Mechanistic studies of intracellular delivery of proteins by cell-penetrating peptides in cyanobacteria

BMC Microbiology

Han-Jung Lee¹  Yue-Wern Huang²  Betty R Liu¹ 

[1]Department of Natural Resources and Environmental Studies, National Dong Hwa University, Hualien 97401, Taiwan

[2]Department of Biological Sciences, Missouri University of Science and Technology, Rolla, MO 65409-1120, USA

关键词: Red fluorescent protein (RFP);    Protein transduction;    Macropinocytosis;    Green fluorescent protein (GFP);    Endocytosis;    Cell-penetrating peptide (CPP);   

Others  :  1144209
DOI  :  10.1186/1471-2180-13-57

 received in 2012-08-11, accepted in 2013-03-08,  发布年份 2013

【 摘 要 】

Background

The plasma membrane plays an essential role in selective permeability, compartmentalization, osmotic balance, and cellular uptake. The characteristics and functions of cyanobacterial membranes have been extensively investigated in recent years. Cell-penetrating peptides (CPPs) are special nanocarriers that can overcome the plasma membrane barrier and enter cells directly, either alone or with associated cargoes. However, the cellular entry mechanisms of CPPs in cyanobacteria have not been studied.

Results

In the present study, we determine CPP-mediated transduction efficiency and internalization mechanisms in cyanobacteria using a combination of biological and biophysical methods. We demonstrate that both Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 strains of cyanobacteria possess red autofluorescence. Green fluorescent protein (GFP), either alone or noncovalently associated with a CPP comprised of nine arginine residues (R9/GFP complexes), entered cyanobacteria. The ATP-depleting inhibitor of classical endocytosis, N-ethylmaleimide (NEM), could block the spontaneous internalization of GFP, but not the transduction of R9/GFP complexes. Three specific inhibitors of macropinocytosis, cytochalasin D (CytD), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and wortmannin, reduced the efficiency of R9/GFP complex transduction, indicating that entry of R9/GFP complexes involves macropinocytosis. Both the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) and membrane leakage analyses confirmed that R9/GFP complexes were not toxic to the cyanobacteria, nor were the endocytic and macropinocytic inhibitors used in these studies.

Conclusions

In summary, we have demonstrated that cyanobacteria use classical endocytosis and macropinocytosis to internalize exogenous GFP and CPP/GFP proteins, respectively. Moreover, the CPP-mediated delivery system is not toxic to cyanobacteria, and can be used to investigate biological processes at the cellular level in this species. These results suggest that both endocytic and macropinocytic pathways can be used for efficient internalization of regular protein and CPP-mediated protein delivery in cyanobacteria, respectively.

