New evidence suggests that the initial photoinduced cleavage of the D1-protein may not occur near the PEST sequence

When isolated reaction centres of photosystem 2 from pea or wheat are exposed to photoinhibitory illumination in the presence of an electron acceptor, breakdown products of the D1-protein are observed having molecular masses ranging from about 24 to 10 kDa. By using antibodies raised to the C-terminal or N-terminal portions of D1 it was shown that the major breakdown fragment of 24 kDa was derived from the C-terminus. This conclusion was supported by phosphorylation studies and from the digestion pattern obtained by lysine specific endoprotease-induced proteolysis. The complementary N-terminal breakdown fragment was found to have an apparent molecular mass of 10 kDa. The implications of these data are discussed in terms of the possible relationship between the 24 kDa C-terminal fragment and the 23.5 kDa breakdown fragment detected in vivo by Greenberg et al. [1987, EMBO J. 6, 2865–2869] and it is suggested, based on limited proteolysis using papain, that the latter may not be derived from the N-terminus as previously thought but also originates from the C-terminus.     

Dissecting a cyanobacterial proteolytic system: efficiency in inducing degradation of the D1 protein of photosystem II in cyanobacteria and plants

A chromatography fraction, prepared from isolated thylakoids of a fatty acid desaturation mutant (Fad6/desA Colon, two colons Km(r)) of the cyanobacterium Synechocystis 6803, could induce an initial cleavage of the D1 protein in Photosystem II (PSII) particles of Synechocystis 6803 mutant and Synechococcus 7002 wild type as well as in supercomplexes of PSII-light harvesting complex II of spinach. Proteolysis was demonstrated both in darkness and in light as a reduction in the amount of full-length D1 protein or as a production of C-terminal initial degradation fragments. In the Synechocystis mutant, the main degradation fragment was a 10-kDa C-terminal one, indicating an initial cleavage occurring in the cytoplasmic DE-loop of the D1 protein. A protein component of 70-90 kDa isolated from the chromatographic fraction was found to be involved in the production of this 10-kDa fragment. In spinach, only traces of the corresponding fragment were detected, whereas a 24-kDa C-terminal fragment accumulated, indicating an initial cleavage in the lumenal AB-loop of the D1 protein. Also in Synechocystis the 24-kDa fragment was detected as a faint band. An antibody raised against the Arabidopsis DegP2 protease recognized a 35-kDa band in the proteolytically active chromatographic fraction, suggesting the existence of a lumenal protease that may be the homologue DegP of Synechocystis. The identity of the other protease cleaving the D1 protein in the DE-loop exposed on the stromal (cytoplasmic) side of the membrane is discussed.     

Degradation pattern of photosystem II reaction center protein D1 in intact leaves. The major photoinhibition-induced cleavage site in D1 polypeptide is located amino terminally of the DE loop.

Photoinhibition-induced degradation of the D1 protein of the photosystem II reaction center was studied in intact pumpkin (Cucurbita pepo L.) leaves. Photoinhibition was observed to cause the cleavage of the D1 protein at two distinct sites. The main cleavage generated an 18-kD N-terminal and a 20-kD C-terminal degradation fragment of the D1 protein. this cleavage site was mapped to be located clearly N terminally of the DE loop. The other, less-frequent cleavage occurred at the DE loop and produced the well-documented 23-kD, N-terminal D1 degradation product. Furthermore, the 23-kD, N-terminal D1 fragment appears to be phosphorylated and can be detected only under severe photoinhibition in vivo. Comparison of the D1 degradation pattern after in vivo photoinhibition to that after in vitro acceptor-side and donor-side photoinhibition, performed with isolated photosystem II core particles, gives indirect evidence in support of donor-side photoinhibition in intact leaves.     

Intermolecular association between caspase-mediated cleavage fragments of phospholipase D1 protects against apoptosis

