis. Additionally, part of its mechanism of action appears to include NAD(+) synthesis.

Comparison of the impact of environmental stress on male and female skin.

Past research on understanding gender differences of skin biology and its response to environmental insults has focused on morphological and gross physiological comparisons. In general it has been found that male skin has a greater susceptibility to being negatively impacted by environmental stressors, in particular ultraviolet radiation. These noted differences in response to environmental insults are probably due to a combination of underlying biologically based differences and variable sun-protection and skin-care product usage between genders. Overall, published data support the hypothesis that male facial skin undergoes significant challenges from environmental insults that lead to a more damaged condition compared with female skin. These changes occur both from acute insults and from the impact of cumulative chronic exposure. Appropriate sun protection should be viewed as an important step in male skin care and grooming habits.

A chloroplast processing enzyme involved in precursor maturation shares a zinc-binding motif with a recently recognized family of metalloendopeptidases.

Nuclear-encoded proteins targeted to the chloroplast are typically synthesized with N-terminal transit peptides which are proteolytically removed upon import. Structurally related proteins of 145 and 143 kDa copurify with a soluble chloroplast processing enzyme (CPE) that cleaves the precursor for the major light-harvesting chlorophyll a/b binding protein and have been implicated in the maturation of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and acyl carrier protein. The 145- and 143-kDa proteins have not been found as a heterodimer and thus may represent functionally independent isoforms encoded by separate genes. Here we describe the primary structure of a 140-kDa polypeptide encoded by cDNAs isolated by using antibodies raised against the 145/143-kDa doublet. The 140-kDa polypeptide contains a transit peptide, and strikingly, a His-Xaa-Xaa-Glu-His zinc-binding motif that is conserved in a recently recognized family of metalloendopeptidases, which includes Escherichia coli protease III, insulin-degrading enzyme, and subunit beta of the mitochondrial processing peptidase. Identity of 25-30%, concentrated near the N terminus of the 140-kDa polypeptide, is found with these proteases. Expression of CPE in leaves is not light dependent. Indeed, transcripts are present in dark-grown plants, and the 145/143-kDa doublet and proteolytic activity are both found in etioplasts, as well as in root plastids. Thus, CPE appears to be a necessary component of the import machinery in photosynthetic and nonphotosynthetic tissues, and it may function as a general stromal processing peptidase in plastids.

Site-directed mutagenesis of Lys36 in human thioredoxin: the highly conserved residue affects reduction rates and growth stimulation but is not essential for the redox protein's biochemical or biological properties.

Previous studies have demonstrated that a recombinant form of the human redox protein thioredoxin can stimulate the growth rate of Swiss 3T3 murine fibroblasts and that this ability to promote cellular proliferation was dependent upon a redox-active form. A site-directed mutagenesis study of the highly conserved Lys36 adjacent to the two active site cysteines of thioredoxin was performed to determine whether the basic residue was essential for the biochemical and mitogenic properties of human thioredoxin. Two mutants were generated in which the lysine residue was replaced with either glutamic acid (K36E) or leucine (K36L). While K36E and K36L were both redox-active in a thioredoxin-specific assay, the mutants exhibited decreased affinities for thioredoxin reductase relative to wild-type thioredoxin since their respective KM values increased by a factor of 5 and 7. Examination of the secondary structure of the variants by circular dichroism spectroscopy revealed that both mutants had minor variations in the overall structural content when compared to thioredoxin, with K36L being most similar to the wild-type protein. Thermal equilibrium denaturation studies of the variants showed that K36E had a TM of 69.5 degrees C. A TM value for thioredoxin and K36L could not be established because the absence of a plateau above 83 degrees C rendered it difficult to establish an upper base line and, hence, the TM. The two mutants were able to stimulate cellular proliferation, albeit with reduced efficiency when compared with wild-type thioredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

The predicted amino acid sequence of human thioredoxin is identical to that of the autocrine growth factor human adult T-cell derived factor (ADF): thioredoxin mRNA is elevated in some human tumors.

The cDNA sequences of thioredoxin obtained by PCR cloning from human colon cancer cells, human lymphoblastoid cells, and human liver have been found to be identical with the cDNA sequence reported for the autocrine growth factor, human adult T-cell leukemia derived factor (ADF). Recombinant human thioredoxin was 95% reduced by dithiothreitol and was a substrate for reduction by human thioredoxin reductase. Human non-small cell primary lung tumors from subjects who were not cigarette smokers at the time of surgery showed significantly increased levels of thioredoxin mRNA compared to thioredoxin mRNA in paired normal human lung tissue. Subjects who were smokers did not show a significant increase in lung tumor thioredoxin mRNA. The results of the study show that human thioredoxin and ADF are identical species and suggest that there may be increased production of thioredoxin (ADF) by some human cancers.

