bluuebunny/crossref_metadata_2025 huggingface dataset:
Context-dependent utility overrides absolute memory as a determinant of choice
Authors: Pompilio Lorena , Kacelnik Alex
Published: December 2009

A core problem of decision theories is that although decisionmakers’ preferences depend on learning, their choices could be driven either by learned representations of the physical properties of each alternative (for instance reward sizes) or of the benefit (utility and fitness) experienced from them. Physical properties are independent of the subject’s state and context, but utility depends on both. We show that starlings’ choices are better explained by memory for context-dependent utility than by representations of the alternatives’ physical properties, even when the decisionmakers’ state is controlled and they have accurate knowledge about the options’ physical properties. Our results support the potential universality of utility-driven preference control.
Simulations of anion transport through OprP reveal the molecular basis for high affinity and selectivity for phosphate
Authors: Pongprayoon Prapasiri , Beckstein Oliver , Wee Chze Ling , Sansom Mark S. P.
Published: December 2009

The outer membrane protein OprP from
Pseudomonas aeruginosa
forms a phosphate selective pore. To understand the mechanism of phosphate permeation and selectivity, we used three simulation techniques [equilibrium molecular dynamics simulations, steered molecular dynamics, and calculation of a potential of mean force (PMF)]. The PMF for phosphate reveals a deep free energy well midway along the OprP channel. Two adjacent phosphate-binding sites (W1 and W2), each with a well depth of ≈8
kT
, are identified close to the L3 loop in the most constricted region of the pore. A dissociation constant for phosphate of 6 μM is computed from the PMF, within the range of reported experimental values. The transfer of phosphate between sites W1 and W2 is correlated with changes in conformation of the sidechain of K121, which serves as a “charged brush” to facilitate phosphate passage between the two subsites. OprP also binds chloride, but less strongly than phosphate, as calculated from a Cl
−
PMF. The difference in affinity and hence selectivity is due to the “Lys-cluster” motif, the positive charges of which interact strongly with a partially dehydrated phosphate ion but are shielded from a Cl
−
by the hydration shell of the smaller ion. Our simulations suggest that OprP does not conform to the conventional picture of a channel with relatively flat energy landscape for permeant ions, but rather resembles a membrane-inserted binding protein with a high specificity that allows access to a centrally located binding site from both the extracellular and the periplasmic spaces.
A cancer-associated DNA polymerase δ variant modeled in yeast causes a catastrophic increase in genomic instability
Authors: Daee Danielle L. , Mertz Tony M. , Shcherbakova Polina V.
Published: December 2009

Accurate DNA synthesis by the replicative DNA polymerases α, δ, and ε is critical for genome stability in eukaryotes. In humans, over 20 SNPs were reported that result in amino–acid changes in Polδ or Polε. In addition, Polδ variants were found in colon–cancer cell lines and in sporadic colorectal carcinomas. Using the yeast-model system, we examined the functional consequences of two cancer-associated Polδ mutations and four polymorphisms affecting well-conserved regions of Polδ or Polε. We show that the R696W substitution in Polδ (analog of the R689W change in the human cancer-cell line DLD-1) is lethal in haploid and homozygous diploid yeast. The cell death results from a catastrophic increase in spontaneous mutagenesis attributed to low-fidelity DNA synthesis by Polδ-R696W. Heterozygotes survive, and the mutation rate depends on the relative expression level of wild-type versus mutant alleles. Based on these observations, we propose that the mutation rate in heterozygous human cells could be regulated by transient changes in gene expression leading to a temporary excess of Polδ-R689W. The similarities between the mutational spectra of the yeast strains producing Polδ-R696W and DLD-1 cells suggest that the altered Polδ could be responsible for a significant proportion of spontaneous mutations in this cancer cell line. These results suggest that the highly error-prone Polδ-R689W could contribute to cancer initiation and/or progression in humans.
Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase
Authors: Jackson C. J. , Foo J.-L. , Tokuriki N. , Afriat L. , Carr P. D. , Kim H.-K. , Schenk G. , Tawfik D. S. , Ollis D. L.
Published: December 2009