Phospholipase D plays an anti-apoptotic role but little is known about dynamics of phospholipase D turnover during apoptosis. We have recently identified phospholipase D1 as a new substrate of caspases which generates the N-terminal and C-terminal fragment of phospholipase D1. In the present study, we tried to investigate whether association of the caspase cleavage fragments may be involved in regulation of apoptosis. Ectopically expressed C-terminal fragment, but not N-terminal fragment of phospholipase D1, is exclusively imported into the nucleus via a nuclear localization sequence; however, endogenous C-terminal fragment of phospholipase D1 from etoposide-induced apoptotic cells and Alzheimer's disease brain tissues with active caspase-3, was localized in the cytosolic fraction as well as the nuclear fraction. Intermolecular association between the two fragments of phospholipase D1 through hydrophobic residues within the catalytic motif inhibited nuclear localization of C-terminal fragment of phospholipase D1, and two catalytic motif and nuclear localization sequence regulated nuclocytoplasmic shuttling of phospholipase D1. Moreover, hydrophobic residues involved in the intermolecular association are also required for both its enzymatic activity and anti-apoptotic function. Taken together, we demonstrate that interdomain association and dissociation of phospholipase D1 might provide new insights into modulation of apoptosis.     

Detection of an In Vivo 8 kD C-terminal Breakdown Product of D1-protein under Visible and UV-B Light

An 8 kD in vivo turnover product of D1 polypeptide was identified using a C-terminal specific anti-D1-antibody in thylakoids isolated from 5–10 days old seedlings of wheat (Triticum aestivum cv HD2329). Eight days old wheat seedlings grown under visible light were irradiated with UV-B (1 MW cm^?2) alone (0–5h) and Tricine SDS-PAGE was run of the isolated thylakoids. UV-B exposure of intact wheat leaves generates a fragment of 24 kD and concomitant increase in 8 kD fragment was also observed. From these results it is concluded that in wheat visible light induces the breakdown of D1 polypeptide into an 8 kD C-terminal thylakold bound fragment and UV-B stress results in an increase in 8 kD breakdown fragment, thus suggesting that visible light/UV-B has a common hot spot on D1 protein in wheat.     

Studies on the mechanism of photosystem II photoinhibition I. A two-step degradation of D1-protein

The role of D1-protein in photoinhibition was examined. Photoinhibition of spinach thylakoids at 20°C caused considerable degradation of D1-protein and a parallel loss of variable fluorescence, Q_B-independent electron flow and Q_B-dependent electron flow. The breakdown of D1-protein as well as the loss of variable fluorescence and Q_B-independent electron flow were largely prevented when thylakoids were photoinhibited at 0°C. The Q_B-dependent electron flow markedly decreased under the same conditions. This inactivation may represent the primary event in photoinhibition and could be the result of some modification at the Q_B-site of D1-protein. Evidence for this comes from fluorescence relaxation kinetics following photoinhibition at 0°C which indicate a partial inactivation of Q_A ^--reoxidation. These results support the idea of D1-protein breakdown during photoinhibition as a two step process consisting of an initial inactivation at the Q_B-site of the protein followed by its degradation. The latter is accompanied by the loss of PS II-reaction centre function.Abbreviations Asc ascorbate - p-BQ 1, 4-benzoquinone - DAD diaminodurene - DPC diphenylcarbazide - DQH₂ duroquinole - Fecy ferricyanide - MV methylviologen - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - SiMo silicomolybdate     

Photosystem II in a mutant of Chlamydomonas reinhardtii lacking the 23 kDa psbP protein shows increased sensitivity to photoinhibition in the absence of chloride

The psbP gene product, the so called 23 kDa extrinsic protein, is involved in water oxidation carried out by Photosystem II. However, the protein is not absolutely required for water oxidation. Here we have studied Photosystem II mediated electron transfer in a mutant of Chlamydomonas reinhardtii, the FUD 39 mutant, that lacks the psbP protein. When grown in dim light the Photosystem II content in thylakoid membranes of FUD 39 is approximately similar to that in the wild-type. The oxygen evolution is dependent on the presence of chloride as a cofactor, which activates the water oxidation with a dissociation constant of about 4 mM. In the mutant, the oxygen evolution is very sensitive to photoinhibition when assayed at low chloride concentrations while chloride protects against photoinhibition with a dissociation constant of about 5 mM. The photoinhibition is irreversible as oxygen evolution cannot be restored by the addition of chloride to inhibited samples. In addition the inhibition seems to be targeted primarily to the Mn-cluster in Photosystem II as the electron transfer through the remaining part of Photosystem II is photoinhibited with slower kinetics. Thus, this mutant provides an experimental system in which effects of photoinhibition induced by lesions at the donor side of Photosystem II can be studied in vivo.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DPC 2,2-diphenylcarbonic dihydrazide - HEPES 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - P₆₈₀ the primary electron donor to PS II - PpBQ phenyl-p-benzoquinone - PS II Photosystem II - Q_A the first quinone acceptor of PS II - Q_B the second quinone acceptor of PS II - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane - Tyr_D accessory electron donor on the D₂-protein - Tyr_Z tyrosine residue, acting as electron carrier between P₆₈₀ and the water oxidizing system     

Electron paramagnetic resonance evidence for binding of Cu(2+) to the C-terminal domain of the murine prion protein.