【 授权许可】

   
2013 Liu et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Ruffing AM: Engineered cyanobacteria: teaching an old bug new tricks. Bioeng Bugs 2011, 2:136-149.
• [2]Herranen M, Battchikova N, Zhang P, Graf A, Sirpio S, Paakkarinen V, Aro EM: Towards functional proteomics of membrane protein complexes in Synechocystis sp. PCC 6803. Plant Physiol 2004, 134:470-481.
• [3]Huang F, Hedman E, Funk C, Kieselbach T, Schroder WP, Norling B: Isolation of outer membrane of Synechocystis sp. PCC 6803 and its proteomic characterization. Mol Cell Proteomics 2004, 3:586-595.
• [4]Shestakov SV, Khyen NT: Evidence for genetic transformation in blue-green alga Anacystis nidulans. Mol Gen Genet 1970, 107:372-375.
• [5]Balasubramanian L, Subramanian G, Nazeer TT, Simpson HS, Rahuman ST, Raju P: Cyanobacteria cultivation in industrial wastewaters and biodiesel production from their biomass: a review. Biotechnol Appl Biochem 2011, 58:220-225.
• [6]Crosthwaite SK: Circadian timekeeping in Neurospora crassa and Synechococcus elongates. Essays Biochem 2011, 49:37-51.
• [7]Machado IMP, Atsumi S: Cyanobacterial biofuel production. J Biotechnol 2012, 162:50-56.
• [8]Green M, Loewenstein PM: Autonomous functional domains of chemically synthesized human immunodeficiency virus Tat trans-activator protein. Cell 1988, 55:1179-1188.
• [9]Frankel AD, Pabo CO: Cellular uptake of the Tat protein from human immunodeficiency virus. Cell 1988, 55:1189-1193.
• [10]Vives E, Brodin P, Lebleu B: A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 1997, 272:16010-16017.
• [11]Wadia JS, Dowdy SF: Protein transduction technology. Curr Opinion Biotechnol 2002, 13:52-56.
• [12]Fonseca SB, Pereira MP, Kelley SO: Recent advances in the use of cell-penetrating peptides for medical and biological applications. Adv Drug Deliv Rev 2009, 61:953-964.
• [13]Tunnemann G, Ter-Avetisyan G, Martin RM, Stockl M, Herrmann A, Cardoso MC: Live-cell analysis of cell penetration ability and toxicity of oligo-arginines. J Pept Sci 2008, 14:469-476.
• [14]Futaki S: Arginine-rich peptides: potential for intracellular delivery of macromolecules and the mystery of the translocation mechanisms. Int J Pharm 2002, 245:1-7.
• [15]Lee CY, Li JF, Liou JS, Charng YC, Huang YW, Lee HJ: A gene delivery system for human cells mediated by both a cell-penetrating peptide and a piggyBac transposase. Biomaterials 2011, 32:6264-6276.
• [16]Dai YH, Liu BR, Chiang HJ, Lee HJ: Gene transport and expression by arginine-rich cell-penetrating peptides in Paramecium. Gene 2011, 489:89-97.
• [17]Chen YJ, Liu BR, Dai YH, Lee CY, Chan MH, Chen HH, Chiang HJ, Lee HJ: A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides. Gene 2012, 493:201-210.
• [18]Liu BR, Lin MD, Chiang HJ, Lee HJ: Arginine-rich cell-penetrating peptides deliver gene into living human cells. Gene 2012, 505:37-45.
• [19]Liou JS, Liu BR, Martin AL, Huang YW, Chiang HJ, Lee HJ: Protein transduction in human cells is enhanced by cell-penetrating peptides fused with an endosomolytic HA2 sequence. Peptides 2012, 37:273-284.
• [20]Liu MJ, Chou JC, Lee HJ: A gene delivery method mediated by three arginine-rich cell-penetrating peptides in plant cells. Adv Stud Biol 2013, 5:71-88.
• [21]Liu BR, Chiang HJ, Huang YW, Chan MH, Chen HH, Lee HJ: Cellular internalization of quantum dots mediated by cell-penetrating peptides. Pharm Nanotechnol 2013, 1:151-161.
• [22]Hu JW, Liu BR, Wu CY, Lu SW, Lee HJ: Protein transport in human cells mediated by covalently and noncovalently conjugated arginine-rich intracellular delivery peptides. Peptides 2009, 30:1669-1678.
• [23]Li JF, Huang Y, Chen RL, Lee HJ: Induction of apoptosis by gene transfer of human TRAIL mediated by arginine-rich intracellular delivery peptides. Anticancer Res 2010, 30:2193-2202.
• [24]Lu SW, Hu JW, Liu BR, Lee CY, Li JF, Chou JC, Lee HJ: Arginine-rich intracellular delivery peptides synchronously deliver covalently and noncovalently linked proteins into plant cells. J Agric Food Chem 2010, 58:2288-2294.
• [25]Gump JM, Dowdy SF: TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol Med 2007, 13:443-448.
• [26]Liu BR, Chou JC, Lee HJ: Cell membrane diversity in noncovalent protein transduction. J Membr Biol 2008, 222:1-15.
• [27]Liu BR, Huang YW, Chiang HJ, Lee HJ: Primary effectors in the mechanisms of transmembrane delivery of arginine-rich cell-penetrating peptides. Adv Stud Biol 2013, 5:11-25.
• [28]Madani F, Lindberg S, Langel U, Futaki S, Graslund A: Mechanisms of cellular uptake of cell-penetrating peptides. J Biophys 2011, 2011:414729.
• [29]Chang M, Chou JC, Chen CP, Liu BR, Lee HJ: Noncovalent protein transduction in plant cells by macropinocytosis. New Phytol 2007, 174:46-56.
• [30]Chen CP, Chou JC, Liu BR, Chang M, Lee HJ: Transfection and expression of plasmid DNA in plant cells by an arginine-rich intracellular delivery peptide without protoplast preparation. FEBS Lett 2007, 581:1891-1897.
• [31]Liu BR, Li JF, Lu SW, Lee HJ, Huang YW, Shannon KB, Aronstam RS: Cellular internalization of quantum dots noncovalently conjugated with arginine-rich cell-penetrating peptides. J Nanosci Nanotechnol 2010, 10:6534-6543.
