Physical Sciences and Mathematics Commons^™

Eye Lens Β-Crystallins Are Predicted By Native Ion Mobility-Mass Spectrometry And Computations To Form Compact Higher-Ordered Heterooligomers, Amber D. Rolland, Takumi Takata, Micah T. Donor, Kirsten J. Lampi, James S. Prell

Faculty Publications - Department of Biological & Molecular Science

Eye lens crystallin proteins maintain the refractive properties of the lens but are not replaced after denucleation. Rolland et al. use native ion mobility-mass spectrometry, kinetics experiments, and computations to reveal that b-crystallins form heterodimers. These likely assemble into compact heterooligomers that enable the very high protein concentrations found in lens tissue.

Regulation Of Kv11.1 Isoform Expression By Polyadenylate Binding Protein Nuclear 1, Matthew R. Stump, Rachel T. Nguyen, Rachel H. Drgastin, Delaney Search, Quiming Gong, Zhengfeng Zhou

Faculty Publications - Department of Biological & Molecular Science

The Kv11.1 voltage-gated potassium channel, encoded by the KCNH2 gene, conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation to generate two C-terminal Kv11.1 isoforms in the heart. Utilization of a poly(A) signal in exon 15 produces the full-length, functional Kv11.1a isoform, while intron 9 polyadenylation generates the C-terminally truncated, nonfunctional Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO isoforms plays an important role in the regulation of Kv11.1 channel function. In this study, we tested the hypothesis that the RNA polyadenylate binding protein nuclear 1 (PABPN1) interacts with a unique 22 nt …

The Allometry Of Daily Energy Expenditure In Hummingbirds: An Energy Budget Approach, Anushu Shankar, Donald R. Powers, Liliana M. Dávalos, Catherine H. Graham

Faculty Publications - Department of Biological & Molecular Science

1. Within-clade allometric relationships represent standard laws of scaling between energy and size, and their outliers provide new avenues for physiological and ecological research. According to the metabolic-level boundaries hypothesis, metabolic rates as a function of mass are expected to scale closer to 0.67 when driven by surface-related processes (e.g. heat or water flux), while volume-related processes (e.g. activity) generate slopes closer to one.

2. In birds, daily energy expenditure (DEE) scales with body mass (M) in the relationship log (DEE)=2.35+0.68×log (M), consistent with surface-level processes driving the relationship. However, taxon-specific patterns differ from the scaling slope of all birds. …

Regulation Of Kv11.1 Potassium Channel C-Terminal Isoform Expression By The Rna-Binding Proteins Hur And Hud, Qiuming Gong, Matthew R. Stump, Zhengfeng Zhou

Faculty Publications - Department of Biological & Molecular Science

The potassium voltage-gated channel subfamily H member 2 (KCNH2) gene encodes the Kv11.1 potassium channel, which conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation and forms a functional, full-length Kv11.1a isoform if exon 15 is polyadenylated or a nonfunctional, C-terminally truncated Kv11.1a-USO isoform if intron 9 is polyadenylated. The molecular mechanisms that regulate Kv11.1 isoform expression are poorly understood. In this study, using HEK293 cells and reporter gene expression, pulldown assays, and RNase protection assays, we identified the RNA-binding proteins Hu antigen R (HuR) and Hu antigen D (HuD) as regulators of Kv11.1 …

Upregulation Of Functional Kv11.1a Isoform Expression By Modified U1 Small Nuclear Rna, Qiuming Gong, Matthew R. Stump, Zhengfeng Zhou

Faculty Publications - Department of Biological & Molecular Science

The KCNH2 or human ether-a go-go-related gene (hERG) encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier potassium current in the heart. The expression of Kv11.1 C-terminal isoforms is directed by the alternative splicing and polyadenylation of intron 9. Splicing of intron 9 leads to the formation of a functional, full-length Kv11.1a isoform and polyadenylation of intron 9 results in the production of a non-functional, C-terminally truncated Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO plays an important role in regulating Kv11.1 channel function. In the heart, only one-third of KCNH2 pre-mRNA is processed to Kv11.1a …

Strad Pseudokinases Regulate Axogenesis And Lkb1 Stability, Biliana O. Veleva-Rotse, James L. Smart, Annette F. Baas, Benjamin Edmonds, Zi-Ming Zhao, Allyson Brown, Lillian R. Klug, Kelly Hansen, Gabrielle Rielly, Alexandria P. Gardner, Krishnaveni Subbiah, Eric A. Gaucher, Hans Clevers, Anthony P. Barnes

Faculty Publications - Department of Biological & Molecular Science

No abstract provided.