Transmissible spongiform encephalopathies in mammals are believed to be caused by scrapie form of prion protein (PrP(Sc)), an abnormal, oligomeric isoform of the monomeric cellular prion protein (PrP(C)). One of the proposed functions of PrP(C) in vivo is a Cu(II) binding activity. Previous studies revealed that Cu(2+) binds to the unstructured N-terminal PrP(C) segment (residues 23-120) through conserved histidine residues. Here we analyzed the Cu(II) binding properties of full-length murine PrP(C) (mPrP), of its isolated C-terminal domain mPrP(121-231) and of the N-terminal fragment mPrP(58-91) in the range of pH 3-8 with electron paramagnetic resonance spectroscopy. We find that the C-terminal domain, both in its isolated form and in the context of the full-length protein, is capable of interacting with Cu(2+). Three Cu(II) coordination types are observed for the C-terminal domain. The N-terminal segment mPrP(58-91) binds Cu(2+) only at pH values above 5.0, whereas both mPrP(121-231) and mPrP(23-231) already show identical Cu(II) coordination in the pH range 3-5. As the Cu(2+)-binding N-terminal segment 58-91 is not required for prion propagation, our results open the possibility that Cu(2+) ions bound to the C-terminal domain are involved in the replication of prions, and provide the basis for further analytical studies on the specificity of Cu(II) binding by PrP.     

Amino acid sequence studies on cyanogen bromide peptides of chicken caldesmon which bind to calmodulin

Three major calmodulin-binding cyanogen bromide peptides (fragments A, B, and D) were isolated from chicken gizzard muscle caldesmon and their amino acid sequences were determined. The molecular masses of fragments A, B, and D were estimated to 16, 12, and 9 kDa, respectively, by SDS-urea polyacrylamide gel electrophoresis. Fragment A was composed of 102 amino acid residues and contained homoserine at the C terminus. The amino acid sequence from the 37th residue of fragment A corresponds to the N-terminal sequence of the 15 kDa peptide which was obtained by thrombin digestion [Mornet, D., Audemard, E., & Derancourt, J. (1988) Biochem. Biophys. Res. Commun. 154, 564-571]. Thrombin 15 kDa peptide binds to F-actin but does not bind to calmodulin. Thus the N-terminal 36 residues and the C-terminal part from the 37th residue of fragment A are supposed to bind to calmodulin and F-actin, respectively. The sequences of fragments B and D were identical, but fragment D was composed of 64 amino acid residues and ended with tryptophan, whereas fragment B was of 98 or 99 amino acid residues and ended with proline. Both fragments B and D are supposed to be the C-terminal peptides of chicken caldesmon. Fragment B had heterogeneous sequences at the C-terminal region. These results can explain the reported heterogeneity of chicken caldesmon in charge and molecular mass.     

Preparation of subfragment-1 from abalone smooth muscle myosin

Myosin subfragment-1 (S1), which has one heavy chain (HC) (93 kDa) and two light chains (LC1 and LC2), was prepared by papain digestion of myosin from abalone-smooth muscle in the presence of Ca2+. The Ca-sensitivity of abalone S1 itself was not lost completely (about 30%). The tryptic digestion of S1 showed that in the presence of EDTA, S1 HC was split into 68, 55, and 23 kDa fragments, as in the presence of Ca2+, but 23 kDa was further degraded into 19 kDa. In contrast to the result in the presence of Ca2+, LCs disappeared in the early stage of reaction and Ca-ATPase activity decreased rapidly to about 70% of that of intact S1. This rapid decrease of Ca-ATPase activity seemed to be accompanied with the digestion of LCs. Therefore, LCs contribute to the protection of 23 kDa fragment from further digestion, to the maintenance of Ca-ATPase activity by stabilizing the structure of S1 to some extent in the presence of Ca2+. Since F-actin suppressed the cleavage of S1 HC to 68 and 23 kDa during tryptic digestion, it might be that 23 and 68 kDa corresponded to 20 kDa (C-terminal fragment) and to 50 + 25 kDa (N-terminal fragment) of skeletal myosin S1, respectively.     

