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51.
Protein body formation in leaves of Nicotiana benthamiana: a concentration‐dependent mechanism influenced by the presence of fusion tags 下载免费PDF全文
Reza Saberianfar Jussi J. Joensuu Andrew J. Conley Rima Menassa 《Plant biotechnology journal》2015,13(7):927-937
Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles originally found in seeds whose function is to accumulate seed storage proteins. It has been shown that PB formation is not limited to seeds and green fluorescent protein (GFP) fused to either elastin‐like polypeptide (ELP) or hydrophobin (HFBI) fusion tags induce the formation of PBs in leaves of N. benthamiana. In this study, we compared the ELP‐ and HFBI‐induced PBs and showed that ELP‐induced PBs are larger than HFBI‐induced PBs. The size of ELP‐ and HFBI‐induced PBs increased over time along with the accumulation levels of their fused protein. Our results show that PB formation is a concentration‐dependent mechanism in which proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. Our results show that the presence of fusion tags is not necessary for the formation of PBs, but affects the distribution pattern and size of PBs. This was confirmed by PBs induced by fluorescent proteins as well as fungal xylanases. We noticed that in the process of PB formation, secretory and ER‐resident molecules are passively sequestered into the lumen of PBs. We propose to use this property of PBs as a tool to increase the accumulation levels of erythropoietin and human interleukin‐10 by co‐expression with PB‐inducing proteins. 相似文献
52.
The AP7 and AP24 proteins represent a class of mineral-interaction polypeptides that are found in the aragonite-containing nacre layer of mollusk shell (H. rufescens). These proteins have been shown to preferentially interfere with calcium carbonate mineral growth in vitro. It is believed that both proteins play an important role in aragonite polymorph selection in the mollusk shell. Previously, we demonstrated the 1-30 amino acid (AA) N-terminal sequences of AP7 and AP24 represent mineral interaction/modification domains in both proteins, as evidenced by their ability to frustrate calcium carbonate crystal growth at step edge regions. In this present report, using free N-terminal, C(alpha)-amide "capped" synthetic polypeptides representing the 1-30 AA regions of AP7 (AP7-1 polypeptide) and AP24 (AP24-1 polypeptide) and NMR spectroscopy, we confirm that both N-terminal sequences possess putative Ca (II) interaction polyanionic sequence regions (2 x -DD- in AP7-1, -DDDED- in AP24-1) that are random coil-like in structure. However, with regard to the remaining sequences regions, each polypeptide features unique structural differences. AP7-1 possesses an extended beta-strand or polyproline type II-like structure within the A11-M10, S12-V13, and S28-I27 sequence regions, with the remaining sequence regions adopting a random-coil-like structure, a trait common to other polyelectrolyte mineral-associated polypeptide sequences. Conversely, AP24-1 possesses random coil-like structure within A1-S9 and Q14-N16 sequence regions, and evidence for turn-like, bend, or loop conformation within the G10-N13, Q17-N24, and M29-F30 sequence regions, similar to the structures identified within the putative elastomeric proteins Lustrin A and sea urchin spicule matrix proteins. The similarities and differences in AP7 and AP24 N-terminal domain structure are discussed with regard to joint AP7-AP24 protein modification of calcium carbonate growth. 相似文献
53.
Shade effect alters leaf pigments and photosynthetic responses in Norway spruce (Picea abies L.) grown under field conditions 总被引:1,自引:0,他引:1
The contents of chlorophyll (Chl) and carotenoids (Car) per fresh mass were lower in shade needles than in sun needles. Ribulose-1,5-bisphosphate
carboxylase (RuBPC) activity and contents of soluble proteins were also significantly lower in shade needles. In isolated
thylakoids, a marked lower rate of whole chain and photosystem (PS) 2 activities were observed in shade needles. Smaller lower
rate of PS1 activity was also observed in shade needles. The artificial exogenous electron donors, diphenyl carbazide (DPC)
and NH2OH, significantly restored the loss of PS2 activity in shade needles. Similar results were obtained when Fv/Fm was evaluated by Chl fluorescence measurements. The marked lower rate of PS2 activity in shade needles was due to the lower
contents of 47, 33, 28–25, 23, and 17 kDa polypeptides. This conclusion was confirmed by immunological studies showing that
the content of the 33 kDa protein of the watersplitting complex was diminished significantly in shade needles. 相似文献
54.
