Bioluminescent RIPoptosome Assay for FADD/RIPK1 Interaction Based on Split Luciferase Assay in a Human Neuroblastoma Cell Line SH-SY5Y.

Different programed cell death (PCD) modalities involve protein-protein interactions in large complexes. Tumor necrosis factor α (TNFα) stimulated assembly of receptor-interacting protein kinase 1 (RIPK1)/Fas-associated death domain (FADD) interaction forms Ripoptosome complex that may cause either apoptosis or necroptosis. The present study addresses the interaction of RIPK1 and FADD in TNFα signaling by fusion of C-terminal (CLuc) and N-terminal (NLuc) luciferase fragments to RIPK1-CLuc (R1C) or FADD-NLuc (FN) in a caspase 8 negative neuroblastic SH-SY5Y cell line, respectively. In addition, based on our findings, an RIPK1 mutant (R1C K612R) had less interaction with FN, resulting in increasing cell viability. Moreover, presence of a caspase inhibitor (zVAD.fmk) increases luciferase activity compared to Smac mimetic BV6 (B), TNFα -induced (T) and non-induced cell. Furthermore, etoposide decreased luciferase activity, but dexamethasone was not effective in SH-SY5Y. This reporter assay might be used to evaluate basic aspects of this interaction as well as for screening of necroptosis and apoptosis targeting drugs with potential therapeutic application.

Inhibition of noncaspase proteases, calpain and proteasome, via ALLN and Bortezomib contributes to cell death through low degradation of pro-/anti-apoptotic proteins and apoptosis induction.

Dysfunction at any regulatory point along the apoptotic signaling pathway is closely related to many diseases including cancers. The apoptotic protein expression level is an important cause of cancer-related death, and the correct degradation of apoptotic proteins is involved in tumor development. Therefore, understanding of a regulatory point that underlying cancer-related death may help the development of new strategies to overcome the clinical challenges. Here, proteasome inhibitor Bortezomib and calpain inhibitor ALLN were examined on protein levels of caspase-3, caspase-9, XIAP, and E3-ligase PARC in HEK293T cells overexpressing XIAP and caspase-9. ATP depletion and caspase-3 activation were as a consequence of Bortezomib and ALLN function. Higher numbers of PI-stained cells provided evidence of cell death by both inhibitors. Western blotting analysis showed that both ALLN and Bortezomib equally inhibited degradation of XIAP, but only ALLN was effective at inhibiting caspase proteolytic degradation. Moreover, treatment of cells with both types of inhibitors significantly increased the level of E3-ligase PARC. Our findings showed that inhibition of proteasome and calpains enhanced the level of anti-apoptotic, XIAP and PARC, and pro-apoptotic, caspase-9 and 3 proteins, which totally promote cell death significantly.

XIAP as a multifaceted molecule in Cellular Signaling.

XIAP, X-linked inhibitor of apoptosis protein, is a member of the inhibitor of apoptosis protein (IAP) family known for its important conserved inhibitory effect on caspase activity. Since the introduction of XIAP almost three decades ago, numerous experimental studies have been performed for a wide range of cellular pathways and mechanisms. In this perspective, we summarize key trends of XIAP as an important regulator of cellular signaling. Experimental research indicates that XIAP as a key molecule of cell death not only suppress caspases and apoptosis, but also regulates inflammatory signaling, mitogenic kinase signaling, cell proliferation as well as cell invasion and metastasis. In this review, we provide basic knowledge of the roles of XIAP, explain its role in necroptosis, anoikis, autophagy and neuronal differentiation. XIAP is involved in regulating intracellular ROS production and copper homeostasis which this review focuses on. A different face of XIAP in response to DNA damage and chronic ER stress is also discussed.

Upregulation of apoptotic protease activating factor-1 expression correlates with anti-tumor effect of taxane drug.