Characterization of truncated forms of abnormal prion protein in Creutzfeldt-Jakob disease

In prion disease, the abnormal conformer of the cellular prion protein, PrP(Sc), deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrP(Sc) to proteolytic digestion in vitro generates a core fragment of 19-21 kDa, named PrP27-30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27-30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2-3 kDa faster than PrP27-30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27-30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27-30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5- or 17-kDa fragments and the previously described 13-kDa PrP(Sc) C-terminal fragment relatively to the PrP27-30 signal significantly differed among CJD subtypes. Furthermore, protease digestion of PrP(Sc) or PrP27-30 in partially denaturing conditions generated an additional truncated fragment of about 16 kDa only in typical sporadic CJD (i.e. MM1). These results show that the physicochemical heterogeneity of PrP(Sc) in CJD extends to abnormal truncated forms of the protein. The findings support the notion of distinct structural "conformers" of PrP(Sc) and indicate that the characterization of truncated PrP(Sc) forms may further improve molecular typing in CJD.     

Studies on the mechanism of photosystem II photoinhibition II. The involvement of toxic oxygen species

In a previous paper it was shown that photoinhibition of reaction centre II of spinach thylakoids was predominantly caused by the degradation of D1-protein. An initial inactivation step at the Q_B-site was distinguished from its breakdown. The present paper deals with the question as to whether this loss of Q_B-function is caused by oxygen radical attack. For this purpose the photoinhibition of thylakoids was induced at 20°C in the presence of either superoxide dismutase and catalase or the antioxidants glutathione and ascorbic acid. This resulted in comparable though not total protection of D1-protein, photochemistry and fluorescence from photoinhibition. The combined action of both the enzymatic and the non-enzymatic radical scavenging systems brought about an even more pronounced protective effect against photoinhibition than did either of the two systems singularly at saturating concentrations. The results signify a major contribution of activated oxygen species to the degradation process of D1-protein and the related phenomena of photoinhibition. Thylakoids treated with hydroxyl radicals generated through a Fenton reaction showed a loss of atrazine binding sites, electron transport capacity and variable fluorescence in a similar manner, though not to the same extent, as usually observed following photoinhibitory treatment.Abbreviations Asc ascorbate - Fecy ferricyanide - GSH reduced glutathione - PQ plastoquinone - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - SOD superoxide dismutase     

Role of visible light in the recovery of photosystem II structure and function from ultraviolet-B stress in higher plants

The effect of visible light on photosystem II reaction centre D1 protein in plants treated with ultraviolet-B light was studied. It was found that a 20 kDa C-terminal fragment of D1 protein generated during irradiation with ultraviolet-B light was stable when plants were incubated in the dark, but was degraded when plants were incubated in visible light. In this condition the recovery of photosynthetic activity was also observed. Even a low level of white light was sufficient to promote both further degradation of the fragment and recovery of activity. During this phase, the D1 protein is the main synthesized thylakoid polypeptide, indicating that other photosystem II proteins are recycled in the recovery process. Although both degradation of the 20 kDa fragment and resynthesis of D1 are light-dependent phenomena, they are not closely related, as degradation of the 20 kDa fragment may occur even in the absence of D1 synthesis. Comparing chemical and physical factors affecting the formation of the fragment in ultraviolet-B light and its degradation in white light, it was concluded that the formation of the fragment in ultraviolet-B light is a photochemical process, whereas the degradation of the fragment in white light is a protease-mediated process.     

Spectrofluorimetric studies on C-terminal 34 kDa fragment of caldesmon

Analysis of the tryptophan fluorescence emission spectra of caldesmon and its 34 kDa C-terminal fragment indicates that all tryptophan residues are located on the surface of the molecule, accessible to solvent. All three tryptophan residues of the 34 kDa fragment and four of the five tryptophan residues of intact protein are accessible to free water, whereas one located in the N-terminal region of molecule is accessible only to bound water molecules. The temperature dependence of the fluorescence parameters indicates higher thermal stability of the 34 kDa fragment than the whole caldesmon molecule. The interaction of the 34 kDa fragment of caldesmon (like that of the intact molecule) with calmodulin is accompanied by a blue shift of the fluorescence emission maximum and an increase in the relative quantum yield. Computer-calculated binding constants show that the binding of calmodulin to the 34 kDa fragment (K = 2.5 x 10(5) M-1) is of two orders of magnitude weaker than that to intact caldesmon (K = 1.4 x 10(7) M-1). The interaction with tropomyosin results in a blue shift of the spectrum of the 34 kDa fragment, yet there is no effect on the spectrum of intact caldesmon. Binding constants of tropomyosin to caldesmon (K = 3.8 x 10(5) M-1) and its 34 kDa fragment (K = 2.3 x 10(5) M-1) are similar. Binding of calmodulin to caldesmon and to the 34 kDa fragment affects their interaction with tropomyosin.     

