Biochemistry and Cell Biology Publications

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CIRCADIAN CLOCK-ASSOCIATED1 Controls Resistance to Aphids by Altering Indole Glucosinolate Production
(Oxford University Press, 2019) Lei, Jiaxin; Jayaprakasha, Guddadarangavvanahally K.; Singh, Jashbir; Uckoo, Rammohan; Borrego, Eli J.; Finlayson, Scott; Kolomiets, Mike; Patil, Bhimanagouda S.; Braam, Janet; Zhu-Salzman, Keyan
CIRCADIAN CLOCK-ASSOCIATED1 (CCA1), a well-known central circadian clock regulator, coordinates plant responses to environmental challenges. Its daily rhythmic expression in Arabidopsis (Arabidopsis thaliana) confers host resistance to the caterpillar Trichoplusia ni. However, it is unclear whether CCA1 plays a role in defense against phloem sap-feeding aphids. In this study, we showed that green peach aphid (Myzus persicae) displayed an intrinsic circadian feeding rhythm. Under constant light, wild-type Columbia-0 (Col-0) Arabidopsis plants coentrained with aphids in the same light/dark cycles exhibited greater antixenotic activity than plants preentrained in the opposite cycle from the aphids. Consistently, circadian mutants cca1-1, cca1-11, lhy-21, ztl-1, ztl-4, and lux-2 suffered more severe damage than Col-0 plants when infested by aphids, suggesting that the Arabidopsis circadian clock plays a defensive role. However, the arrhythmic CCA1 overexpression line (CCA1-OX) displayed strong antixenotic and antibiotic activities despite its loss of circadian regulation. Aphids feeding on CCA1-OX plants exhibited lower reproduction and smaller body size and weight than those on Col-0. Apparently, CCA1 regulates both clock-dependent and -independent defense responses. Systematic investigation based on bioinformatics analyses indicated that resistance to aphids in CCA1-OX plants was due primarily to heightened basal indole glucosinolate levels. Interestingly, aphid feeding induced alternatively spliced intron-retaining CCA1a/b transcripts, which are normally expressed at low levels, whereas expression of the major fully spliced CCA1 transcript remained largely unchanged. We hypothesize that posttranscriptional modulation of CCA1 expression upon aphid infestation maximizes the potential of circadian-mediated defense and stress tolerance while ensuring normal plant development.
OncomiR-10b hijacks the small molecule inhibitor linifanib in human cancers
(Springer Nature, 2018) Monroig-Bosque, Paloma del C.; Shah, Maitri Y.; Fu, Xiao; Fuentes-Mattei, Enrique; Ling, Hui; Ivan, Cristina; Nouraee, Nazila; Huang, Beibei; Chen, Lu; Pileczki, Valentina; Redis, Roxana S.; Jung, Eun-Jung; Zhang, Xin; Lehrer, Michael; Nagvekar, Rahul; Mafra, Ana Carolina P.; Monroig-Bosque, Maria del Mar; Irimie, Alexandra; Rivera, Carlos; Dumitru, Calin Dan; Berindan-Neagoe, Ioana; Nikonowicz, Edward P.; Zhang, Shuxing; Calin, George A.
The pervasive role of microRNAs (miRNAs) in cancer pathobiology drives the introduction of new drug development approaches such as miRNA inhibition. In order to advance miRNA-therapeutics, meticulous screening strategies addressing specific tumor targets are needed. Small molecule inhibitors represent an attractive goal for these strategies. In this study, we devised a strategy to screen for small molecule inhibitors that specifically inhibit, directly or indirectly, miR-10b (SMIRs) which is overexpressed in metastatic tumors. We found that the multi-tyrosine kinase inhibitor linifanib could significantly inhibit miR-10b and reverse its oncogenic function in breast cancer and liver cancer both in vitro and in vivo. In addition, we showed that the efficacy of linifanib to inhibit tyrosine kinases was reduced by high miR-10b levels. When the level of miR-10b is high, it can "hijack" the linifanib and reduce its kinase inhibitory effects in cancer resulting in reduced anti-tumor efficacy. In conclusion, our study describes an effective strategy to screen for small molecule inhibitors of miRNAs. We further propose that miR-10b expression levels, due to the newly described "hijacking" effect, may be used as a biomarker to select patients for linifanib treatment.