Drug resistance is a multifactorial process involving a variety of mechanisms and genes. Taxane drug class like Docetaxel is not effective for all types' breast cancers and presents a huge clinical challenge. To improve cancer treatment outcome, it is important to distinguish which proteins can kill the cancer cells and whether the expression levels of these proteins affect treatment. Cancer cells are wildly known to be protected from apoptosis, due to low level of apoptotic protease activating factor-1 (Apaf-1) compared with normal cells. Apaf-1 is an essential protein that defines whether cytochrome c released form mitochondria remains stable or degrades. According to this hypothesis, increasing of Apaf-1 expression in MCF7 breast cancer cells was performed and Docetaxel efficacy examined. The immunoassay techniques were used to investigate Apaf-1 and cytochrome c levels, and different apoptosis assay methods applied to better understand the effect of Apaf-1 expression levels in cellular response to apoptotic stimuli by Docetaxel. Our results determined that cytoplasmic cytochrome c level elevated along with increasing Apaf-1 and MCF7 cells were sensitised to Docetaxel, suggesting that loss of Apaf-1 may cause Docetaxel-resistance in breast cancer cells through less apoptosome formation. ROS level increased in cells transfected with Apaf-1 and induced mitochondrial permeability for cytochrome c release, which subsequently promoted apoptosome formation, intrinsic apoptosis and ATP depletion.

Treating MCF7 breast cancer cell with proteasome inhibitor Bortezomib restores apoptotic factors and sensitizes cell to Docetaxel.

Chemoresistance is the leading cause of limiting long-term treatment success in cancer cells. Anticancer drugs usually kill cells through apoptosis induction and defects in this signaling pathway lead to chemoresistance. Apoptotic protease activating factor 1 regulates cellular stress evoked by chemotherapeutic agents through facilitating apoptosome assembling but can be degraded by proteasome. This study examined the role of proteasome inhibitor Bortezomib in the cytotoxic effects of Docetaxel on MCF7 cells response and its correlation with Apaf-1 expression level. MTT assay, caspase 3/7 activity assay, propidium iodide staining, adenosine triphosphate and reactive oxygen species amount measurements were utilized to demonstrate the role of Bortezomib in Docetaxel efficacy with and without Apaf-1 overexpressing. Meanwhile, two-dimensional cell migration assay was performed by scratch wound assay. The combination of Docetaxel with Bortezomib was significantly more cytotoxic compared single drug, more effectively delayed cell growth, reduced ATP level and increased ROS production. In Apaf-1 overexpressing, Docetaxel was more efficient in preventing cell migration, however, Docetaxel plus Bortezomib were not significantly effective; and fluorescence images supported the interpretation. Our findings demonstrated MCF7 resistance to Docetaxel is due in part to low Apaf-1 level and Apaf-1 overexpression resulted in the increase of cell susceptibility to Docetaxel stimulus. We assume that proteasome inhibitor may restore apoptotic proteins like Apaf-1 and prevent the degradation of cytosolic cytochrome c released by Docetaxel, consequently triggering intrinsic apoptosis and promoting cancer cell death. Collectively, treating MCF7 breast cells with proteasome inhibitor sensitizes cells to Docetaxel-induced apoptosis and possibly overcomes chemoresistance.

Efficient Stable Cell Line Generation of Survivin as an In Vitro Model for Specific Functional Analysis in Apoptosis and Drug Screening.

Recognizing proteins that lead to a decreased efficiency of treatment in cancer cells constitutes a main goal for biomedical and biotechnological research and applications. Establishing recombinant cells that overexpress a gene of interest stably is important for treatment studies and drug/compound screening. Survivin is an anti-apoptotic protein which can be a potential candidate for regulating cell death and survival. To investigate the association between survivin increment and apoptosis rate, survivin-reconstituted HEK (HEK-S) cell was developed as in vitro model. RT-PCR and Western blot demonstrated that survivin was constitutively overexpressed in HEK-S cells. Both morphological observation and survival assay showed that HEK-S cells were significantly resistant to apoptotic stimuli. Survivin overexpression led to a decrease in caspase 3/7 activity, whereas YM155 led to a corresponding enhance of caspase activity. ROS level was decreased but ATP content increased in HEK-S cells. Also, HEK-S showed less red- fluorescence and reduced cell proliferation compared to HEK after stimulation. Resistance to laser irradiation was clearly visible as compared with control. Moreover, scratching analysis demonstrated the ability of survivin to cause neighboring cells to increase resistance to drug, whereas YM155 enhanced apoptotic rate and declined invasion in HEK-S cells.