Studies of the strand-annealing activity of mammalian hnRNP complex protein A1.

A1 is a major core protein of the mammalian hnRNP complex, and as a purified protein of approximately 34 kDa, A1 is a strong single-stranded nucleic acid binding protein. Several lines of evidence suggest that the protein is organized in discrete domains consisting of an N-terminal segment of approximately 22 kDa and a C-terminal segment of approximately 12 kDa. Each of these domains as a purified fragment is capable of binding to both ssDNA and RNA. We report here that A1 and its C-terminal domain fragment are capable of potent strand-annealing activity for base-pair complementary single-stranded polynucleotides of both RNA and DNA. This effect is not stimulated by ATP. Compared with A1 and the C-terminal fragment, the N-terminal domain fragment has negligible annealing activity. These results indicate that A1 has biochemical activity consistent with a strand-annealing role in relevant reactions, such as pre-mRNA splicing.     

Phospholipid transfer protein (PLTP) causes proteolytic cleavage of apolipoprotein A-I

Plasma phospholipid transfer protein (PLTP) is a factor that plays an important role in HDL metabolism. In this study we present data suggesting that PLTP has an inherent protease activity. After incubation of HDL3 in the presence of purified plasma PLTP, the d < 1.25 g/ml particles (fusion particles) contained intact 28.2 kDa apoA-I while the d > 1.25 g/ml fraction (apoA-I-PL complexes) contained, in addition to intact apoA-I, a cleaved 23 kDa form of apoA-I. Purified apoA-I was also cleaved by PLTP and produced a similar 23 kDa apoA-I fragment. The cleavage of apoA-I increased as a function of incubation time and the amount of PLTP added. The process displayed typically an 8-10 h lag or induction period, after which the cleavage proceeded in a time-dependent manner. This lag-phase was necessary for the development of the cleavage activity during incubation at 37 degrees C. The specific apoA-I cleavage activity of different PLTP preparations varied between 0.4-0.8 microg apoA-I degraded/h per 1000 nmol per h of PLTP activity. The 23 kDa apoA-I fragment reacted with monoclonal antibodies specific for the N-terminal part of apoA-I, indicating that the apoA-I cleavage occurred in the C-terminal portion. The apoA-I cleavage products were further characterized by mass spectrometry. The 23 kDa fragment yielded a mass of 22.924 kDa, demonstrating that the cleavage occurs in the C-terminal portion of apoA-I between amino acid residues 196 (alanine) and 197 (threonine). The intact apoA-I and the 23 kDa fragment revealed identical N-terminal amino acid sequences. The cleavage of apoA-I could be inhibited with APMSF and chymostatin, suggesting that it is due to a serine esterase-type of proteolytic activity. Recombinant PLTP produced in CHO cells or using the baculovirus-insect cell system caused an apoA-I cleavage pattern identical to that obtained with plasma PLTP. The present results raise the question of whether PLTP-mediated proteolytic cleavage of apoA-I might affect plasma HDL metabolism by generating a novel kinetic compartment of apoA-I with an increased turnover rate.     

Reconstitution of photosynthetic water splitting after salt-washing of oxygen-evolving Photosystem-II particles

Oxygen-evolving Photosystem-II particles were isolated after treatment of spinach chloroplasts with Triton X-100. Treatment of these particles with 2 M NaCl released polypeptides of 24 and 16 kDa concomitant with a loss of the water-splitting activity. Readdition of the concentrated 24-16 kDa protein fraction restored water splitting in the salt-washed particles, the extent of reconstitution being dependent upon the intensity of continuous light during the assay. Under flash illumination, the salt-washed particles transported the normal number of electrons from water to DCIP on the first two flashes but much less reduction occurred on all subsequent flashes; addition of reconstituting protein only slightly prevented this loss process. Absorbance difference spectroscopy revealed that the salt-washed particles were at least able to perform the normal S₁-S₂ transition. The results suggest that removal of the 24-16 kDa protein affects the efficiency of the higher S-state transitions. Additional components may be required for optimal reconstitution and interference with the secondary electron acceptor mechanism after salt washing was detected.     