Understanding molecular recognition of promiscuity of thermophilic methionine adenosyltransferase sMAT from Sulfolobus solfataricus
(Wiley, 2014) Wang, Fengbin; Singh, Shanteri; Zhang, Jianjun; Huber, Tyler D.; Helmich, Kate E.; Sunkara, Manjula; Hurley, Katherine A.; Goff, Randal D.; Bingman, Craig A.; Morris, Andrew J.; Thorson, Jon S.; Phillips, George N.Jr.
Deficiency in Perlecan/HSPG2 During Bone Development Enhances Osteogenesis and Decreases Quality of Adult Bone in Mice
(Springer, 2014) Lowe, Dylan A.; Lepori-Bui, Nadia; Fomin, Peter V.; Sloofman, Laura G.; Zhou, Xiaozhou; Farach-Carson, Mary C.; Wang, Liyun; Kirn-Safran, Catherine B.
Perlecan/HSPG2 (Pln) is a large heparan sulfate proteoglycan abundant in the extracellular matrix of cartilage and the lacunocanalicular space of adult bones. Although Pln function during cartilage development is critical, evidenced by deficiency disorders including Schwartz–Jampel Syndrome and dyssegmental dysplasia Silverman-Handmaker type, little is known about its function in development of bone shape and quality. The purpose of this study was to understand the contribution of Pln to bone geometric and mechanical properties. We used hypomorph mutant mice that secrete negligible amount of Pln into skeletal tissues and analyzed their adult bone properties using micro-computed tomography and three-point-bending tests. Bone shortening and widening in Pln mutants was observed and could be attributed to loss of growth plate organization and accelerated osteogenesis that was reflected by elevated cortical thickness at older ages. This effect was more pronounced in Pln mutant females, indicating a sex-specific effect of Pln deficiency on bone geometry. Additionally, mutant females, and to a lesser extent mutant males, increased their elastic modulus and bone mineral densities to counteract changes in bone shape, but at the expense of increased brittleness. In summary, Pln deficiency alters cartilage matrix patterning and, as we now show, coordinately influences bone formation and calcification.
A promoter-level mammalian expression atlas
(Nature Publishing Group, 2014) FANTOM Consortium; RIKEN PMI; CLST (DGT)
Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.
Structure, Dynamics, and Specificity of Endoglucanase D from Clostridium cellulovorans
(Elsevier, 2013) Bianchetti, Christopher M.; Brumm, Phillip; Smith, Robert W.; Dyer, Kevin; Hura, Greg L.; Rutkoski, Thomas J.; Phillips, George N.Jr.
The enzymatic degradation of cellulose is a critical step in the biological conversion of plant biomass into an abundant renewable energy source. An understanding of the structural and dynamic features that cellulases utilize to bind a single strand of crystalline cellulose and hydrolyze the β-1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme comprising an N-terminal catalytic domain and a C-terminal carbohydrate-binding module, which is attached via a flexible linker. Here, we present the 2.1-Å-resolution crystal structures of full-length EngD with and without cellotriose bound, solution small-angle X-ray scattering (SAXS) studies of the full-length enzyme, the characterization of the active cleft glucose binding subsites, and substrate specificity of EngD on soluble and insoluble polymeric carbohydrates. SAXS data support a model in which the linker is flexible, allowing EngD to adopt an extended conformation in solution. The cellotriose-bound EngD structure revealed an extended active-site cleft that contains seven glucose-binding subsites, but unlike the majority of structurally determined endocellulases, the active-site cleft of EngD is partially enclosed by Trp162 and Tyr232. EngD variants, which lack Trp162, showed a significant reduction in activity and an alteration in the distribution of cellohexaose degradation products, suggesting that Trp162 plays a direct role in substrate binding.