Upregulation Of Functional Kv11.1 Isoform Expression By Inhibition Of Intronic Polyadenylation With Antisense Morpholino Oligonucleotides, Qiuming Gong, Matthew R. Stump, Zhengfeng Zhou

Faculty Publications - Department of Biological & Molecular Science

The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative processing; intron 9 splicing leads to the formation of a functional, full-length Kv11.1a isoform, while polyadenylationwithin intron 9 generates a non-functional, Cterminally truncated Kv11.1a-USO isoform. The relative expression of Kv11.1 isoforms plays an important role in the regulation of Kv11.1 channel function and the pathogenesis of long QT syndrome. In this study,we identified cis-acting elements that are required for KCNH2 intron 9 poly(A) signal activity. Mutation of these elements decreased Kv11.1a-USO expression and increased the expression of …

Position Of Premature Termination Codons Determines Susceptibility Of Herg Mutations To Nonsense-Mediated Mrna Decay In Long Qt Syndrome, Qiuming Gong, Matthew R. Stump, Zhengfeng Zhou

Faculty Publications - Department of Biological & Molecular Science

The degradation of human ether-a-go-go-related gene (hERG, KCNH2) transcripts containing premature termination codon (PTC)mutations by nonsense-mediatedmRNA decay (NMD) is an importantmechanismof long QT syndrome type 2 (LQT2). The mechanisms governing the recognition of PTC-containing hERG transcripts asNMD substrates have not been established. We used a minigene system to study two frameshift mutations, R1032Gfs*25 and D1037Rfs*82. R1032Gfs*25 introduces a PTC in exon 14, whereas D1037Rfs*82 causes a PTC in the last exon (exon 15). We showed that R1032Gfs*25, but not D1037Rfs*82, reduced the level of mutant mRNA compared to thewild-type minigene in an NMD-dependent manner. The deletion of intron 14 prevented …

Early Lqt2 Nonsense Mutation Generates N-Terminally Truncated Herg Channels With Altered Gating Properties By The Reinitiation Of Translation, Matthew R. Stump, Qiuming Gong, Jonathan D. Packer, Zhengfeng Zhou

Faculty Publications - Department of Biological & Molecular Science

Mutations in the human ether-a-go-go-related gene (hERG) result in long QT syndrome type 2 (LQT2). The hERG gene encodes a K+ channel that contributes to the repolarization of the cardiac action potential. We have previously shown that hERG mRNA transcripts that contain premature termination codon mutations are rapidly degraded by nonsense-mediated mRNA decay (NMD). In this study, we identified a LQT2 nonsense mutation, Q81X, which escapes degradation by the reinitiation of translation and generates N-terminally truncated channels. RNA analysis of hERG minigenes revealed equivalent levels of wild-type and Q81X mRNA while the mRNA expressed from minigenes containing the LQT2 frameshift …

Isoform-Specific Dominant-Negative Effects Associated With Herg1 G628s Mutation In Long Qt Syndrome, Matthew R. Stump, Qiuming Gong, Zhengfeng Zhou

Faculty Publications - Department of Biological & Molecular Science

Background: Mutations in the human ether-a-go-go-related gene 1 (hERG1) cause type 2 long QT syndrome (LQT2). The hERG1 gene encodes a K+ channel with properties similar to the rapidly activating delayed rectifying K+ current in the heart. Several hERG1 isoforms with unique structural and functional properties have been identified. To date, the pathogenic mechanisms of LQT2 mutations have been predominantly described in the context of the hERG1a isoform. In the present study, we investigated the functional consequences of the LQT2 mutation G628S in the hERG1b and hERG1aUSO isoforms.