Characterization of functional domains of the hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata

CEL-III is a Ca(2+)-dependent, galactose/N-acetylgalactosamine (GalNAc)-specific lectin isolated from the marine invertebrate Cucumaria echinata. This lectin exhibits strong hemolytic activity and cytotoxicity through pore formation in target cell membranes. The amino acid sequence of CEL-III revealed the N-terminal two-thirds to have homology to the B-chains of ricin and abrin, which are galactose-specific plant toxic lectins; the C-terminal one-third shows no homology to any known proteins. To examine the carbohydrate-binding ability of the N-terminal region of CEL-III, the protein comprising Pyr1-Phe283 was expressed in Escherichia coli cells. The expressed protein showed both the ability to bind to a GalNAc-immobilized column as well as hemagglutinating activity for rabbit erythrocytes, confirming that the N-terminal region has binding activity for specific carbohydrates. Since the C-terminal region could not be expressed in E. coli cells, a fragment containing this region was produced by limited proteolysis of the native protein by trypsin. The resulting C-terminal 15 kDa fragment of CEL-III exhibited a tendency to self-associate, forming an oligomer. When mixed with erythrocytes, the oligomer of the C-terminal fragment caused hemagglutination, probably due to hydrophobic interaction with cell membranes, while the monomeric fragment did not. Chymotryptic digestion of the preformed CEL-III oligomer induced upon lactose binding also yielded an oligomer of the C-terminal fragment comprising six molecules of the 16 kDa fragment. These results suggest that after binding to cell surface carbohydrate chains, CEL-III oligomerizes through C-terminal domains, leading to the formation of ion-permeable pores by hydrophobic interaction with the cell membrane.     

Quality control of Photosystem II: cleavage and aggregation of heat-damaged D1 protein in spinach thylakoids

Moderate heat stress (40 degrees C, 30 min) on spinach thylakoids induced cleavage of the D1 protein, producing an N-terminal 23-kDa fragment, a C-terminal 9-kDa fragment, and aggregation of the D1 protein. A homologue of Arabidopsis FtsH2 protease, which is responsible for degradation of the damaged D1 protein, was abundant in the stroma thylakoids. Two processes occurred in the thylakoids in response to heat stress: dephosphorylation of the D1 protein in the stroma thylakoids, and aggregation of the phosphorylated D1 protein in the grana. Heat stress also induced the release of the extrinsic PsbO, P and Q proteins from Photosystem II, which affected D1 degradation and aggregation significantly. The cleavage and aggregation of the D1 protein appear to be two alternative processes influenced by protein phosphorylation/dephosphorylation, distribution of FtsH, and intactness of the thylakoids.     

Cathepsin D processes human prolactin into multiple 16K-like N-terminal fragments: study of their antiangiogenic properties and physiological relevance

16K prolactin (PRL) is the name given to the 16-kDa N-terminal fragment obtained by proteolysis of rat PRL by tissue extracts or cell lysates, in which cathepsin D was identified as the candidate protease. Based on its antiangiogenic activity, 16K PRL is potentially a physiological inhibitor of tumor growth. Full-length human PRL (hPRL) was reported to be resistant to cathepsin D, suggesting that antiangiogenic 16K PRL may be physiologically irrelevant in humans. In this study, we show that hPRL can be cleaved by cathepsin D or mammary cell extracts under the same conditions as described earlier for rat PRL, although with lower efficiency. In contrast to the rat hormone, hPRL proteolysis generates three 16K-like fragments, which were identified by N-terminal sequencing and mass spectrometry as corresponding to amino acids 1-132 (15 kDa), 1-147 (16.5 kDa), and 1-150 (17 kDa). Biochemical and mutagenetic studies showed that the species-specific digestion pattern is due to subtle differences in primary and tertiary structures of rat and human hormones. The antiangiogenic activity of N-terminal hPRL fragments was assessed by the inhibition of growth factor-induced thymidine uptake and MAPK activation in bovine umbilical endothelial cells. Finally, an N-terminal hPRL fragment comigrating with the proteolytic 17-kDa fragment was identified in human pituitary adenomas, suggesting that the physiological relevance of antiangiogenic N-terminal hPRL fragments needs to be reevaluated in humans.