To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier—either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies “open” and “closed” CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.
DNA methylation of developmental genes in pediatric medulloblastomas identified by denaturation analysis of methylation differences
Authors: Diede Scott J. , Guenthoer Jamie , Geng Linda N. , Mahoney Sarah E. , Marotta Michael , Olson James M. , Tanaka Hisashi , Tapscott Stephen J.
Published: December 2009

DNA methylation might have a significant role in preventing normal differentiation in pediatric cancers. We used a genomewide method for detecting regions of CpG methylation on the basis of the increased melting temperature of methylated DNA, termed denaturation analysis of methylation differences (DAMD). Using the DAMD assay, we find common regions of cancer-specific methylation changes in primary medulloblastomas in critical developmental regulatory pathways, including Sonic hedgehog (Shh), Wingless (Wnt), retinoic acid receptor (RAR), and bone morphogenetic protein (BMP). One of the commonly methylated loci is the
PTCH1-1C
promoter, a negative regulator of the Shh pathway that is methylated in both primary patient samples and human medulloblastoma cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) increases the expression of
PTCH1
and other methylated loci. Whereas genetic mutations in
PTCH1
have previously been shown to lead to medulloblastoma, our study indicates that epigenetic silencing of
PTCH1
, and other critical developmental loci, by DNA methylation is a fundamental process of pediatric medulloblastoma formation. This finding warrants strong consideration for DNA demethylating agents in future clinical trials for children with this disease.
The HD-exchange motions of ribosomal protein S6 are insensitive to reversal of the protein-folding pathway
Authors: Haglund Ellinor , Lind Jesper , Öman Tommy , Öhman Anders , Mäler Lena , Oliveberg Mikael
Published: December 2009

An increasing number of protein structures are found to encompass multiple folding nuclei, allowing their structures to be formed by several competing pathways. A typical example is the ribosomal protein S6, which comprises two folding nuclei (σ1 and σ2) defining two competing pathways in the folding energy landscape: σ1 → σ2 and σ2 → σ1. The balance between the two pathways, and thus the order of folding events, is easily controlled by circular permutation. In this study, we make use of this ability to manipulate the folding pathway to demonstrate that the dynamic motions of the S6 structure are independent of how the protein folds. The HD-exchange protection factors remain the same upon complete reversal of the folding order. The phenomenon arises because the HD-exchange motions and the high-energy excitations controlling the folding pathway occur at separated free-energy levels: the Boltzmann distribution of unproductive unfolding attempts samples all unfolding channels in parallel, even those that end up in excessively high barriers. Accordingly, the findings provide a simple rationale for how to interpret native-state dynamics without the need to invoke fluctuations off the normal unfolding reaction coordinate.
Latent regeneration abilities persist following recent evolutionary loss in asexual annelids
Authors: Bely Alexandra E. , Sikes James M.
Published: December 2009

Regeneration abilities have been repeatedly lost in many animal phyla. However, because regeneration research has focused almost exclusively on highly regenerative taxa or on comparisons between regenerating and nonregenerating taxa that are deeply diverged, virtually nothing is known about how regeneration loss occurs. Here, we show that, following a recent evolutionary loss of regeneration, regenerative abilities can remain latent and still be elicited. Using comparative regeneration experiments and a molecular phylogeny, we show that ancestral head regeneration abilities have been lost three times among naidine annelids, a group of small aquatic worms that typically reproduce asexually by fission. In all three lineages incapable of head regeneration, worms consistently seal the wound but fail to progress to the first stage of tissue replacement. However, despite this coarse-level convergence in regeneration loss, further investigation of two of these lineages reveals marked differences in how much of the regeneration machinery has been abolished. Most notably, in a species representing one of these two lineages, but not in a representative of the other, amputation within a narrow proliferative region that forms during fission can still elicit regeneration of an essentially normal head. Thus, the presence at the wound site of elements characteristic of actively growing tissues, such as activated stem cells or growth factors, may permit blocks to regeneration to be circumvented, allowing latent regeneration abilities to be manifested.
Angiosperm genome comparisons reveal early polyploidy in the monocot lineage
Authors: Tang Haibao , Bowers John E. , Wang Xiyin , Paterson Andrew H.
Published: December 2009

