Chii-Shen Yang

The α-Helical Domain of Gαt Determines Specific Interaction with Regulator of G Protein Signaling 9

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein α subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the α subunit of transducin, Gαt. Here we report another unique property of RGS9: high specificity for Gαt. The core (RGS) domain of RGS9 (RGS9) stimulates Gαt GTPase activity by 10-fold and Gαi1 GTPase activity by only 2-fold at a concentration of 10 μm. Using chimeric Gαt/Gαi1 subunits we demonstrated that the α-helical domain of Gαt imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Gα subunits as measured by a change in fluorescence of a mutant Gαt (Chi6b) selectively labeled at Cys-210.K d values for RGS9 complexes with Gαtand Gαi1 calculated from the direct binding and competition experiments were 185 nm and 2 μm, respectively. The γ subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pγ to increase the affinity of RGS9 for Gαt. A distinct, nonoverlapping pattern of RGS and Pγ interaction with Gαt suggests a unique mechanism of effector-mediated GAP function of the RGS9.

The Cytoplasmic Membrane-proximal Domain of the HtrII Transducer Interacts with the E-F Loop of Photoactivated Natronomonas pharaonis Sensory Rhodopsin II

The structures of the cytoplasmic loops of the phototaxis receptor sensory rhodopsin II (SRII) and the membrane-proximal cytoplasmic domain of its bound transducer HtrII were examined in the dark and in the light-activated state by fluorescent probes and cysteine cross-linking. Light decreased the accessibility of E-F loop position 154 in the SRII-HtrII complex, but not in free SRII, consistent with HtrII proximity, which was confirmed by tryptophans placed within a 5-residue region identified in the HtrII membrane-proximal domain that exhibited Förster resonance energy transfer to a fluorescent probe at position 154 in SRII. The Förster resonance energy transfer was eliminated in the signaling deficient HtrII mutant G83F without loss of affinity for SRII. Finally, the presence of SRII and HtrII reciprocally inhibit homodimer disulfide cross-linking reactions in their membrane-proximal domains, showing that each interferes with the others self-interaction in this region. The results demonstrate close proximity between SRII-HtrII in the membrane-proximal domain, and in addition, light stimulation of the SRII inhibition of HtrII cross-linking was observed, indicating that the contact is enhanced in the photoactivated complex. A mechanism is proposed in which photoactivation alters the SRII-HtrII interaction in the membrane-proximal region during the signal relay process.

A Novel Six-Rhodopsin System in a Single Archaeon

Microbial rhodopsins, a diverse group of photoactive proteins found in Archaea, Bacteria, and Eukarya, function in photosensing and photoenergy harvesting and may have been present in the resource-limited early global environment. Four different physiological functions have been identified and characterized for nearly 5,000 retinal-binding photoreceptors, these being ion transporters that transport proton or chloride and sensory rhodopsins that mediate light-attractant and/or -repellent responses. The greatest number of rhodopsins previously observed in a single archaeon had been four. Here, we report a newly discovered six-rhodopsin system in a single archaeon, Haloarcula marismortui, which shows a more diverse absorbance spectral distribution than any previously known rhodopsin system, and, for the first time, two light-driven proton transporters that respond to the same wavelength. All six rhodopsins, the greatest number ever identified in a single archaeon, were first shown to be expressed in H. marismortui, and these were then overexpressed in Escherichia coli. The proteins were purified for absorption spectra and photocycle determination, followed by measurement of ion transportation and phototaxis. The results clearly indicate the existence of a proton transporter system with two isochromatic rhodopsins and a new type of sensory rhodopsin-like transducer in H. marismortui.

Codon changed immobilization antigen (iAg), a potent DNA vaccine in fish against Cryptocaryon irritans infection.