Perlecan-Containing Pericellular Matrix Regulates Solute Transport and Mechanosensing Within the Osteocyte Lacunar-Canalicular System
(Wiley, 2014) Wang, Bin; Lai, Xiaohan; Price, Christopher; Thompson, William R.; Li, Wen; Quabili, Tonima R.; Tseng, Wei-Ju; Liu, Xiaowei Sherry; Zhang, Hong-Yi; Pan, Jun; Kirn-Safran, Catherine B.; Farach-Carson, Mary C.; Wang, Liyun
The pericellular matrix (PCM), a thin coating surrounding nearly all mammalian cells, plays a critical role in many cell-surface phenomena. In osteocytes, the PCM is believed to control both “outside-in” (mechanosensing) and “inside-out” (signaling molecule transport) processes. However, the osteocytic PCM is challenging to study in situ because it is thin (∼100 nm) and enclosed in mineralized matrix. To this end, we recently developed a novel tracer velocimetry approach that combined fluorescence recovery after photobleaching (FRAP) imaging with hydrodynamic modeling to quantify the osteocytic PCM in young murine bone. In this study, we applied the technique to older mice expressing or deficient for perlecan/HSPG2, a large heparan-sulfate proteoglycan normally secreted in osteocytic PCM. The objectives were (1) to characterize transport within an altered PCM; (2) to test the sensitivity of our approach in detecting the PCM alterations; and (3) to dissect the roles of the PCM in osteocyte mechanosensing. We found that: (1) solute transport increases in the perlecan-deficient (hypomorphic [Hypo]) mice compared with control mice; (2) PCM fiber density decreases with aging and perlecan deficiency; (3) osteocytes in the Hypo bones are predicted to experience higher shear stress (+34%), but decreased fluid drag force (−35%) under 3-N peak tibial loading; and (4) when subjected to tibial loading in a preliminary in vivo experiment, the Hypo mice did not respond to the anabolic stimuli as the CTL mice did. These findings support the hypothesis that the PCM fibers act as osteocyte's sensing antennae, regulating load-induced cellular stimulations and thus bone's sensitivity and in vivo bone adaptation. If this hypothesis is further confirmed, osteocytic PCM could be new targets to develop osteoporosis treatments by modulating bone's intrinsic sensitivity to mechanical loading and be used to design patient-specific exercise regimens to promote bone formation.
The crystal structure of BlmI as a model for nonribosomal peptide synthetase peptidyl carrier proteins
(Wiley, 2014) Lohman, Jeremy R.; Ma, Ming; Cuff, Marianne E.; Bigelow, Lance; Bearden, Jessica; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.Jr.; Shen, Ben
Carrier proteins (CPs) play a critical role in the biosynthesis of various natural products, especially in nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymology, where the CPs are referred to as peptidyl-carrier proteins (PCPs) or acyl-carrier proteins (ACPs), respectively. CPs can either be a domain in large multifunctional polypeptides or standalone proteins, termed Type I and Type II, respectively. There have been many biochemical studies of the Type I PKS and NRPS CPs, and of Type II ACPs. However, recently a number of Type II PCPs have been found and biochemically characterized. In order to understand the possible interaction surfaces for combinatorial biosynthetic efforts we crystallized the first characterized and representative Type II PCP member, BlmI, from the bleomycin biosynthetic pathway from Streptomyces verticillus ATCC 15003. The structure is similar to CPs in general but most closely resembles PCPs. Comparisons with previously determined PCP structures in complex with catalytic domains reveals a common interaction surface. This surface is highly variable in charge and shape, which likely confers specificity for interactions. Previous nuclear magnetic resonance (NMR) analysis of a prototypical Type I PCP excised from the multimodular context revealed three conformational states. Comparison of the states with the structure of BlmI and other PCPs reveals that only one of the NMR states is found in other studies, suggesting the other two states may not be relevant. The state represented by the BlmI crystal structure can therefore serve as a model for both Type I and Type II PCPs.
A novel in vivo model for evaluating functional restoration of a tissue-engineered salivary gland
(Wiley, 2014) Pradhan-Bhatt, Swati; Harrington, Daniel Anton; Duncan, Randall L.; Farach-Carson, Mary C.; Jia, Xinqiao; Witt, Robert L.
Objectives/Hypothesis: To create a novel model for development of a tissue-engineered salivary gland from human salivary gland cells that retains progenitor cell markers useful for treatment of radiation-induced xerostomia. Study Design: A three-dimensional (3D) hyaluronic acid (HA)-based hydrogel scaffold was used to encapsulate primary human salivary gland cells and to obtain organized acini-like spheroids. Hydrogels were implanted into rat models, and cell viability and receptor expression were evaluated. Methods: A parotid gland surgical resection model for xenografting was developed. Salivary cells loaded in HA hydrogels formed spheroids and in vitro were implanted in the three-fourths resected parotid bed of athymic rats. Implants were removed after 1 week and analyzed for spheroid viability and phenotype retention. Results: Spheroids in 3D stained positive for HA receptors CD168/RHAMM and CD44, which is also a progenitor cell marker. The parotid gland three-fourths resection model was well-tolerated by rodent hosts, and the salivary cell/hydrogel scaffolds were adherent to the remaining parotid gland, with no obvious signs of inflammation. A majority of human cells in the extracted hydrogels demonstrated robust expression of CD44. Conclusions: A 3D HA-based hydrogel scaffold that supported long-term culture of salivary gland cells into organized spheroids was established. An in vivo salivary gland resection model was developed that allowed for integration of the 3D HA hydrogel scaffold with the existing glandular parenchyma. The expression of CD44 among salivary cultures may partially explain their regenerative potential, and the expression of CD168/RHAMM along with CD44 may aid the development of these 3D spheroids into regenerated salivary glands.
