Mario Kuo - Academia.edu (original) (raw)

Papers by Mario Kuo

Experimental and Molecular Therapeutics

Oncotarget

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) binds to death receptors an... more Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) binds to death receptors and induces apoptosis in various cancer cell lines while sparing normal cells. Recombinant TRAIL has shown good safety and efficacy profiles in preclinical cancer models. However, clinical success has been limited due to poor PK and development of resistance to death receptor-induced apoptosis. We have addressed these issues by creating a fusion protein of TRAIL and arginine deiminase (ADI). The fusion protein benefits from structural and functional synergies between its two components and has an extended half-life in vivo. ADI downregulates survivin, upregulates DR5 receptor and sensitizes cancer cells to TRAIL induced apoptosis. ADI-TRAIL fusion protein was efficacious in a number of cell lines and synergized with some standard of care drugs. In an HCT116 xenograft model ADI-TRAIL localized to the tumor and induced dose-dependent tumor regression, the fusion protein was superior to rhTRAIL administered at the same molar amounts.

Oncotarget, Jan 16, 2016

The biological role of BMP-9 signaling in liver cancer remains dubious. To explore the potential ... more The biological role of BMP-9 signaling in liver cancer remains dubious. To explore the potential use of BMP-9 signaling for anti-cancer therapy, we used recombinant human BMP-9, which we referred to as MB109, to study the effect on growth of fifteen hepatocellular carcinoma (HCC) cell lines. MB109 effectively inhibits the proliferation of nine HCC cells in vitro. The anti-proliferative effect was found to be induced by turning on p21 signaling, which caused survivin suppression and G0/G1 cell cycle arrest. ID3 was identified to be the mediator of the MB109-induced p21 expression. Blocking the activity of p38 MAPK diminished ID3 and p21 expression, indicating that MB109 signals through a p38 MAPK/ID3/p21 pathway to arrest cell cycle progression. Moreover, prolonged MB109 treatment suppressed the expression of five prominent liver cancer stem cell (LCSC) markers, including CD44, CD90, AFP, GPC3 and ANPEP. Xenograft model confirmed the anti-tumor and LCSC-suppression capability of MB10...

Bacterial Ion Channels and Their Eukaryotic Homologs, 2005

Seeing is believing. Out of sight, out of mind. The mind has evolved to deal with" sizab... more Seeing is believing. Out of sight, out of mind. The mind has evolved to deal with" sizable" parents, peers, predators, and preys, so it is wired to gauge objects by our own size, revering large things, downplaying small ones, and ignoring the invisibles. Except in ...

The EMBO Journal, 2003

Although Kch of Escherichia coli is thought to be a K + channel by sequence homology, there is li... more Although Kch of Escherichia coli is thought to be a K + channel by sequence homology, there is little evidence that it actually conducts K + ions in vitro or in vivo. We isolated gain-of-function (GOF) Kch mutations that render bacteria speci®cally sensitive to K + ions. Millimolar added K + , but not Na + or sorbitol, blocks the initiation or continuation of mutant growth in liquid media. The mutations are mapped at the RCK (or KTN) domain, which is considered to be the cytoplasmic sensor controlling the gate. Additional mutations directed to the K +-®lter sequence rescue the GOF mutant. The apparent K +-speci®c conduction through the`loose-cannon' mutant channel suggests that the wild-type Kch channel also conducts, albeit in a regulated manner. Changing the internal ATG does not erase the GOF toxicity, but removes kch's short second product, suggesting that it is not required for channel function in vivo. The mutant phenotypes are better explained by a perturbation of membrane potential instead of internal K + concentration. Possible implications on the normal function of Kch are discussed.

Protein & Peptide Letters, 2010

Two-component systems (TCS) involving the His-Asp phosphotransfer are commonly utilized for signa... more Two-component systems (TCS) involving the His-Asp phosphotransfer are commonly utilized for signal transduction in prokaryotes in which the two essential components are a sensor histidine kinase (HK) receptor and a response regulator (RR). Despite great efforts in structural and functional characterization of signal perception mechanisms, the exact signaling mechanisms remain elusive for many TCSs. Mimicking the natural TCS signaling pathways, chimeric receptor kinases and response regulators have been constructed through the process of swapping modular domains of related TCSs. To design chimeras with new signaling pathways, domains from different proteins that have little relationship at the primary structural level but carrying desirable functional properties can be conjoined to engineer novel TCSs. These chimeras maintain the ability to respond to environmental stimulants by regulating protein phosphorylation to produce downstream output signals. Depending on the nature of external signals, chimeric TCSs can serve as a novel tool not only to examine the natural signaling mechanisms in TCSs, but also for industrial and clinical applications.