Biochemical, structural, and biological properties of human thioredoxin active site peptides.

The human redox protein thioredoxin is an autocrine growth factor for some cancer cells. Redox activity is essential for this function but other required structural features of thioredoxin are not known. Two 8-mer peptides (I and II) and one 14-mer peptide (III) were designed based on the amino acid sequence of the redox active site of thioredoxin. Peptide I and peptide III contained the wild-type sequence of thioredoxin while peptide II contained serine residues in place of the catalytically active cysteines. Circular dichroism spectroscopy indicated that all three peptides were comprised mainly of random coil, with peptide III containing slightly more ordered secondary structure. Peptides I and III were substrates for thioredoxin reductase with KM values of 890 and 265 microM, respectively. The redox inactive peptide II could not compete with thioredoxin for reduction by thioredoxin reductase in a coupled insulin reduction assay. However, peptide II was a competitive inhibitor for the reduction of 5, 5'-dithiobis-(2-nitrobenzoic acid) by thioredoxin reductase. All three peptides gave only background levels of stimulation of the proliferation of Swiss 3T3 murine fibroblasts when compared to the stimulation caused by thioredoxin. These results suggest that while the ability of thioredoxin to stimulate cellular proliferation is redox-dependent, more information than that contained in the redox active site domain alone defined by 14 amino acids is required.

Site-directed mutagenesis of active site cysteines in human thioredoxin produces competitive inhibitors of human thioredoxin reductase and elimination of mitogenic properties of thioredoxin.

Thioredoxin and thioredoxin reductase comprise a redox system ubiquitous in all organisms. To better understand the thiol chemistry of the mammalian thioredoxin-thioredoxin reductase redox system, mutants of human thioredoxin were produced by site-directed mutagenesis in which the two active site cysteines were replaced by serine residues, individually (C32S and C35S) and in combination (C32S/C35S). C35S and C32S/C35S were found to be competitive inhibitors of the reduction of human thioredoxin by human thioredoxin reductase with Ki values of 1.8 and 6.7 microM, respectively. C32S did not inhibit thioredoxin reductase, apparently due to aggregation of the oxidized C32S species. Examination of the three mutant forms of thioredoxin by circular dichroism spectroscopy indicated that there were significant differences in the secondary structures when compared with thioredoxin. There were detectable changes in the circular dichroism spectra when thioredoxin, C35S, and C32S/C35S were bound to thioredoxin reductase, whereas C32S with thioredoxin reductase underwent only a small spectral change. Recombinant human thioredoxin stimulated DNA synthesis and the proliferation of murine fibroblasts. The ability of thioredoxin to stimulate cell proliferation could not be duplicated by either dithiothreitol or glutathione. C32S, C35S, and C32S/C35S failed to stimulate cell proliferation, showing that the redox active form of thioredoxin is necessary for eliciting growth stimulation.

The thioredoxin/thioredoxin reductase redox system and control of cell growth.

Thioredoxin is a redox protein that is important for a variety of intracellular functions, possibly including regulation of transcription factor activity. We have shown that human thioredoxin has the same predicted amino acid sequence as adult T-cell-derived leukemic cell growth factor. Recombinant human thioredoxin stimulates the proliferation of Swiss murine 3T3 fibroblasts with an EC50 of 100 nM and the proliferation of a number of human cancer cells. Site-directed mutagenesis of the active-site cysteines of thioredoxin has shown that redox activity is necessary for the stimulation of cell proliferation. Added 125I-thioredoxin is taken up by cells in culture and could have intracellular action. A series of alkyl 2-imidazolyl disulfides have been shown to be competitive inhibitors of human thioredoxin reductase with Ki values of 3.3 to 8.6 microM. The compounds inhibited Swiss 3T3 serum-dependent proliferation with IC50 values of 2.0 to 4.0 microM, and one compound inhibited Swiss 3T3 thioredoxin-dependent proliferation with an IC50 value of 60 nM.

Reversible inhibition of human thioredoxin reductase activity by cytotoxic alkyl 2-imidazolyl disulfide analogues.