p62/SQSTM1 is required for cell survival of apoptosis-resistant bone metastatic prostate cancer cell lines
(Wiley, 2014) Chang, Megan A.; Morgado, Micaela; Warren, Curtis R.; Hinton, Cimona V.; Farach-Carson, Mary C.; Delk, Nikki A.
BACKGROUND: Bone marrow stromal cell (BMSC) paracrine factor(s) can induce apoptosis in bone metastatic prostate cancer (PCa) cell lines. However, the PCa cells that escape BMSC-induced apoptosis can upregulate cytoprotective autophagy. METHODS: C4-2, C4-2B, MDA PCa 2a, MDA PCa 2b, VCaP, PC3, or DU145 PCa cell lines were grown in BMSC conditioned medium and analyzed for mRNA and/or protein accumulation of p62 (also known as sequestome-1/SQSTM1), Microtubule-associated protein 1 light chain 3B (LC3B), or lysosomal-associated membrane protein 1 (LAMP1) using quantitative polymerase chain reaction (QPCR), Western blot, or immunofluorescence. Small interfering RNA (siRNA) was used to determine if p62 is necessary PCa cell survival. RESULTS: BMSC paracrine signaling upregulated p62 mRNA and protein in a subset of the PCa cell lines. The PCa cell lines that were insensitive to BMSC-induced apoptosis and autophagy induction had elevated basal p62 mRNA and protein. In the BMSC-insensitive PCa cell lines, siRNA knockdown of p62 was cytotoxic and immunostaining showed peri-nuclear clustering of autolysosomes. However, in the BMSC-sensitive PCa cell lines, p62 siRNA knockdown was not appreciably cytotoxic and did not affect autolysosome subcellular localization. CONCLUSIONS: A pattern emerges wherein the BMSC-sensitive PCa cell lines are known to be osteoblastic and express the androgen receptor, while the BMSC-insensitive PCa cell lines are characteristically osteolytic and do not express the androgen receptor. Furthermore, BMSC-insensitive PCa may have evolved a dependency on p62 for cell survival that could be exploited to target and kill these apoptosis-resistant PCa cells in the bone.
Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin
(Elsevier, 2014) Mollan, Todd L.; Jia, Yiping; Banerjee, Sambuddha; Wu, Gang; Kreulen, R.Timothy; Tsai, Ah-Lim; Olson, John S.; Crumbliss, Alvin L.; Alayash, Abdu I.
Haptoglobin (Hp) is an abundant and conserved plasma glycoprotein, which binds acellular adult hemoglobin (Hb) dimers with high affinity and facilitates their rapid clearance from circulation after hemolysis. Humans possess three main phenotypes of Hp, designated Hp 1-1, Hp 2-1, and Hp 2-2. These variants exhibit diverse structural configurations and have been reported to be functionally nonequivalent. We have investigated the functional and redox properties of Hb–Hp complexes prepared using commercially fractionated Hp and found that all forms exhibit similar behavior. The rate of Hb dimer binding to Hp occurs with bimolecular rate constants of ~0.9 μM−1 s−1, irrespective of the type of Hp assayed. Although Hp binding does accelerate the observed rate of HbO2 autoxidation by dissociating Hb tetramers into dimers, the rate observed for these bound dimers is three- to fourfold slower than that of Hb dimers free in solution. Co-incubation of ferric Hb with any form of Hp inhibits heme loss to below detectable levels. Intrinsic redox potentials (E1/2) of the ferric/ferrous pair of each Hb–Hp complex are similar, varying from +54 to +59 mV (vs NHE), and are essentially the same as reported by us previously for Hb–Hp complexes prepared from unfractionated Hp. All Hb–Hp complexes generate similar high amounts of ferryl Hb after exposure to hydrogen peroxide. Electron paramagnetic resonance data indicate that the yields of protein-based radicals during this process are approximately 4 to 5% and are unaffected by the variant of Hp assayed. These data indicate that the Hp fractions examined are equivalent to one another with respect to Hb binding and associated stability and redox properties and that this result should be taken into account in the design of phenotype-specific Hp therapeutics aimed at countering Hb-mediated vascular disease.