The immobilization antigen (iAg) DNA sequence from Chiayi isolate of Cryptocaryon irritans was computationally reviewed to replace the stop codons with suitable amino acids and its GC content was intensified. The plasmid construct comprising the codon changed iAg (optiAg/optimized iAg) was successfully expressed in the bacterial strain BL21 and also in grouper fin cells (GF-1). Results of immobilization assay, ELISA and western blot of C. irritans theront and recombinant iAg by grouper antiserum against optiAg DNA indicated that the codon changed iAg retains the native conformation. The DNA vaccine construct pcDNA3.1-optiAg was encapsulated in water-oil-water triple layer emulsions measuring 19 μm diameters and was used for the immunization experiment. In trial I experiment, grouper fish were immunized twice via intramuscular injection with the pcDNA3.1-optiAg and were challenged with C. irritans at 8-day post immunization (dpi), which resulted in 46% relative percent survival (RPS). In trial II, single immunization with pcDNA3.1-optiAg boosted with recombinant iAg protein, resulted in 40% RPS. The data from this study reveal that codon change in iAg not only accomplished the expression of iAg protein in both prokaryotic and eukaryotic cell systems, but also optiAg was proved as immunogenic due to the protection it confers to the immunized fish against C. irritans infection. Hence, it is concluded that iAg can be a potent DNA vaccine in fish against infection of the ciliated protozoan, C. irritans.

Evaluation of allelic strength of human TET2 mutations and cooperation between Tet2 knockdown and oncogenic Nras mutation.