Effects of doxorubicin and docetaxel on susceptibility to apoptosis in high expression level of survivin in HEK and HEK-S cell lines as in vitro models.

A major problem in the cancer treatment is the inherent resistance to chemotherapy. Identifying proteins that, once introduced in cancer cells, lead to a decreased efficiency of treatment outcome constitutes a major goal for biomedical research and applications. Survivin is a protein of IAPs family which its high expression can be a potential candidate for regulating cell death and survival in cancer therapy. To investigate the association of survivin increment and resistance to drug, survivin-reconstituted HEK (HEK-S) and HEK cells were used as in vitro models for the doxorubicin and docetaxel cellular response. Both morphological observation and survival assay exhibited that survivin reconstitution cells were significantly resistant to apoptotic stimuli by both drugs. It was observed that survivin overexpression has led to a decrease in caspase 3/7 activity and ROS level of cells but an increase in ATP content. Also, survivin-reconstituted cell displayed less red fluorescence compared to control after stimulation by drugs. Moreover, wound healing assay showed the ability of survivin to cause neighbouring cells to increase resistance to induction. These findings demonstrated survivin could be a potential target that can be inhibited the function of different drugs with various mechanisms in chemotherapy.

A Luminescent Amine-Functionalized Metal-Organic Framework Conjugated with Folic Acid as a Targeted Biocompatible pH-Responsive Nanocarrier for Apoptosis Induction in Breast Cancer Cells.

Folic acid amine-functionalized metal-organic framework (FOLA@NH2-Eu:TMU-62) with luminescent properties loaded with 5-fluorouracil (5-Fu), as an anticancer medication, was used to construct a new cancer targeted drug delivery system in the present study. The 5-Fu release from this targeted carrier along with MTT assay and trypan blue dye exclusion test results also exhibited pH-controlled characteristics of the given carrier in acidic environments, which is very suitable for targeting solid tumors. Then, the inhibitory action of 5-Fu-loaded FOLA@NH2-Eu:TMU-62 for Michigan Cancer Foundation-7 (MCF7) cell migration was explored according to scratch wound healing assays. Based on the results, the FOLA@NH2-Eu:TMU-62 carrier was not toxic for MCF-10A normal cells, but it was significantly toxic for MCF-7 breast cancer ones, revealing that the FOLA@NH2-Eu:TMU-62 carrier could be utilized in accurate cancer treatments through apoptotic pathways with higher reactive oxygen species compared with 5-Fu alone. This cancer-targeted design of FOLA@NH2-Eu:TMU-62 could thus pave the way for synergistic effects of targeting as well as organized release capabilities.

Effect of mutation at positively charged residues (K329 and R330) in a flexible region of firefly luciferase on structure and kinetic properties.

Firefly luciferase as a bioluminescent enzyme has many applications in various fields from scientific research to commercial goals. This enzyme is relatively unstable with low functional capacity due to rapid inactivation in physiological temperature, low in vitro stability and high susceptibility to proteolytic degradation. Based on previous studies, two regions 206-220 and 329-341 on N-domain of Photinus pyralis luciferase are known accessible and flexible. Flexible regions may lead to protein instability. Here, the effect of mutation at positively charged residues Lys(K)329 and Arg(R)330 on the stability of luciferase was studied. Furthermore, the role of these mutations on the structure and function was evaluated. Introducing of these point mutations did not affect the orientation of critical residues in bioluminescence color determination. The kinetic studies showed that thermostability and Km value for luciferin in both mutants were decreased as compared to wild type. However, optimum pH and optimum temperature showed no significant changes in both mutants. Moreover, the structural data revealed an increase in tryptophan fluorescence intensity and secondary structure content for R330Q in compared with wild type, while intrinsic fluorescence and far-UV CD intensity in K329I mutant was decreased.