Modeling delay in genetic networks: From delay birth-death processes to delay stochastic differential equations
(AIP Publishing, 2014) Gupta, Chinmaya; López, José Manuel; Azencott, Robert; Bennett, Matthew R.; Josić, Krešimir; Ott, William; Institute of Biosciences and Bioengineering
Delay is an important and ubiquitous aspect of many biochemical processes. For example, delay plays a central role in the dynamics of genetic regulatory networks as it stems from the sequential assembly of first mRNA and then protein. Genetic regulatory networks are therefore frequently modeled as stochastic birth-death processes with delay. Here, we examine the relationship between delay birth-death processes and their appropriate approximating delay chemical Langevin equations. We prove a quantitative bound on the error between the pathwise realizations of these two processes. Our results hold for both fixed delay and distributed delay. Simulations demonstrate that the delay chemical Langevin approximation is accurate even at moderate system sizes. It captures dynamical features such as the oscillatory behavior in negative feedback circuits, cross-correlations between nodes in a network, and spatial and temporal information in two commonly studied motifs of metastability in biochemical systems. Overall, these results provide a foundation for using delay stochastic differential equations to approximate the dynamics of birth-death processes with delay.
The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome
(Public Library of Science, 2015) Hurst, Laurence D.; Ghanbarian, Avazeh T.; Forrest, Alistair R.R.; FANTOM Consortium; Huminiecki, Lukasz
X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X’s gene content, gene expression, and evolution.
The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin
(Elsevier, 2016) Sjodt, Megan; Macdonald, Ramsay; Spirig, Thomas; Chan, Albert H.; Dickson, Claire F.; Fabian, Marian; Olson, John S.; Gell, David A.; Clubb, Robert T.
Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdHN2N3, A326–D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix.
Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of β-Aryl Ether Bonds in Lignin
(American Society for Biochemistry and Molecular Biology, 2016) Helmich, Kate E.; Pereira, Jose Henrique; Gall, Daniel L.; Heins, Richard A.; McAndrew, Ryan P.; Bingman, Craig A.; Deng, Kai; Holland, Keefe C.; Noguera, Daniel R.; Simmons, Blake A.; Sale, Kenneth L.; Ralph, John; Donohue, Timothy J.; Adams, Paul D.; Phillips, George N.Jr.
Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent β-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because β-aryl ether bonds account for 50-70% of all interunit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.
Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of β-Aryl Ether Bonds in Lignin
(American Society for Biochemistry and Molecular Biology, 2016) Helmich, Kate E.; Pereira, Jose Henrique; Gall, Daniel L.; Heins, Richard A.; McAndrew, Ryan P.; Bingman, Craig A.; Deng, Kai; Holland, Keefe C.; Noguera, Daniel R.; Simmons, Blake A.; Sale, Kenneth L.; Ralph, John; Donohue, Timothy J.; Adams, Paul D.; Phillips, George N.Jr.
Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent β-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because β-aryl ether bonds account for 50–70% of all interunit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.
Structural Basis for the Stereochemical Control of Amine Installation in Nucleotide Sugar Aminotransferases
(American Chemical Society, 2015) Wang, Fengbin; Singh, Shanteri; Xu, Weijun; Helmich, Kate E.; Miller, Mitchell D.; Cao, Hongnan; Bingman, Craig A.; Thorson, Jon S.; Phillips, George N.Jr.
Sugar aminotransferases (SATs) are an important class of tailoring enzymes that catalyze the 5'-pyridoxal phosphate (PLP)-dependent stereo- and regiospecific installation of an amino group from an amino acid donor (typically L-Glu or L-Gln) to a corresponding ketosugar nucleotide acceptor. Herein we report the strategic structural study of two homologous C4 SATs (Micromonospora echinospora CalS13 and Escherichia coli WecE) that utilize identical substrates but differ in their stereochemistry of aminotransfer. This study reveals for the first time a new mode of SAT sugar nucleotide binding and, in conjunction with previously reported SAT structural studies, provides the basis from which to propose a universal model for SAT stereo- and regiochemical control of amine installation. Specifically, the universal model put forth highlights catalytic divergence to derive solely from distinctions within nucleotide sugar orientation upon binding within a relatively fixed SAT active site where the available ligand bound structures of the three out of four representative C3 and C4 SAT examples provide a basis for the overall model. Importantly, this study presents a new predictive model to support SAT functional annotation, biochemical study and rational engineering.