The TET2 (tet methylcytosine dioxygenase 2) gene encodes a methylcytosine dioxygenase that catalyses the hydrolysis of 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5hmC) and promotes DNA demethylation through passive and active mechanisms (Shih, et al 2012). Loss-of-function mutations in TET2 are identified in patients with myeloid and lymphoid malignancies, and are particularly frequent in patients with chronic myelomonocytic leukaemia (CMML) (36–58%) (Shih, et al 2012). Consistent with the patient sequencing analysis, conditional knockout of Tet2 in mice dysregulates haematopoietic stem cell (HSC) function and promotes development of a myeloid malignancy closely resembling human CMML (Cimmino, et al 2011). Despite the high mutation frequency, the prognostic importance of TET2 mutations is unclear in many cases (Shih, et al 2012). We postulated that this could be due to the differential allelic strengths of distinct TET2 mutations (e.g. amorphic versus hypomorphic) and/or the influence of other concurrent genetic alterations. Although nonsense and frameshift mutations are found spread over the entire TET2 sequence, the majority of missense mutations occur in the two conserved regions of TET2 protein (Figure S1): a cysteine-rich region within 1134-1444 amino acids and a catalytic domain (double strand β helix, DSBH) in 1842-1921 amino acids (Fig. 1A). To determine the allelic strengths of distinct TET2 mutations, we characterized five missense mutations prevalent in the COSMIC database in the context of full length human TET2. Two of them (C1193W and R1261G) are located in the cysteine-rich domain and have not been examined before. The other three mutations (I1873T, H1881Q, and R1896S) are located in the DSBH domain and their equivalent mutations were previously evaluated in mouse Tet2 (Ko, et al 2010). Transient expression of full-length wild-type human TET2 in HEK293T cells showed a predominant nuclear localization (~75%) and concomitant detection of 5hmC in the nucleus (Fig. 1B–1D). We observed that in ~25% of TET2-expressing cells, TET2 protein was distributed in both cytoplasm and nucleus and 5hmC staining was diminished (Figure 1B and 1C). These results suggest that intracellular localization of TET2 influences production of 5hmC. In contrast, the mutant proteins containing C1193W, R1261G, I1873T or H1881Q mutations maintained their nuclear localization but 5hmC levels were not detectable, suggesting that these mutations are amorphic. TET2R1896S mutant only showed partial loss of function, suggesting that this mutation is hypomorphic (Fig. 1D). Importantly, our results of human TET2I1873T and TET2R1896S are not consistent with those obtained from equivalent mouse Tet2 mutants, which did not show diminished 5hmC staining (Ko, et al 2010). This could be due to the differences between human TET2 and mouse TET2, emphasizing the importance of validating discoveries from mouse genes in human genes. Nonetheless, our data indicate that leukaemia-associated TET2 mutations lead to complete or partial loss of TET2 function, providing a rational to further stratify leukaemia patients based on their specific TET2 mutations in future prognostic studies. Fig 1 Missense mutations at the Cys-rich and DSBH regions of human TET2 attenuate its catalytic function Recent work identified a significant synergy between loss-of-TET2 and loss-of other epigenetic regulators (Ezh2 (Muto, et al 2013) and Asxl1 (Abdel-Wahab, et al 2013) ) or NOTCH inactivation (loss of Ncstn) (Lobry, et al 2013) in mice. These results suggest that TET2 mutations might indicate a poor prognosis outcome in CMML patients with concurrent EZH2, ASXL1, or NCSTN mutations. However, the prognostic importance of TET2 mutations in patients with other concurrent mutations, for example, RAS signalling pathway mutations, has not been evaluated. Given the high mutation rate of TET2 in CMML patients, we set out to find and characterize additional gene mutations concurrent with TET2 mutations. We performed whole exome sequence analysis of 5 CMML patients with a normal karyotype and at different stages of CMML development, including 2 collected from patients transformed to acute myeloid leukaemia (AML) with antecedent of CMML, 1 with Type II CMML, and 2 with Type I CMML (Fig. 2A). All of them contained TET2 mutations, including 4 frameshift and 11 missense mutations. Among the missense mutations, L1721W and I1762V were reported before (Kohlmann, et al 2011, Nibourel, et al 2010), L1340R was found in the COSMIC database (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/), while N7S, Q129K, and N196K have not been described but are absent from the SNP database (http://snp-nexus.org/index.html). Detection of more than one TET2 mutation in individual patients suggests the presence of multiple leukaemic clones. The mutation frequency of TET2 in our small cohort is much higher than previously reported (Shih, et al 2012). This could be due to the small sample size and the selection of myeloproliferative variant of CMML (indicated by high white blood cell and monocyte counts) and transformed AML in our study. Fig 2 Tet2 knockdown does not promote NrasG12D/+-induced CMML Consistent with the mouse studies, our sequencing results revealed that all patients carried ASXL1 mutations and two patients carried EZH2 mutations. However, NCSTN mutations were not detected in any of the patients (Fig. 2A). In addition, four patients carried canonical oncogenic mutations in NRAS or KRAS, and one patient carried the KRASV7E mutation, which has not been reported in human cancers. However, V7 codon was recently suggested as a key residual in regulating oncogenic Kras activity (Maurer, et al 2012). Our finding of concurrent TET2 mutations with oncogenic RAS mutations in CMML patients is consistent with our data-mining result of the COSMIC database (Table S1) and other reports (Table S2). To determine whether loss-of-Tet2 co-operates with oncogenic Ras to promote CMML development, we knocked down Tet2 expression in NrasG12D/+ bone marrow cells (Fig. 2B–2D). Compared with recipients transplanted with control cells expressing a scrambled shRNA, recipients transplanted with control cells expressing Tet2 shRNA (Ko, et al 2010) developed CMML-like phenotypes after a prolonged latency, consistent with previous reports of Tet2 knockout mice (Cimmino, et al 2011). To our surprise, knockdown of Tet2 did not accelerate NrasG12D/+ induced CMML (Fig. 2B) or further promote CMML phenotypes (Fig. 2C and 2D). All CMML mice displayed comparably enlarged spleen and significantly higher percentage of monocytes (Mac1+ Gr1−) and neutrophils (Mac1+ Gr1+) in peripheral blood compared to controls. It is likely that Tet2 knockdown does not result in long-term abrogation of Tet2 expression. Alternatively, oncogenic Ras signalling might alter the subcellular localization of TET2 protein to promote Tet2 loss-of-function during CMML development as shown in BCR-ABL1-driven chronic myeloid leukaemia (Mancini, et al 2012). Thus, further downregulation of Tet2 expression in NrasG12D/+ bone marrow cells does not significantly accelerate CMML progression. It is also possible that the order of mutational acquisition is important. However, current technologies do not allow us to assess this possibility under physiological conditions. In summary, our results provide a rationale to further stratify leukaemia patients based on their specific TET2 mutations and presence of specific additional genetic mutations in future prognostic studies.