The effects of ovarian cancer cell-derived exosomes on vascular endothelial growth factor expression in endothelial cells.

Ovarian carcinoma is considered as a major clinical challenge worldwide. Exosomes, nano-sized intraluminal vesicles of multivesicular bodies, are secreted by most types of cells and play an important role in intercellular communication. Cancer cell-derived exosomes can develop cancer progression and metastasis by manipulating the local and distant biological environments. Angiogenesis is an important contributor to tumor progression. Vascular endothelial growth factor (VEGF) is the most potent pro-angiogenic protein and induces proliferation, sprouting, and vessel formation by endothelial cells. In this study, exosomes derived from ovarian epithelial cancer cells OVACAR-3 (exo-OVCAR-3) were successfully isolated and characterized by scanning electron microscopy in terms of size and morphology. Cellular internalization of exosomes labeled with PKH fluorescent dye was monitored by a fluorescence microscope. Our results elucidated that exosomes treatment (100 µg/ml) had a promoting effect on VEGF expression and secretion in endothelial cells. Furthermore, we demonstrated that exo-OVCAR-3 caused an increase in the proliferation and migration rate of endothelial cells. It seems that inducing VEGF by exo-OVCAR-3 can influence the vascular behavior of endothelial cells in vitro.

Experimental and theoretical study of IBC domain from human IP3R2; molecular cloning, bacterial expression and protein purification.

IP3 is a ubiquitous second messenger in eukaryotic cells that triggers Ca2+-release from intracellular stores. IP3 binds to intracellular IP3-receptor (IP3R) and induces conformational change within the ligand-binding domain which regulates Ca2+-release; hence, both IP3 and IP3R are key components of the signal transduction mechanism. Here we present cDNA cloning of IP3-binding core (IBC) domain encoding only residues 224-604 of human IP3R type 2 that binds to IP3 with high affinity. RNA extraction, RT-PCR, PCR and cloning were carried out, and then the cloned DNA was checked by sequencing. Thereafter, expression vector pET-28a harboring the correct gene was transformed into different E. coli (DE3) strains and investigated its protein expression under various conditions. Finally, the IBC expression was induced at 20 °C for 20 h into BL21 strain at LB medium with 4 mM lactose and 0.5 mM IPTG, and then confirmed by western blotting. After protein purification, structural study was recorded in absence and presence of its ligand. Far-CD and intrinsic fluorescence spectra analysis of the purified protein with and without IP3 ligand showed change in secondary and tertiary IBC structure. Moreover, bioinformatics study demonstrated that the ligand binding site residues R269, K508 and R511 are conserved.

Tris-chelated complexes of nickel(II) with bipyridine derivatives: DNA binding and cleavage, BSA binding, molecular docking, and cytotoxicity.

Two nickel(II) complexes with substituted bipyridine ligand of the type [Ni(NN)3](ClO4)2, where NN is 4,4'-dimethyl-2,2'-bipyridine (dimethylbpy) (1) and 4,4'-dimethoxy-2,2'-bipyridine (dimethoxybpy) (2), have been synthesized, characterized, and their interaction with DNA and bovine serum albumin (BSA) studied by different physical methods. X-ray crystal structure of 1 shows a six-coordinate complex in a distorted octahedral geometry. DNA-binding studies of 1 and 2 reveal that both complexes sit in DNA groove and then interact with neighboring nucleotides differently; 2 undergoes a partial intercalation. This is supported by molecular-docking studies, where hydrophobic interactions are apparent between 1 and DNA as compared to hydrogen bonding, hydrophobic, and π-π interactions between 2 and DNA minor groove. Moreover, the two complexes exhibit oxidative cleavage of supercoiled plasmid DNA in the presence of hydrogen peroxide as an activator in the order of 1 > 2. In terms of interaction with BSA, the results of spectroscopic methods and molecular docking show that 1 binds with BSA only via hydrophobic contacts while 2 interacts through hydrophobic and hydrogen bonding. It has been extensively demonstrated that the nature of the methyl- and methoxy-groups in ligands is a strong determinant of the bioactivity of nickel(II) complexes. This may justify the above differences in biomolecular interactions. In addition, the in vitro cytotoxicity of the complexes on human carcinoma cells lines (MCF-7, HT-29, and U-87) has been examined by MTT assay. According to our observations, 1 and 2 display cytotoxicity activity against selected cell lines. Communicated by Ramaswamy H. Sarma.