Although the timing and extent of a whole-genome duplication occurring in the common lineage of most modern cereals are clear, the existence or extent of more ancient genome duplications in cereals and perhaps other monocots has been hinted at, but remain unclear. We present evidence of additional duplication blocks of deeper hierarchy than the pancereal rho (ρ) duplication, covering at least 20% of the cereal transcriptome. These more ancient duplicated regions, herein called σ, are evident in both intragenomic and intergenomic analyses of rice and sorghum. Resolution of such ancient duplication events improves the understanding of the early evolutionary history of monocots and the origins and expansions of gene families. Comparisons of syntenic blocks reveal clear structural similarities in putatively homologous regions of monocots (rice) and eudicots (grapevine). Although the exact timing of the σ-duplication(s) is unclear because of uncertainties of the molecular clock assumption, our data suggest that it occurred early in the monocot lineage after its divergence from the eudicot clade.
Gatekeeper residues in the major curlin subunit modulate bacterial amyloid fiber biogenesis
Authors: Wang Xuan , Zhou Yizhou , Ren Juan-Jie , Hammer Neal D. , Chapman Matthew R.
Published: December 2009

Amyloid fibers are filamentous protein structures commonly associated with neurodegenerative diseases. Unlike disease-associated amyloids, which are the products of protein misfolding,
Escherichia coli
assemble membrane-anchored functional amyloid fibers called curli. Curli fibers are composed of two proteins, CsgA and CsgB. In vivo, the polymerization of the major curli subunit protein, CsgA, is dependent on CsgB-mediated nucleation. The amyloid core of CsgA features five imperfect repeats (R1–R5), and R1 and R5 govern CsgA responsiveness to CsgB nucleation and self-seeding by CsgA fibers. Here, the specificity of bacterial amyloid nucleation was probed, revealing that certain aspartic acid and glycine residues inhibit the intrinsic aggregation propensities and nucleation responsiveness of R2, R3, and R4. These residues function as “gatekeepers” to modulate CsgA polymerization efficiency and potential toxicity. A CsgA molecule lacking gatekeeper residues polymerized in vitro significantly faster than wild-type CsgA and polymerized in vivo in the absence of the nucleation machinery, resulting in mislocalized fibers. This uncontrolled polymerization was associated with cytotoxicity, suggesting that incorrectly regulated CsgA polymerization was detrimental to the cell.
Role of the translocation partner in protection against AID-dependent chromosomal translocations
Authors: Jankovic Mila , Robbiani Davide F. , Dorsett Yair , Eisenreich Thomas , Xu Yang , Tarakhovsky Alexander , Nussenzweig Andre , Nussenzweig Michel C.
Published: December 2009