Insight into a single halobacterium using a dual‐bacteriorhodopsin system with different functionally optimized pH ranges to cope with periplasmic pH changes associated with continuous light illumination

The light‐driven outward proton transporter assists energy production via an ATP synthase system best exemplified by the bacteriorhodopsin (BR) from Halobacterium salinarum, HsBR. As the only archaea able to survive in the resource‐limited ecosystem of the Dead Sea, Haloarcula marismortui has been reported to have a unique dual‐BR system, consisting of HmBRI and HmBRII, instead of only a single BR in a cell (solo‐BR). The contribution of this dual‐BR system to survival was investigated. First, native H. marismortui and H. salinarum cells were tested in water that had been adjusted to mimic the conditions of Dead Sea water. These archaea were shown to accumulate protons and reduce pH in their periplasmic regions, which disabled further proton transportation functionality in H. salinarum but not in H. marismortui. Then, pH‐dependent photocurrent measurements using purified BR proteins demonstrated that HsBR and HmBRI were functional at pH > 5.0 and that HmBRII was functional at pH > 4.0. Our results indicate that the dual‐HmBR system is composed of two BRs with different optimal functional pH ranges and together they maintain light‐driven proton transport activity under pH > 4.0, which might contribute the survival of H. marismortui under the acidic pH of the Dead Sea.

Ser(262) determines the chloride-dependent colour tuning of a new halorhodopsin from Haloquadratum walsbyi.

Light is an important environmental signal for all organisms on earth because it is essential for physiological signalling and the regulation of most biological systems. Halophiles found in salt-saturated ponds encode various archaeal rhodopsins and thereby harvest various wavelengths of light either for ion transportation or as sensory mediators. HR (halorhodopsin), one of the microbial rhodopsins, senses yellow light and transports chloride or other halides into the cytoplasm to maintain the osmotic balance during cell growth, and it exists almost ubiquitously in all known halobacteria. To date, only two HRs, isolated from HsHR (Halobacterium salinarum HR) and NpHR (Natronomonas pharaonis HR), have been characterized. In the present study, two new HRs, HmHR (Haloarcula marismortui HR) and HwHR (Haloquadratum walsbyi HR), were functionally overexpressed in Escherichia coli, and the maximum absorbance (λmax) of the purified proteins, the light-driven chloride uptake and the chloride-binding affinity were measured. The results showed them to have similar properties to two HRs reported previously. However, the λmax of HwHR is extremely consistent in a wide range of salt/chloride concentrations, which had not been observed previously. A structural-based sequence alignment identified a single serine residue at 262 in HwHR, which is typically a conserved alanine in all other known HRs. A Ser262 to alanine replacement in HwHR eliminated the chloride-independent colour tuning, whereas an Ala246 to serine mutagenesis in HsHR transformed it to have chloride-independent colour tuning similar to that of HwHR. Thus Ser262 is a key residue for the mechanism of chloride-dependent colour tuning in HwHR.