Proceedings of the National Academy of Sciences, 2007

Proceedings of the National Academy of Sciences, 2002

YggB and MscL are the major mechanosensitive channels in Escherichia coli, and each can rescue th... more YggB and MscL are the major mechanosensitive channels in Escherichia coli, and each can rescue the double knockout mutant from osmotic downshock. However, the role of MscL in wild-type bacteria is in question, not only because cells without MscL survive severe osmotic downshocks, but because 1.8 times more suction is required to gate MscL than YggB under patch clamp. Here, we extend previous evidence [Ajouz, B., Berrier, C., Garrigues, A., Besnard, M. & Ghazi, A. (1998) J. Biol. Chem. 273, 26670-26674] to show that downshock gates MscL in vivo even in the presence of YggB. We have made this determination by engineering a channel we can structurally modify in vivo (Leu-193 Cys MscL). MscLs with charges in their constrictions are known to open easily and transiently to substates and stop cell growth. In this study, we use downshock to stretch this region open to allow attachment of a charged thiosulfonate reagent MTSET ؉ , thereby creating a toxic channel. Therefore, channel opening can be monitored by loss of colony forming units. By this measure, we find that an Ϸ800 mmol͞kg downshock from 1,200 mmol͞kg medium opens Leu-193 Cys MscL in the presence of YggB, but a downshock of only Ϸ400 mmol͞kg is required in the absence of YggB. In parallel, Leu-193 Cys MscL, stretched open by large sustained suction in the presence of MTSET ؉ in voltage-clamped patches, subsequently flickers open with little suction. These observations show that MscL opening is triggered by a specific downshock, even in the presence of YggB, that YggB buffers MscL gating in vivo, and that residue 19 becomes exposed upon channel opening.

PLoS Biology, 2008

The RCK-containing MthK channel undergoes two inactivation processes: activation-coupled desensit... more The RCK-containing MthK channel undergoes two inactivation processes: activation-coupled desensitization and acidinduced inactivation. The acid inactivation is mediated by the C-terminal RCK domain assembly. Here, we report that the desensitization gating is governed by a desensitization domain (DD) of the cytoplasmic N-terminal 17 residues. Deletion of DD completely removes the desensitization, and the process can be fully restored by a synthetic DD peptide added in trans. Mutagenesis analyses reveal a sequence-specific determinant for desensitization within the initial hydrophobic segment of DD. Proton nuclear magnetic resonance (1 H NMR) spectroscopy analyses with synthetic peptides and isolated RCK show interactions between the two terminal domains. Additionally, we show that deletion of DD does not affect the acid-induced inactivation, indicating that the two inactivation processes are mutually independent. Our results demonstrate that the short N-terminal DD of MthK functions as a complete moveable module responsible for the desensitization. Its interaction with the C-terminal RCK domain may play a role in the gating process.

Microbial Cell Factories, 2014

Background: The development of chemical refolding of transforming growth factor-beta (TGF-β) supe... more Background: The development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods. Results: Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320-Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC 50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. Conclusion: MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of aggregation suppressors and the concentrations of protein, salt and detegent. These results add to the current understanding of producing recombinant TGF-β superfamily ligands in the microbial E. coli system. An application of the technique to produce a large number of synthetic TGF-β chimeras for activity screen is also discussed.