• [32]Xu Y, Liu BR, Chiang HJ, Lee HJ, Shannon KS, Winiarz JG, Wang TC, Chiang HJ, Huang YW: Nona-arginine facilitates delivery of quantum dots into cells via multiple pathways. J Biomed Biotechnol 2010, 2010:948543.
• [33]Liu BR, Huang YW, Winiarz JG, Chiang HJ, Lee HJ: Intracellular delivery of quantum dots mediated by a histidine- and arginine-rich HR9 cell-penetrating peptide through the direct membrane translocation mechanism. Biomaterials 2011, 32:3520-3537.
• [34]Hou YW, Chan MH, Hsu HR, Liu BR, Chen CP, Chen HH, Lee HJ: Transdermal delivery of proteins mediated by non-covalently associated arginine-rich intracellular delivery peptides. Exp Dermatol 2007, 16:999-1006.
• [35]Schulze K, Lopez DA, Tillich UM, Frohme M: A simple viability analysis for unicellular cyanobacteria using a new autofluorescence assay, automated microscopy, and ImageJ. BMC Biotechnol 2011, 11:118. BioMed Central Full Text
• [36]Tillich UM, Lehmann S, Schulze K, Duhring U, Frohme M: The optimal mutagen dosage to induce point-mutations in Synechocystis sp. PCC6803 and its application to promote temperature tolerance. PLoS One 2012, 7:e49467.
• [37]Hajek J, Vaczi P, Bartak M, Jahnova L: Interspecific differences in cryoresistance of lichen symbiotic algae of genus Trebouxia assessed by cell viability and chlorophyll fluorescence. Cryobiology 2012, 64:215-222.
• [38]Sato M, Murata Y, Mizusawa M: A simple and rapid dual-fluorescence viability assay for microalgae. Microbiol Cult Collect 2004, 20:53-59.
• [39]Zeder M, Van den Wyngaert S, Koster O, Felder KM, Pernthaler J: Automated quantification and sizing of unbranched filamentous cyanobacteria by model-based object-oriented image analysis. Appl Environ Microbiol 2010, 76:1615-1622.
• [40]Kroth PG: Protein transport into secondary plastids and the evolution of primary and secondary plastids. Int Rev Cytol 2002, 221:191-255.
• [41]Karapetyan NV: Non-photochemical quenching of fluorescence in cyanobacteria. Biochemistry (Mosc) 2007, 72:1127-1135.
• [42]Chang M, Chou JC, Lee HJ: Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells. Plant Cell Physiol 2005, 46:482-488.
• [43]Liu K, Lee HJ, Leong SS, Liu CL, Chou JC: A bacterial indole-3-acetyl-L-aspartic acid hydrolase inhibits mung bean (Vigna radiata L.) seed germination through arginine-rich intracellular delivery. J Plant Growth Regul 2007, 26:278-284.
• [44]Ma DX, Shi NQ, Qi XR: Distinct transduction modes of arginine-rich cell-penetrating peptides for cargo delivery into tumor cells. Int J Pharm 2011, 419:200-208.
• [45]Koksharova OA, Wolk CP: Genetic tools for cyanobacteria. Appl Microbiol Biotechnol 2002, 58:123-137.
• [46]Ruffing AM, Jones HDT: Physiological effects of free fatty acid production in genetically engineered Synechococcus elongates PCC 7942. Biotechnol Bioeng 2012, 109:2190-2199.
• [47]Chang M, Hsu HY, Lee HJ: Dye-free protein molecular weight markers. Electrophoresis 2005, 26:3062-3068.
• [48]Nazarenko LV, Andreev IM, Lyukevich AA, Pisareva TV, Los DA: Calcium release from Synechocystis cells induced by depolarization of the plasma membrane: MscL as an outward Ca2+ channel. Microbiology 2003, 149:1147-1153.
• [49]Karnauchov I, Herrmann RG, Pakrasi HB, Klosgen RB: Transport of CtpA protein from the cyanobacterium Synechocystis 6803 across the thylakoid membrane in chloroplasts. Eur J Biochem 1997, 249:497-504.
• [50]Stamatakis K, Papageorgiou GC: The osmolality of the cell suspension regulates phycobilisome-to-photosystem I excitation transfers in cyanobacteria. Biochim Biophys Acta 2001, 1506:172-181.
• [51]Toyomasu T, Tsukahara M, Kaneko A, Niida R, Mitsuhashi W, Dairi T, Kato N, Sassa T: Fusicoccins are biosynthesized by an unusual chimera diterpene synthase in fungi. Proc Natl Acad Sci USA 2007, 104:3084-3088.
• [52]Liu BR, Huang YW, Chiang HJ, Lee HJ: Cell-penetrating peptide-functionized quantum dots for intracellular delivery. J Nanosci Nanotechnol 2010, 10:7897-7905.
• [53]Nanbo A, Imai M, Watanabe S, Noda T, Takahashi K, Neumann G, Halfmann P, Kawaoka Y: Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner. PLoS Pathog 2010, 6:e1001121.
• [54]Hansen SH, Olsson A, Casanova JE: Wortmannin, an inhibitor of phosphoinositide 3-kinase, inhibits transcytosis in polarized epithelial cells. J Biol Chem 1995, 270:28425-28432.
• [55]Mellerick DM, Liu H: Methanol exposure interferes with morphological cell movements in the Drosophila embryo and causes increased apoptosis in the CNS. J Neurobiol 2004, 60:308-318.
• [56]Li J, Song L: Applicability of the MTT assay for measuring viability of cyanobacteria and algae, specifically for Microcystis aeruginosa (Chroococcales, Cyanobacteria). Phycologia 2007, 46:593-599.
• [57]Adav SS, Lin JC, Yang Z, Whiteley CG, Lee DJ, Peng XF, Zhang ZP: Stereological assessment of extracellular polymeric substances, exo-enzymes, and specific bacterial strains in bioaggregates using fluorescence experiments. Biotechnol Adv 2010, 28:255-280.
• [58]Wang YH, Chen CP, Chan MH, Chang M, Hou YW, Chen HH, Hsu HR, Liu K, Lee HJ: Arginine-rich intracellular delivery peptides noncovalently transport protein into living cells. Biochem Biophys Res Commun 2006, 346:758-767.
• [59]Wang YH, Hou YW, Lee HJ: An intracellular delivery method for siRNA by an arginine-rich peptide. J Biochem Biophys Methods 2007, 70:579-586.