Structural and Functional Studies of a Newly Grouped Haloquadratum walsbyi Bacteriorhodopsin Reveal the Acid-resistant Light-driven Proton Pumping Activity

Background: Most bacteriorhodopsins demonstrate red-shifted spectrum in acidic condition. Results: Structures of Haloquadratum walsbyi bacteriorhodopsin explain stable action spectra from pH 2 to 8. Conclusion: The extracellular hydrogen-bonding network assists in the maintenance of protonation status in the Haloquadratum walsbyi bacteriorhodopsin retinal-binding pocket. Significance: A bacteriorhodopsin subfamily has a stable optical property, and its structure is useful for protein engineering in optogenetic tools. Retinal bound light-driven proton pumps are widespread in eukaryotic and prokaryotic organisms. Among these pumps, bacteriorhodopsin (BR) proteins cooperate with ATP synthase to convert captured solar energy into a biologically consumable form, ATP. In an acidic environment or when pumped-out protons accumulate in the extracellular region, the maximum absorbance of BR proteins shifts markedly to the longer wavelengths. These conditions affect the light-driven proton pumping functional exertion as well. In this study, wild-type crystal structure of a BR with optical stability under wide pH range from a square halophilic archaeon, Haloquadratum walsbyi (HwBR), was solved in two crystal forms. One crystal form, refined to 1.85 Å resolution, contains a trimer in the asymmetric unit, whereas another contains an antiparallel dimer was refined at 2.58 Å. HwBR could not be classified into any existing subgroup of archaeal BR proteins based on the protein sequence phylogenetic tree, and it showed unique absorption spectral stability when exposed to low pH values. All structures showed a unique hydrogen-bonding network between Arg82 and Thr201, linking the BC and FG loops to shield the retinal-binding pocket in the interior from the extracellular environment. This result was supported by R82E mutation that attenuated the optical stability. The negatively charged cytoplasmic side and the Arg82–Thr201 hydrogen bond may play an important role in the proton translocation trend in HwBR under acidic conditions. Our findings have unveiled a strategy adopted by BR proteins to solidify their defenses against unfavorable environments and maintain their optical properties associated with proton pumping.

A Unique Light-Driven Proton Transportation Signal in Halorhodopsin from Natronomonas pharaonis

Halorhodopsin (HR) is a seven-transmembrane retinylidene protein from haloarchaea that is commonly known to function as a light-driven inward chloride pump. However, previous studies have indicated that despite the general characteristics that most HRs share, HRs from distinct species differ in many aspects. We present indium-tin-oxide-based photocurrent measurements that reveal a light-induced signal generated by proton release that is observed solely in NpHR via purified protein-based assays, demonstrating that indeed HRs are not all identical. We conducted mutagenesis studies on several conserved residues that are considered critical for chloride stability among HRs. Intriguingly, the photocurrent signals were eliminated after specific point mutations. We propose an NpHR light-driven, cytoplasmic-side proton circulation model to explain the unique light-induced photocurrent recorded in NpHR. Notably, the photocurrent and various photocycle intermediates were recorded simultaneously. This approach provides a high-resolution method for further investigations of the proton-assisted chloride translocation mechanism.

Phototaxis of Haloarcula marismortui Revealed Through a Novel Microbial Motion Analysis Algorithm

Haloarcula marismortui has been described to be nonmotile prior to the recent identification of flagellar filaments, suggesting the motile nature of H. marismortui. Here we observed the locomotion of freshly cultured H. marismortui cells and tracked the swimming trajectories via ImageJ. Trajectories of H. marismortui are intrinsically noisy, posing difficulties in motion analysis with previously established algorithms. By introducing the concept of “window vector,” a Microsoft Excel‐VBA‐implemented microbial motion analysis algorithm reported here was able to (1) discriminate nonswimming objects from swimming cells without empirical customization by applying a power‐law relationship and (2) reduce the noise caused by Brownian motion, thus enhancing the accuracy of swim reversal identification. Based on this motion analysis algorithm, two recently identified sensory rhodopsins, HmSRI and HmSRII, were shown to mediate photoattractant and photorepellent responses, respectively, revealing the phototactic activity of H. marismortui, the only archaeon showing such phenomenon other than Halobacterium salinarum.