The Journal of General Physiology, 2005

Journal of Biological Chemistry, 2007

Prokaryotic ion channels have been valuable in providing structural models for understanding ion ... more Prokaryotic ion channels have been valuable in providing structural models for understanding ion filtration and channel-gating mechanisms. However, their functional examinations have remained rare and usually been carried out by incorporating purified channel protein into artificial lipid membranes. Here we demonstrate the utilization of Escherichia coli to host the functional analyses by examining a putative cyclic nucleotide-gated K ؉ channel cloned from Magnetospirillum magnetotacticum, MmaK. When expressed in wild-type E. coli cells, MmaK renders the host sensitive to millimolar concentrations of externally applied K ؉ , indicating MmaK forms a functional K ؉ conduit in the E. coli membrane in vivo. After enlarging these cells into giant spheroplasts, macro-and microscopic MmaK currents are readily detected in excised E. coli membrane patches by a patch clamp. We show that MmaK is indeed gated by submicromolar cAMP and ϳ10-fold higher concentration of cGMP and manifests as an inwardly rectified, K ؉-specific current with a 10.8 pS unitary conductance at ؊100 mV. Additionally, MmaK is inactivated by slightly acidic pH only from the cytoplasmic side. Our in vitro biophysical characterizations of MmaK correlate with its in vivo phenotype in E. coli, implicating its critical role as an intracellular cAMP and pH sensor for modulating bacterial membrane potential. Exemplified by MmaK functional studies, we establish that E. coli and its giant spheroplast provide a convenient and versatile system to express foreign channels for biophysical analyses that can be further dovetailed with microbial genetics. Recent sequencing of bacterial genomes reveals that ion channels evolved as early as three billion years ago. K ϩ channels, for example, are widely spread in all life forms, Bacteria, Archaea, and Eukarya (1, 2). Because prokaryotic channel genes can often be heterologously expressed in Escherichia coli at high yield, the channel proteins so produced have laid an inroad to determine their crystal structures. Beginning with the prelude of MacKinnon and Doyle (3) crystal structures of these channels have raised our understanding of the molecular bases of ion channels as illustrated in several atomic structures of prokaryotic K ϩ channels (4-9). Functional interpretation of prokaryotic channel structures by electrophysiological methods, however, has not been straightforward. The main technical barrier is that the prokaryotic channel activities are often difficult to analyze under the existing methodology. A common strategy has been reconstitution of the purified channel protein into artificial lipids for bilayer lipid membrane measurement (5, 10, 11), a process that relies on the chance survival of the channel during detergent extraction and lipid reconstitution. In some cases, the reconstituted channel activities can only be demonstrated with the low resolution 86 Rb ϩ uptake assay (9, 12-14). An often overlooked opportunity to capture these channels in action is the very membrane of the E. coli cells in which they are commonly overproduced. Although the rod of this bacterium (0.75-m diameter, 2 m in length) is about the dimension of a patch clamp pipette tip, there are genetic and pharmacological ways of generating giant E. coli some ten times its original size. In 1987, Martinac et al. (15) first described the enlargement of E. coli into giant spheroplast for direct patch clamp examination of the native mechanosensitive channels (16). Besides mechanosensitive channels, however, this pioneering method has seldom been extended to study the activities of other foreign channels. We have optimized the methods of functional preparation of giant E. coli spheroplast as well as patch clamp of the enlarged membrane to study MthK, the RCK (regulating the conductance of K ϩ)-containing K ϩ channel from Methanobacterium thermoautotrophicum (5). The success in detecting the ensemble current of MthK in E. coli membrane led us to discover its hitherto unknown properties, including deactivation, desensitization, acidic inactivation, and Cd 2ϩ activation (17). In this report, we illustrate the optimized method of giant spheroplast preparation and gigOhm seal formation in detail by functional expression and biophysical characterization of a bacterial cyclic nucleotide-gated K ϩ channel, MmaK 2 from M. magnetotacticum. Related extension of microbial genetics to ion channel research is also discussed. EXPERIMENTAL PROCEDURES Molecular Biology and Phenotype Analysis-The mmaK gene (NCBI 46202428) from M. magnetotacticum (genomic DNA from Dr. B. Martinac, University of Queensland, Austra-* This work was supported by National Institutes of Health Grants GM74821

FEMS Microbiology Reviews, 2005

The deep roots and wide branches of the K +-channel family are evident from genome surveys and la... more The deep roots and wide branches of the K +-channel family are evident from genome surveys and laboratory experimentation. K +-channel genes are widespread and found in nearly all the free-living bacteria, archaea and eukarya. The conservation of basic structures and mechanisms such as the K + filter, the gate, and some of the gateÕs regulatory domains have allowed general insights on animal K + channels to be gained from crystal structures of prokaryotic channels. Since microbes are the great majority of lifeÕs diversity, it is not surprising that microbial genomes reveal structural motifs beyond those found in animals. There are open-reading frames that encode K +-channel subunits with unconventional filter sequences, or regulatory domains of different sizes and numbers not previously known. Parasitic or symbiotic bacteria tend not to have K + channels, while those showing lifestyle versatility often have more than one K +-channel gene. It is speculated that prokaryotic K + channels function to allow adaptation to environmental and metabolic changes, although the actual roles of these channels in prokaryotes are not yet known. Unlike enzymes in basic metabolism, K + channel, though evolved early, appear to play more diverse roles than revealed by animal research. Finding and sorting out these roles will be the goal and challenge of the near future.