Chitosan Immobilization on Bio-MOF Nanostructures: A Biocompatible pH-Responsive Nanocarrier for Doxorubicin Release on MCF-7 Cell Lines of Human Breast Cancer.

In this work, a bio-metal-organic framework (Bio-MOF) coated with a monodispersed layer of chitosan (CS; CS/Bio-MOF) was synthesized and applied as a pH-responsive and target-selective system for delivery of doxorubicin (DOX) in the treatment of breast cancer. The efficiency of the nanocarrier in loading and releasing DOX was assessed at different pH levels. To monitor the in vitro drug release behavior of the drug-loaded carrier, the carrier was immersed in a phosphate buffered saline solution (PBS, pH 7.4) at 37 °C. According to the observations, the nanocarrier presents a slow and continuous release profile as well as a noticeable drug loading capacity. In addition, the carrier demonstrates a pH-responsive and target-selective behavior by releasing a high amount of DOX at pH 6.8, which is indicative of tumor cells and inflamed tissues and releasing a substantially lower amount of DOX at higher pH values. In addition, the results indicated that pH is effective on DOX uptake by CS/Bio-MOF. A 3.6 mg amount of DOX was loaded into 10 mg of CS/Bio-MOF, resulting in a 21.7% removal at pH 7.4 and 93.0% at pH 6.8. The collapsing and swelling of the CS layers coated on the surface of the Bio-MOFs were found to be responsible for the observed pH dependence of DOX delivery. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the trypan blue test were performed on the MCF-7 (breast cancer) cell line in the presence of the CS/Bio-MOF carrier to confirm its biological compatibility. In addition, Annexin V staining was conducted to evaluate the cytotoxicity of the free and loaded DOX molecules. On the basis of the obtained optical microscopy, MTT assay, fluorescence microscopy, and dyeing results, the CS/Bio-MOF carrier greatly enhances cellular uptake of the drug by the MCF-7 cells and, therefore, apoptosis of the cells due to its biocompatibility and pH-responsive behavior.

Design of a remote-control drug delivery implantable chip for cancer local on demand therapy using ionic polymer metal composite actuator.

Since the local, on demand, cancer therapy is a challenging clinical issue today, this paper presents the design, fabrication and characterization of a remotely controlled single reservoir drug delivery chip using Ionic Polymer Metal Composite (IPMC) actuator. Here, Drug release was externally programmed and controlled wirelessly on demand by a communication circuit. The transmitter and receiver circuits were designed to control the release/sealed status remotely even from 7 cm distance while the transmitter and receiver were coupled magnetically. IPMC here was used as the moving cap of the reservoir, that in release mode, lets the drug out on demand with a low received power of 20 mW. The novel simple design could release the whole content of the drug which is remarkable in comparison with the designs which need complicated optimizations of diffuser, nuzzle and IPMC diaphragm pump, leading to an incomplete release. To make sure that there is no leakage in the sealed mode, IPMC was attached to a polydimethylsiloxane (PDMS) support film. Biocompatibility of all the components of the chip were tested by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay.

Mutation of conserved residues K329 and R330 on the surface of firefly luciferase: Effect on proteolytic degradation.