Methods: A double-stable, mammalian expression system was developed to characterize …

Reciprocal Regulation Of Trek-1 Channels By Arachidonic Acid And Crh In Mouse Corticotropes, Andy K. Lee, James L. Smart, Marcelo Rubinstein, Malcolm J. Low, Amy Tse

Faculty Publications - Department of Biological & Molecular Science

Arachidonic acid (AA) is generated in the anterior pituitary gland upon stimulation by the ACTH secretagogue, CRH. Using the patch clamp technique,we examined the action of AA on the excitability of single pituitary corticotropes obtained from a transgenic mouse strain that expresses the enhanced green fluorescent protein driven by the proopiomelanocortin promoter. CRH evoked depolarization, but AA caused hyperpolarization. Under voltage clamp condition, AA caused a rapid inhibition of the delayed rectifier K current and then increased a background K current. Inhibition of AA metabolism did not prevent the activation of the K current by AA, suggesting a direct action …

Inhibition Of Nonsense-Mediated Mrna Decay By Antisense Morpholino Oligonucleotides Restores Functional Expression Of Herg Nonsense And Frameshift Mutations In Long-Qt Syndrome, Qiuming Gong, Matthew R. Stump, Zhengfeng Zhou

Faculty Publications - Department of Biological & Molecular Science

Mutations in the human ether-a-go-go-related gene (hERG) cause long-QT syndrome type 2 (LQT2). We previously described a homozygous LQT2 nonsense mutation Q1070X in which the mutant mRNA is degraded by nonsense-mediated mRNA decay (NMD) leading to a severe clinical phenotype. The degradation of the Q1070X transcript precludes the expression of truncated but functional mutant channels. In the present study, we tested the hypothesis that inhibition of NMD can restore functional expression of LQT2 mutations that are targeted by NMD. We showed that inhibition of NMD by RNA interference-mediated knockdown of UPF1 increased Q1070X mutant channel protein expression and hERG current …

Alternative Splicing And Polyadenylation Contribute To The Generation Of Herg1 C-Terminal Isoforms, Qiuming Gong, Matthew R. Stump, A. Russell Dunn, Vivianne Deng, Zhengfeng Zhou

Faculty Publications - Department of Biological & Molecular Science

The human ether-a-go-go-related gene 1 (hERG1) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel. Several hERG1 isoforms with different N- and C-terminal ends have been identified. The hERG1a, hERG1b, and hERG1-3.1 isoforms contain the full-length C terminus, whereas the hERG1_USOisoforms, hERG1a_USO and hERG1b_USO, lack most of the C-terminal domain and contain a unique C-terminal end. The mechanisms underlying the generation of hERG1_USOisoforms are not understood. We show that hERG1 isoforms with different C-terminal ends are generated by alternative splicing and polyadenylation of hERG1 pre-mRNA. We identified an …

Mutational Studies Uncover Non-Native Structure In The Dimeric Kinetic Intermediate Of The H2a–H2b Heterodimer, Matthew R. Stump, Lisa M. Gloss

Faculty Publications - Department of Biological & Molecular Science

The folding pathway of the histone H2A–H2B heterodimer minimally includes an on-pathway, dimeric, burst-phase intermediate, I2. The partially folded H2A and H2B monomers populated at equilibrium were characterized as potential monomeric kinetic intermediates. Folding kinetics were compared for initiation from isolated, folded monomers and the heterodimer unfolded in 4 M urea. The observed rates were virtually identical above 0.4Murea, exhibiting a log-linear relationship on the final denaturant concentration. Below ∼0.4 M urea (concentrations inaccessible from the 4-M urea unfolded state), a rollover in the rates was observed; this suggests that a component of the I2 ensemble contains non-native structure that …

Mutational Analysis Of The Stability Of The H2a And H2b Histone Monomers, Matthew R. Stump, Lisa M. Gloss