The thioredoxin/thioredoxin reductase system is important for several aspects of the regulation of cellular proliferation by both intracellular and extracellular mechanisms. The effects of n-butyl 2-imidazolyl disulfide (III-2), 1-methylpropyl 2-imidazolyl disulfide (IV-2), and n-decyl 2-imidazolyl disulfide (VII-2) on purified human placental thioredoxin reductase activity were examined. The analogues were competitive inhibitors with DTNB for reduction by thioredoxin reductase, with Ki values for III-2, IV-2, and VII-2 being 3.3, 13.0, and 8.6 microM, respectively. The inhibition was noncompetitive with reduced nicotinamide adenine dinucleotide phosphate (NADPH). None of the analogues was a suicide substrate inhibitor of the flavoenzyme. III-2 and VII-2 were metabolized by thioredoxin reductase at about half the rate of DTNB, whereas IV-2 was not detectably metabolized. The second order rate constants for the reactions of III-2 and IV-2 with reduced GSH were 931 and 91 M-1 s-1, respectively. The lower reactivity of IV-2 with reduced GSH and the lack of the analogue's metabolism by thioredoxin reductase may be due to the more sterically hindered structure of this analogue. The 50% inhibitory concentrations (IC50 values) for the inhibition of serum-dependent cellular proliferation of Swiss 3T3 murine fibroblasts by III-2, IV-2, and VII-2 were 2.0, 3.5, and 4.0 microM, respectively. IV-2 was considerably more potent as an inhibitor of the thioredoxin-dependent cellular proliferation of Swiss 3T3 fibroblasts, showing an IC50 value of 60 nM. Thus, inhibition of cellular proliferation by alkyl 2-imidazolyl disulfide analogues may involve interaction with thioredoxin, thioredoxin reductase, or an alternative target that is redox-regulated by thioredoxin.

A comment on the absence of calcium regulation of human thioredoxin reductase.

It has been previously suggested that human thioredoxin reductase activity is regulated by calcium. However, the activity of a purified form of human placental thioredoxin reductase was found to not be affected by mM concentrations of calcium, well above intra- and extracellular physiological levels. Furthermore, the suggestion that an E-F hand is present in Escherichia coli thioredoxin reductase is strongly contested. These current results suggest that human thioredoxin reductase is not regulated by calcium.

Purification of human thioredoxin reductase: properties and characterization by absorption and circular dichroism spectroscopy.

The flavoenzyme thioredoxin reductase (TR) and its natural substrate thioredoxin comprise a redox system generally found in all organisms. In order to better understand the biochemistry of this redox system, TR was purified (> 4000-fold) from human placenta as a dimer of 60-kDa subunits. The molecular size of native TR was determined to be 160 kDa by gel filtration chromatography whereas migration on a sucrose gradient gave a molecular mass of 130 kDa. The pI of TR was determined to be 4.85. The temperature optima for DTNB and insulin reduction by TR were 52 and 40 degrees C, respectively. Preincubation of TR at 60 degrees C for up to 1 h showed no decrease in the enzymatic rates when assayed at 28 degrees C, while temperatures above 65 degrees C resulted in an irreversible loss of activity. Circular dichroism (CD) spectra of TR indicated that the secondary structural changes at 60 degrees C were only partly reversible at 28 degrees C. CD studies showed the flavoenzyme had a TM of 63 degrees C and above 45 degrees C began to exhibit changes in the secondary structure. Equilibrium denaturation of TR by temperature and guanidine hydrochloride suggested that FAD was not displaced during inactivation of TR and that the tertiary structure was primarily disrupted prior to denaturation of the secondary structure. The results of this study show that purified human TR is a relatively thermostable flavoenzyme whose tightly bound FAD group is not displaced by elevated temperatures up to 60 degrees C or by relatively low concentrations of guanidine hydrochloride.

Identification of two structurally related proteins involved in proteolytic processing of precursors targeted to the chloroplast.

Two proteins of 145 and 143 kDa were identified in pea which co-purify with a chloroplast processing activity that cleaves the precursor for the major light-harvesting chlorophyll binding protein (preLHCP). Antiserum generated against the 145/143 kDa doublet recognizes only these two polypeptides in a chloroplast soluble extract. In immunodepletion experiments the antiserum removed the doublet, and there was a concomitant loss of cleavage of preLHCP as well as of precursors for the small subunit of Rubisco and the acyl carrier protein. The 145 and 143 kDa proteins co-eluted in parallel with the peak of processing activity during all fractionation procedures, but they were not detectable as a homo- or heterodimeric complex. The 145 and 143 kDa proteins were used separately to affinity purify immunoglobulins; each preparation recognized both polypeptides, indicating that they are antigenically related. Wheat chloroplasts contain a soluble species similar in size to the 145/143 kDa doublet.

Precursor for the light-harvesting chlorophyll a/b-binding protein synthesized in Escherichia coli blocks import of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase.