Periodic, Quasi-periodic and Chaotic Dynamics in Simple Gene Elements with Time Delays
(Macmillan Publishers Limited, 2016) Suzuki, Yoko; Lu, Mingyang; Ben-Jacob, Eshel; Onuchic, José Nelson; Center for Theoretical Biological Physics
Regulatory gene circuit motifs play crucial roles in performing and maintaining vital cellular functions. Frequently, theoretical studies of gene circuits focus on steady-state behaviors and do not include time delays. In this study, the inclusion of time delays is shown to entirely change the time-dependent dynamics for even the simplest possible circuits with one and two gene elements with self and cross regulations. These elements can give rise to rich behaviors including periodic, quasi-periodic, weak chaotic, strong chaotic and intermittent dynamics. We introduce a special power-spectrum-based method to characterize and discriminate these dynamical modes quantitatively. Our simulation results suggest that, while a single negative feedback loop of either one- or two-gene element can only have periodic dynamics, the elements with two positive/negative feedback loops are the minimalist elements to have chaotic dynamics. These elements typically have one negative feedback loop that generates oscillations, and another unit that allows frequent switches among multiple steady states or between oscillatory and non-oscillatory dynamics. Possible dynamical features of several simple one- and two-gene elements are presented in details. Discussion is presented for possible roles of the chaotic behavior in the robustness of cellular functions and diseases, for example, in the context of cancer.
Laser Pulse Duration Is Critical For the Generation of Plasmonic Nanobubbles
(American Chemical Society, 2014) Lukianova-Hleb, Ekaterina Y.; Volkov, Alexey N.; Lapotko, Dmitri O.
Plasmonic nanobubbles (PNBs) are transient vapor nanobubbles generated in liquid around laser-overheated plasmonic nanoparticles. Unlike plasmonic nanoparticles, PNBs’ properties are still largely unknown due to their highly nonstationary nature. Here we show the influence of the duration of the optical excitation on the energy efficacy and threshold of PNB generation. The combination of picosecond pulsed excitation with the nanoparticle clustering provides the highest energy efficacy and the lowest threshold fluence, around 5 mJ cm–2, of PNB generation. In contrast, long excitation pulses reduce the energy efficacy of PNB generation by several orders of magnitude. Ultimately, the continuous excitation has the minimal energy efficacy, nine orders of magnitude lower than that for the picosecond excitation. Thus, the duration of the optical excitation of plasmonic nanoparticles can have a stronger effect on the PNB generation than the excitation wavelength, nanoparticle size, shape, or other “stationary” properties of plasmonic nanoparticles.
Modular, Multi-Input Transcriptional Logic Gating with Orthogonal LacI/GalR Family Chimeras
(American Chemical Society, 2014) Shis, David L.; Hussain, Faiza; Meinhardt, Sarah; Swint-Kruse, Liskin; Bennett, Matthew R.
In prokaryotes, the construction of synthetic, multi-input promoters is constrained by the number of transcription factors that can simultaneously regulate a single promoter. This fundamental engineering constraint is an obstacle to synthetic biologists because it limits the computational capacity of engineered gene circuits. Here, we demonstrate that complex multi-input transcriptional logic gating can be achieved through the use of ligand-inducible chimeric transcription factors assembled from the LacI/GalR family. These modular chimeras each contain a ligand-binding domain and a DNA-binding domain, both of which are chosen from a library of possibilities. When two or more chimeras have the same DNA-binding domain, they independently and simultaneously regulate any promoter containing the appropriate operator site. In this manner, simple transcriptional AND gating is possible through the combination of two chimeras, and multiple-input AND gating is possible with the simultaneous use of three or even four chimeras. Furthermore, we demonstrate that orthogonal DNA-binding domains and their cognate operators allow the coexpression of multiple, orthogonal AND gates. Altogether, this work provides synthetic biologists with novel, ligand-inducible logic gates and greatly expands the possibilities for engineering complex synthetic gene circuits.

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