Gomes SA Fonseca de Souza AL Silva BA Kiffer-Moreira T Santos-Mallet JR Santos AL Meyer-Fernandes JR 《Experimental parasitology》2006,112(4):253-262
Trypanosoma rangeli is a parasite of a numerous wild and domestic animals, presenting wide geographical distribution and high immunological cross-reactivity with Trypanosoma cruzi, which may lead to misdiagnosis. T. rangeli has a complex life cycle, involving distinct morphological and functional forms in the vector. Here, we characterized the cell surface polypeptides and the phosphatase activities in short and long epimastigotes forms of T. rangeli, using intact living parasites. The surface protein profile revealed by the incubation of parasites with biotin showed a preferential expression of the 97, 70, 50, 45, 25-22, and 15 kDa biotinylated polypeptides in the long forms, in contrast to the 55 and 28 kDa biotinylated polypeptides synthesized by the short epimastigotes. Additionally, flow cytometry analysis showed that the short forms had relatively lower biotin surface binding than long ones. The involvement of phosphatases with the trypanosomatid differentiation has been proposed. In this sense, T. rangeli living parasites were able to hydrolyze the artificial substrate p-nitrophenylphosphate at a rate of 25.57+/-2.03 and 10.09+/-0.93 nmol p-NPP x h(-1) x 10(7) cells for the short and long epimastigotes, respectively. These phosphatase activities were linear with time for at least 60 min and the optimum pH lies in the acid range. Classical inhibitors of acid phosphatases, such as ammonium molybdate, sodium fluoride, and zinc chloride, showed a significant decrease in these phosphatase activities, with different patterns of inhibition. Additionally, these phosphatase activities presented different kinetic parameters (Km and Vmax) and distinct sensitivities to divalent cations. Both epimastigotes were unable to release phosphatase to the extracellular environment. Cytochemical analysis demonstrated the localization of these enzymes on the parasite surfaces (cell body and flagellum) and in intracellular vacuoles, resembling acidocalcisomes. 相似文献
55.
Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system. 相似文献
56.
Song Y McFarland DC Velleman SG 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2012,161(1):44-52
Syndecan-4 core protein is composed of extracellular, transmembrane, and cytoplasmic domains. The cytoplasmic domain functions in transmitting signals into the cell through the protein kinase C alpha (PKCα) pathway. The glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains attached to the extracellular domain influence cell proliferation. The current study investigated the function of syndecan-4 cytoplasmic domain in combination with GAG and N-glycosylated chains in turkey muscle cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) responsiveness, and PKCα membrane localization. Syndecan-4 or syndecan-4 without the cytoplasmic domain and with or without the GAG and N-glycosylated chains were transfected or co-transfected with a small interfering RNA targeting syndecan-4 cytoplasmic domain into turkey muscle satellite cells. The overexpression of syndecan-4 mutants increased cell proliferation but did not change differentiation. Syndecan-4 mutants had increased cellular responsiveness to FGF2 during proliferation. Syndecan-4 increased PKCα cell membrane localization, whereas the syndecan-4 mutants decreased PKCα cell membrane localization compared to syndecan-4. However, compared to the cells without transfection, syndecan-4 mutants increased cell membrane localization of PKCα. These data indicated that the syndecan‐4 cytoplasmic domain and the GAG and N-glycosylated chains are critical in syndecan-4 regulating satellite cell proliferation, responsiveness to FGF2, and PKCα cell membrane localization. 相似文献
57.
Stefania Mazzini Leonardo Scaglioni Rosanna Mondelli Raniero Rocchi Laura Biondi Marina Gobbo 《Journal of peptide science》2005,11(8):452-462
The conformational properties of the homo oligomers of increasing chain length Boc-(Asn)(n)-NHMe (n = 2, 4, 5), (GlcNAc-beta-Asn)(n)-NHMe (n = 2, 4, 5, 8) and Boc-[GlcNAc(Ac)(3)-beta-Asn](n)-NHMe (n = 2, 4, 5) were studied by using NOE experiments and molecular dynamic calculations (MD). Sequential NOEs and medium range NOEs, including (i,i+2) interactions, were detected by ROESY experiments and quantified. The calculated inter-proton distances are longer than those characteristic of beta-turn secondary structures. Owing to the large conformational motions expected for linear peptides, MD simulations were performed without NMR constraints, with explicit water and by applying different treatments of the electrostatic interactions. In agreement with the NOE results, the simulations showed, for all peptides, the presence of both folded and unfolded structures. The existence of significant populations of beta-turn structures can be excluded for all the examined compounds, but two families of structures were more often recognized. The first one with sinusoidal or S-shaped forms, and another family of large turns together with some more extended conformations. Only the glycosylated pentapeptide shows in vacuo a large amount of structures with helical shaped form. The results achieved in water and in DMSO are compared and discussed, together with the effect of the glycosylation. 相似文献
58.
Gu Ruisheng Liu Qiunlu Pei Dong Jiang Xiangning 《Plant Cell, Tissue and Organ Culture》2004,78(3):261-265
Tolerance of Populus euphratica suspended cells to ionic and osmotic stresses implemented respectively by NaCl and PEG (6000) was characterized by monitoring
cell growth, morphological features, ion compartmentation and polypeptide patterns. The cells grew and proliferated when submitted
to stresses of 137 mM NaCl or 250 g l−1 PEG, and survived at 308 mM of NaCl, showing tolerance to saline and particularly osmotic stress. They were resistant to
plasmolysis and had dense cytoplasms, large nuclei and nucleoli, and evident cytoplasmic strands under high saline and osmotic
stress. The sequestration of Cl− into the vacuoles was observed in the cells stressed with 137 and 223 mM NaCl. The cellular protein profile was modified
by high salt and osmotic stress and showed 28 kDa polypeptides up-regulated by both NaCl and PEG, and 66 and 25 kDa polypeptides
up-regulated only by high NaCl stress. The salt tolerance of P. euphratica cells might be related to their capacity of adapting to higher osmotic stress by maintaining cell integrity, sequestrating
Cl− into vacuoles and modulating polypeptides that reflect cellular metabolic adaptations. 相似文献
59.