When synthesized in Escherichia coli, the light-harvesting chlorophyll a/b-binding protein (LHCP) precursor accumulates in inclusion-like bodies (Abad, M. S., Oblong, J. E., and Lamppa, G. K. (1991) Plant Physiol. 96, 1220-1227). In this study we show that after solubilization in 6 M urea and dialysis into 20 mM Tris-HCl (pH 8.0) the recombinant LHCP precursor (preLHCP) was not found as a monomer (31 kDa), but instead produced a heterogeneous population of oligomeric complexes, ranging from 60-300 kDa as determined by gel filtration chromatography. Circular dichroism analysis indicated that the oligomers had folded structure, and that it was composed of both alpha-helix and beta-sheet. Approximately half of recombinant preLHCP found in these complexes was cleavable at the transit peptide-mature protein junction by a soluble chloroplast-processing enzyme in an organelle-free reaction. At 1.5 microM the recombinant precursor inhibited the import of radiolabeled preLHCP and the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase generated by reticulocyte lysate translations. When chloroplasts were preincubated with the precursor, followed by their reisolation, import was still blocked, providing evidence that competition between recombinant preLHCP and these substrates occurred at the chloroplast per se. Recombinant preLHCP was visualized on the envelope by immunofluorescence microscopy, and its presence there was mediated by a thermolysin-sensitive factor.

Soluble Chloroplast Enzyme Cleaves preLHCP Made in Escherichia coli to a Mature Form Lacking a Basic N-Terminal Domain.

We have investigated the specificity of a chloroplast soluble processing enzyme that cleaves the precursor of the major light-harvesting chlorophyll a/b binding protein (LHCP). The precursor of LHCP (preLHCP) was synthesized in Escherichia coli and recovered from inclusion-like bodies. It was found to be a substrate for proteolytic cleavage by the soluble enzyme in an organelle-free reaction, yielding a 25 kilodalton peptide. This peptide co-migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the smaller of the forms (25 and 26 kilodalton) produced when either the E. coli-synthesized precursor, or preLHCP made in a reticulocyte lysate, was imported into chloroplasts. N-Terminal sequence analysis of the E. coli-generated precursor showed that it lacked an N-terminal methionine. N-Terminal sequencing of the 25 kilodalton peptide produced in the organelle-free reaction indicated that processing occurred between residues 40 and 41, removing a basic domain (RKTAAK) thought to be at the N-terminus of all LHCP molecules of type I associated with photosystem II. To determine if the soluble enzyme involved also cleaves other precursor polypeptides, or is specific to preLHCP, it was partially purified, and the precursors for Rubisco small subunit, plastocyanin, Rubisco activase, heat shock protein 21, and acyl carrier protein were tested as substrates. All of these precursors were cleaved by the same chromatographic peak of activity that processes preLHCP in the organelle-free reaction.

Loss of efficient import and thylakoid insertion due to N- and C-terminal deletions in the light-harvesting chlorophyll a/b binding protein.

C-terminally truncated precursors of wheat light-harvesting chlorophyll a/b binding protein (LHCP) were synthesized to investigate the origin of the two forms (about 25 kD and 26 kD) of the mature protein observed upon in vitro import into the chloroplast. Precursors p delta 13 and p delta 27, lacking 13 and 27 amino acids, respectively, were successfully imported, and both gave rise to two smaller forms proportional to the size of their deletions. These results demonstrate that there are two N-terminal sites that are cleaved during import of the LHCP precursor, undoubtedly contributing to the heterogeneity of LHCP found in vivo. Significantly, p delta 27 yielded only 50% of mature LHCP when compared with wild type. Although the products of p delta 27 import were localized to the thylakoids, in contrast to p delta 13 they were not correctly inserted into the membranes, indicating that residues essential for this step are missing. p delta 27 is distinguished from p delta 13 by lacking the carboxy end of a domain highly conserved between LHCP of photosystems II and I. Other specific precursor mutants with larger C-terminal deletions were not efficiently transported into the organelle in time course experiments, nor did they insert directly into the thylakoids using chloroplast lysates, in an assay independent of translocation across the envelope. In addition, the mutant p delta 18n, lacking the first 18 amino acids of mature LHCP, was only found bound to the chloroplast envelope. However, both p delta 18n and the mature protein, i.e., LHCP, synthesized in vitro without its 34-amino acid transit peptide inserted into the thylakoids in chloroplast lysates. The overall conformation of the mutant precursor polypeptides was probed using the chloroplast soluble processing enzyme in an organelle-free reaction optimized for the LHCP precursor and the more general protease trypsin. A tightly folded, protease-resistant conformation was not apparent to explain the loss of efficient import.