Faculty Publications - Department of Biological & Molecular Science

The eukaryotic histone heterodimer H2A–H2B folds through an obligatory dimeric intermediate that forms in a nearly diffusion-limited association reaction in the stopped-flow dead time. It is unclear whether there is partial folding of the isolated monomers before association. To address the possible contributions of structure in the monomers to the rapid association, we characterized H2A and H2B monomers in the absence of their heterodimeric partner. By far-UV circular dichroism, the H2A and H2B monomers are 15% and 31% helical, respectively—significantly less than observed in X-ray crystal structures. Acrylamide quenching of the intrinsic Tyr fluorescence was indicative of tertiary structure. The …

Central Dysregulation Of The Hypothalamic-Pituitary-Adrenal Axis In Neuron-Specific Proopiomelanocortin-Deficient Mice, James L. Smart, Virgine Tolle, Veronica Otero-Corchon, Malcolm J. Low

Faculty Publications - Department of Biological & Molecular Science

No abstract provided.

Preservation Of Eumelanin Hair Pigmentation In Proopiomelanocortin-Deficient Mice On A Nonagouti (A/A) Genetic Background, Andrzej Slominski, Przemyslaw M. Plonka, Alexander Pisarchik, James L. Smart, Virgine Tolle, Jacobo Wortsman, Malcolm J. Low

Faculty Publications - Department of Biological & Molecular Science

No abstract provided.

The Anorexigenic Fatty Acid Synthase Inhibitor, C75, Is A Nonspecific Neuronal Activator, Kanji A. Takahashi, James L. Smart, Hongyan Liu, Roger D. Cone

Faculty Publications - Department of Biological & Molecular Science

C75, a recently derived compound that potently suppresses feeding and induces weight loss, has been proposed to act mainly by inhibiting fatty acid synthase (FAS) in central neurons that control feeding. For example, normal, fasting- associated, hypothalamic increases in neuropeptide Y (NPY)/Agouti-related protein (AGRP) expression and decreases in proopiomelanocortin (POMC)/cocaine and amphetamine regulated transcript (CART) expression were reported to be blocked by C75. Using loose-patch extracellular recording in acute slices, we tested the effect of C75 on anorexigenic POMC neurons and orexigenic NPY neurons of the hypothalamic arcuate nucleus, which were identified by promoter-driven GFP expression, as well as on …

Hypothalamic Proopiomelanocortin Neurons Are Glucose Responsive And Express Katp Channels, Nurhadi Ibrahim, Martha A. Bosch, James L. Smart, Jian Qiu, Marcelo Rubinstein, Oline K. Rønnekleiv, Malcolm J. Low, Martin J. Kelly

Faculty Publications - Department of Biological & Molecular Science

Hypothalamic proopiomelanocortin (POMC) neurons are critical for controlling homeostatic functions in the mammal. We used a transgenic mouse model in which the POMC neurons were labeled with enhanced green fluorescent protein to perform visualized, whole-cell patch recordings from prepubertal female hypothalamic slices. The mouse POMC-enhanced green fluorescent protein neurons expressed the same endogenous conductances (a transient outward K current and a hyperpolarization-activated, cation current) that have been described for guinea pig POMC neurons. In addition, the selective -opioid receptor agonist DAMGO induced an outward current (maximum of 12.8 1.2 pA), which reversed at K equilibrium potential (EK), in the majority …

Intrinsically Bent Dna In The Promoter Regions Of The Yeast Gal1–10 And Gal80 Genes, Ralph C. Bash, Jeff Vargason, P. Shing Ho, D. Lohr

Faculty Publications - Department of Biological & Molecular Science

Circular permutation analysis has detected fairly strong sites of intrinsic DNA bending on the promoter regions of the yeast GAL1–10 and GAL80 genes. These bends lie in functionally suggestive locations. On the promoter of the GAL1–10 structural genes, strong bends bracket nucleosome B, which lies between the UASG and the GAL1 TATA. These intrinsic bends could help position nucleosome B. Nucleosome B plus two other promoter nucleosomes protect the TATA and start site elements in the inactive state of expression but are completely disrupted (removed) when GAL1–10 expression is induced. The strongest intrinsic bend (;70°) lies at the downstream edge …