Effects of NaCl Stress on the Structure,Pigment Complex Composition,and Photosynthetic Activity of Mangrove Bruguiera parviflora Chloroplasts 总被引:7,自引:1,他引:6
Exposure of two-month-old seedlings of Bruguiera parviflora to NaCl stress (0 to 400 mM) for 45 d under hydroponic culture caused notable disorganisation of the thylakoid structure
of chloroplasts in NaCl-treated leaves as revealed from transmission electron microscopy. The absorption spectra of treated
and control thylakoid samples were similar having a red peak at 680 nm and Soret peaks at 439 and 471 nm in the blue region
of the spectrum. The spectra of treated samples differed from control samples by gradual decrease in absorbance of 100, 200,
and 400 mM NaCl treated samples at 471 and 439 nm, which could be due to scattering of radiation in these samples. Thus, absorption
characteristics of thylakoid membranes indicated no major alterations in the structural integrity of the photosynthetic membranes
during salt stress in B. parviflora. Analysis of pigment protein complexes of thylakoids on non-denaturing gel showed that CP1 complex consisting of photosystem
(PS) 1 reaction centre decreased marginally by 19% and the CP47 constituting the core antenna of PS2 declined significantly
by 30% in 400 mM NaCl treated samples in respect to control. This decrease in structural core antenna might cause inefficient
photon harvesting capacity. However, CP43 content did not alter. An increase in CP2/CP1 ratio from 3.2 in control to 4.0 in
400 mM NaCl treated samples indicated significant structural changes in the thylakoids of salt treated plants. Haem staining
of thylakoids revealed significant losses in cytochrome (Cyt)f and Cyt b
6 contents by NaCl stress. However, Cyt b
559 content remained nearly constant in both control and NaCl treated samples. SDS-PAGE of thylakoid proteins showed that the
intensity of many of Coomassie stained polypeptide bands ranging from 15–22 and 28–66 kDa regions decreased significantly
in NaCl treated samples as compared to control. Electron transport activity of thylakoids, measured in terms of DCPIP photoreduction,
was 22% lower in 400 mM NaCl treated plants than in the control ones. Hence, NaCl induces oxidative stress in chloroplasts
causing structural alterations in thylakoids. These structural alterations might be responsible for declined efficiency of
photosystems and reduced electron transport activity.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
60.
Richard Hahin Ziyi Chen Danhui Wang Giridher Reddy Long Mao 《Cell biochemistry and biophysics》2002,37(3):169-186
We have purified a new toxin (BmK 17[4]) from Asian scorption (Buthus martensii Karsch) venom that possesses a distinctive structural motif in its N-terminal (positions 8–12) that is similarly found in two other
previously described α-like toxins. BmK 17[4] prolongs action potentials (APs) in frog nerve and was purified using gel filtration,
ion exchange, fast protein liquid chromatography (FPLC), and high-performance liquid chromatography (HPLC). BmK 17[4] significantly
prolonged frog APs but it did not alter APs from an insect ventral nerve cord at similar doses. When applied to voltage-clamped
frog muscle single fibers, BmK 17[4] prolonged fast inactivation. Because the polypeptide prolongs APs when both K+ and Ca2+ channels were blocked, BMK 17[4] acts to selectively alter Na+ channel inactivation. The N-terminal sequence of BmK 17[4] was found to be VRDAYIAKPENCVYXC—. The molar mass of BmK 17[4]
was determined by LC/MS/MS to be 7097 Daltons. The N-terminal motif (KPENC), which introduces a reverse turn in residues 8–12,
does not appear in previously characterized BmK α-toxins and may be characteristic of α-like toxins. Sequence similarity database
searches were used to test whether the N-terminal sequences of α-like polypeptide toxins from B. martensii Karsch possess a distinctive structural motif in its 5-residue reverse turn (α-turn) that is conserved. Sequence similarities with
putative polypetides encoded by cDNAs obtained from a cDNA library [Zhu, S. Y., Li, W. X., Zenq, X. C., et al. (2000) Nine
novel precursors of Buthus martensii scorpiox alpha-toxin homologues. Toxicon
38, 1653–1661] from BmK venom glands showed that an active polypeptide toxin cleaved from the putative propolypeptide toxin
BmK M9 is likely identical to BmK 17[4]. Sequence comparisons with toxins and putative toxins from B. martensii Karsch and other species revealed that a group of these toxins possess a common structural motif in their α-turn. A neighbor-joining
phylogenetic analysis suggests that there are two phylogenetic sister groups of related BmK polypeptides; one possesses the
KPENC motif and the other possesses a modifed version (KPHNC) of it. 相似文献