Aquaculture, 2012

ATP-binding cassette (ABC) transporters are integral membrane proteins which carry diverse substr... more ATP-binding cassette (ABC) transporters are integral membrane proteins which carry diverse substrates across biological membranes. In this study, we report the cloning of a full-length, 2122-bp cDNA sequence encoding a gene of the ABC transporter subfamily G (ABCG) from the shrimp Fenneropenaeus chinensis. The deduced protein, FcABCG, contains 633 amino acid residues with a theoretical molecular weight of 70.57 kDa. Phylogenetic analysis showed that the FcABCG clusters with the shrimp Litopenaeus vannamei ABCG in the same clade and is more closely related to the insect ABC transporter/white proteins than to the ABCG2 of the fish Salmo salar. High-level mRNA expression of FcABCG was detected in the tissues of hepatopancreas and intestine using a real-time quantitative reverse transcriptase PCR. Furthermore, exposure to alkaline pH stress conditions and norfloxacin diets result in the activation of the FcABCG gene in F. chinensis. These results suggest that the ABCG transporter of F. chinensis might play a role in coping high pH stress and in exporting xenobiotics.

Applied Microbiology and Biotechnology, 2011

Microbial phytases are widely used as feed additive to increase phytate phosphorus utilization an... more Microbial phytases are widely used as feed additive to increase phytate phosphorus utilization and to reduce fecal phytates and inorganic phosphate (iP) outputs. To facilitate the process of application, we engineered an Escherichia coli appA phytase gene into the chloroplast genome of the model microalga, Chlamydomonas reinhardtii, and isolated homoplasmic plastid transformants. The catalytic activity of the recombinant E. coli AppA can be directly detected in the whole-cell lysate, termed Chlasate, prepared by freeze-drying the transgenic cell paste with liquid nitrogen. The E. coli AppA in the Chlasate has a pH and temperature optima of 4.5 and 60°C, respectively, which are similar to those described in the literature. The phytase-expressed Chlasate contains 10 phytase units per gram dry matter at pH 4.5 and 37°C. Using this transgenic Chlasate at 500 U/kg of diet for young broiler chicks, the fecal phytate excretion was reduced, and the iP was increased by 43% and 41%, respectively, as compared to those of the chicks fed with only the basal diet. The effectiveness of the Chlasate to break down the dietary phytates is compatible with the commercial Natuphos fungal phytase. Our data provide the first evidence of functional expression of microbial phytase in microalgae and demonstrate the proof of concept of using transgenic microalgae as a food additive to deliver dietary enzymes with no need of protein purification.

Biomaterials, 2014

BMP-9, whose expression is highest in liver cells, has been demonstrated to regulate expression o... more BMP-9, whose expression is highest in liver cells, has been demonstrated to regulate expression of enzymes involved in glucose homeostasis. However, the underlying mechanism of this effect has yet to be elucidated. We observed that MB109, a recombinant BMP-9 derivative, enhanced brown adipogenesis of human adipose tissue derived stem cells. With this observation of the cell culture system, we hypothesized that MB109 may be able to improve glucose metabolism by regulating expression of brown adipogenic genes. Systemic intraperitoneal injection of MB109 (200 mg/kg/wk) suppressed weight gaining of high fat diet-induced obese mice by reducing sizes of white adipocytes and decreased 16 h fasting blood glucose levels without changing food consumption or apparent behavioral performances. MB109 induced expression of brown adipogenic genes in the subcutaneous but not in the visceral fat tissues from the mice fed with high fat diet. In addition, systematic injection of MB109 enhanced fatty acid synthase expression in the liver of obese mice, which may help attenuate an obesity-associated increase of blood glucose levels. Our results demonstrate a role of BMP-9 in brown adipogenesis and suppressing pathophysiology of high fat diet-induced obesity, presumably through the activin receptor like kinase 1 signaling pathway.