Firefly luciferase is highly susceptible to proteolytic digestion that reduces its half-life and leads to loss in sensitivity. Due to the protease contamination in most in vitro and in vivo environments, it has interest to generate some mutations that may lead to improved susceptibility to digestion. Some important conserved residues (including K206, R213, R218, K329, R330 and R337) on accessible and flexible regions on the surface of Photinus pyralis luciferase have been suggested that susceptible to trypsinolysis. In current study, two mutants (K329I and R330Q) are designed to investigate the impact of these conserved sites on the protease stability and flexibility. This study showed that these mutations did not cause resistance against trypsin digestion. K329I mutant was more susceptible to trypsin, but no difference in the digestion pattern was observed. This point mutation brought about structural flexibility, which revealed by quenching and extrinsic fluorescence. The experimental and theoretical studies demonstrated that R330Q mutagenesis didn't have any noticeable effect on the tryptic sites and flexibility. Moreover, the results of proteolysis experiment showed that the primary sites for trypsin digestion are still exposed after both mutations.

Insufficient Apaf-1 expression in early stages of neural differentiation of human embryonic stem cells might protect them from apoptosis.

Recent evidence suggests that mitochondrial apoptosis regulators and executioners may regulate differentiation, without being involved in cell death. However, the involved factors and their roles in differentiation and apoptosis are still not fully determined. In the present study, we compared mitochondrial pathway of cell death during early neural differentiation from human embryonic stem cells (hESCs). Our results demonstrated that ROS generation, cytosolic cytochrome c release, caspases activation and rise in p53 protein level occurred upon either neural or apoptosis induction in hESCs. However, unlike apoptosis, no remarkable increase in apoptotic protease activating factor-1 (Apaf-1) level at early stages of differentiation was observed. Also the caspase-like activity of caspase-9 and caspase-3/7 were seen less than apoptosis. The results suggest that low levels of Apaf-1 as an adaptor protein might be considered as a possible regulatory barrier by which differentiating cells control cell death upon rise in ROS production and cytochrome c release from mitochondria. Better understanding of mechanisms via which mitochondria-mediated apoptotic pathway promote neural differentiation can result in development of novel therapeutic approaches.

Increase of Bacillus badius Phenylalanine dehydrogenase specificity towards phenylalanine substrate by site-directed mutagenesis.

Phenylalanine dehydrogenase (PheDH) is a key enzyme in medical diagnostic for determining the amount of phenylalanine to detect phenylketonuria (PKU) disease. However, determination of phenylalanine can be usually disturbed in presence of tyrosine in blood samples. Position N145 of B.sphaericus PheDH, has been previously showed a crucial role in substrate binding, which corresponded by position V144 in B. badius PheDH. In this study, the PheDH of B. badius due to reasonable activity was cloned and subjected to site-directed mutagenesis at mentioned position, followed by kinetic and structural studies to find more exclusive mutants. The results showed that the V144L mutant considerably increases specificity toward phenylalanine and decreases toward l-tyrosine, while in V144N mutant, the specificity reduces toward phenylalanine and increases toward tyrosine. Moreover, concerning the mutated V144D, significantly reduced kcat and also decreased km value for phenylalanine relative to that of wild type. The Phe/Tyr specificity constant in V144L increased more than 4-fold compared to wild type, makes it to be a suitable candidate for more specific identification of PKU. Finally, docking and molecular dynamic simulation on wild type and mutants clarified the structural basis behind more specificity of V144L mutant for phenylalanine substrate.

MicroRNA-100 shuttled by mesenchymal stem cell-derived exosomes suppresses in vitro angiogenesis through modulating the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells.

BACKGROUND: Human mesenchymal stem cells (MSCs) have been shown to be involved in the formation and modulation of tumor stroma and in interacting with tumor cells, partly through their secretome. Exosomes are nano-sized intraluminal multi-vesicular bodies secreted by most types of cells and have been found to mediate intercellular communication through the transfer of genetic information via coding and non-coding RNAs to recipient cells. Since exosomes are considered as protective and enriched sources of shuttle microRNAs (miRNAs), we hypothesized that exosomal transfer of miRNAs from MSCs may affect tumor cell behavior, particularly angiogenesis. METHODS: Exosomes derived from MSCs were isolated and characterized by scanning electron microscopy analyses, dynamic light scattering measurements, and Western blotting. Fold changes in miR-100 expression levels were calculated in exosomes and their corresponding donor cells by qRT-PCR. The effects of exosomal transfer of miR-100 from MSCs were assessed by qRT-PCR and Western blotting of the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells. The quantification of secreted VEGF protein was determined by enzyme-linked immunosorbent assay. The putative paracrine effects of MSC-derived exosomes on tumor angiogenesis were explored by in vitro angiogenesis assays including endothelial cell proliferation, migration and tube formation assays. RESULTS: We found that MSC-derived exosomes induce a significant and dose-dependent decrease in the expression and secretion of vascular endothelial growth factor (VEGF) through modulating the mTOR/HIF-1α signaling axis in breast cancer-derived cells. We also found that miR-100 is enriched in MSC-derived exosomes and that its transfer to breast cancer-derived cells is associated with the down-regulation of VEGF in a time-dependent manner. The putative role of exosomal miR-100 transfer in regulating VEGF expression was substantiated by the ability of anti-miR-100 to rescue the inhibitory effects of MSC-derived exosomes on the expression of VEGF in breast cancer-derived cells. In addition, we found that down-regulation of VEGF mediated by MSC-derived exosomes can affect the vascular behavior of endothelial cells in vitro. CONCLUSIONS: Overall, our findings suggest that exosomal transfer of miR-100 may be a novel mechanism underlying the paracrine effects of MSC-derived exosomes and may provide a means by which these vesicles can modulate vascular responses within the microenvironment of breast cancer cells.

Bifunctional role of leucine 300 of firefly luciferase in structural rigidity.

Firefly luciferase is susceptible to thermal inactivation, thereby its intracellular half-life decreased. Previous reports indicated that L300R mutation (LRR mutant) in E354R/Arg356 double mutant (ERR mutant) from Lampyris turkestanicus luciferase has increased its thermal stability and rigidity through induction of some ionic bonds with Asp 270 and 271. Disruption of the deduced ionic bonds in an ultra-rigid mutant of firefly luciferase did not reverse the flexibility of the protein. In this study, we investigated the effects of this residue to find the truth behind an extraordinary increase in thermal stability and rigidity of luciferase after replacement of leucine 300 by arginine based on previous reports. For this purpose, L300R, L300K and L300E mutations were performed to compare the effects of these mutations on the native firefly luciferase. In spite of increase of intrinsic fluorescence of the mutants a slight increase in thermostability and retention of kinetic properties was observed. Based on our results, we can conclude that L300R mutation in LRR mutant accompanying with alteration in a flexible loop (352-359) increased thermostability and rigidity of luciferase.

Luciferin-Regenerating Enzyme Crystal Structure Is Solved but its Function Is Still Unclear.

Contribution of luciferin-regenerating enzyme (LRE) for in vitro recycling of D-luciferin has been reported. According to crystal structure of LRE, it is a beta-propeller protein which is a type of all ß-protein architecture. In this overview, reinvestigation of the luciferase-based LRE assays and its function is reported. Until now, sequence of LRE genes from four different species of firefly has been reported. In spite of previous reports, T-LRE (from Lampyris turkestanicus) was cloned and expressed in Escherichia coli as well as Pichia pastoris in a nonsoluble form as inclusion body. According to recent investigations, bioluminescent signal of soluble T-LRE-luciferase-coupled assay increased and then reached an equilibrium state in the presence of D-cysteine. In addition, the results revealed that both D- and L-cysteine in the absence of T-LRE caused a significant increase in bioluminescence intensity of luciferase over a long time. Based on activity measurements and spectroscopic results, D-cysteine increased the activity of luciferase due to its redox potential and induction of conformational changes in structure and kinetics properties. In conclusion, in spite of previous reports on the effect of LRE (at least T-LRE) on luciferase activity, most of the increase in luciferase activity is caused by direct effect of D-cysteine on structure and activity of firefly luciferase. Moreover, bioinformatics analysis cannot support the presence of LRE in peroxisome of photocytes in firefly lanterns.