Chromosome translocations between Ig (
Ig
) and non-
Ig
genes are frequently associated with B-cell lymphomas in humans and mice. The best characterized of these is
c-myc/IgH
translocation, which is associated with Burkitt’s lymphoma. These translocations are caused by activation-induced cytidine deaminase (AID), which produces double-strand DNA breaks in both genes.
c-myc/IgH
translocations are rare events, in part because ATM, p53, and p19 actively suppress them. To further define the mechanism of protection against the accumulation of cells that bear
c-myc/IgH
translocation, we assayed B cells from mice that carry mutations in cell-cycle and apoptosis regulator proteins that act downstream of p53. We find that PUMA, Bim, and PKCδ are required for protection against
c-myc/IgH
translocation, whereas Bcl-XL and BAFF enhance
c-myc/IgH
translocation. Whether these effects are general or specific to
c-myc/IgH
translocation and whether AID produces dsDNA breaks in genes other than
c-myc
and
Ig
is not known. To examine these questions, we developed an assay for translocation between
IgH
and
Ig
β, both of which are somatically mutated by AID.
Ig
β
/IgH
, like
c-myc/IgH
translocations, are AID-dependent, and AID is responsible for lesions on
IgH
and the non-
IgH
translocation partners. However, ATM, p53, and p19 do not protect against
Igβ/IgH
translocations. Instead, B cells are protected against
Ig
β
/IgH
translocations by a BAFF- and PKCδ-dependent pathway. We conclude that AID-induced double-strand breaks in non-
Ig
genes other than
c-myc
lead to their translocation, and that at least two nonoverlapping pathways protect against translocations in primary B cells.
The transcriptional coactivator PGC-1α mediates exercise-induced angiogenesis in skeletal muscle
Authors: Chinsomboon Jessica , Ruas Jorge , Gupta Rana K. , Thom Robyn , Shoag Jonathan , Rowe Glenn C. , Sawada Naoki , Raghuram Srilatha , Arany Zoltan
Published: December 2009

Peripheral arterial disease (PAD) affects 5 million people in the US and is the primary cause of limb amputations. Exercise remains the single best intervention for PAD, in part thought to be mediated by increases in capillary density. How exercise triggers angiogenesis is not known. PPARγ coactivator (PGC)-1α is a potent transcriptional co-activator that regulates oxidative metabolism in a variety of tissues. We show here that PGC-1α mediates exercise-induced angiogenesis. Voluntary exercise induced robust angiogenesis in mouse skeletal muscle. Mice lacking PGC-1α in skeletal muscle failed to increase capillary density in response to exercise. Exercise strongly induced expression of PGC-1α from an alternate promoter. The induction of PGC-1α depended on β-adrenergic signaling. β-adrenergic stimulation also induced a broad program of angiogenic factors, including vascular endothelial growth factor (VEGF). This induction required PGC-1α. The orphan nuclear receptor ERRα mediated the induction of VEGF by PGC-1α, and mice lacking ERRα also failed to increase vascular density after exercise. These data demonstrate that β-adrenergic stimulation of a PGC-1α/ERRα/VEGF axis mediates exercise-induced angiogenesis in skeletal muscle.
JNK-mediated turnover and stabilization of the transcription factor p45/NF-E2 during differentiation of murine erythroleukemia cells
Authors: Lee Tung-Liang , Shyu Yu-Chiau , Hsu Pang-Hung , Chang Chiung-Wen , Wen Shau-Ching , Hsiao Wei-Yuan , Tsai Ming-Daw , Shen Che-Kun James
Published: December 2009

Regulation of the homeostatic concentrations of specific sets of transcription factors is essential for correct programming of cell proliferation and differentiation. We have characterized the signal transduction pathways regulating the catabolisis of p45/NF-E2, a bZIP factor activating the erythroid and megakaryocytic gene transcription. Through use of different approaches including nano-scale proteomics, we show that activated-JNK, or Phospho-JNK (P-JNK), physically interacts with p45/NF-E2 and phosphorylates its Ser157 residue. This reaction leads to the poly-ubiquitination of p45/NF-E2 at one or more of six Lys residues, one of which being also a sumoylation site, and its degradation through the proteasome pathway. Significantly, this regulatory pathway of p45/NF-E2 by P-JNK exists only in uninduced murine erythroleukemia (MEL) cells but not in differentiated MEL cells in which JNK is inactivated on DMSO induction. Based on the above data and analysis of the chromatin-binding kinetics of p45/NF-E2 and the erythroid gene repressor Bach1 during the early phase of MEL differentiation, we suggest a model for the regulation of erythroid maturation. In the model, the posttranslational modifications and turnover of p45/NF-E2, as mediated by P-JNK, contribute to the control of its homeostatic concentration and consequently, its regulatory functions in the progression of erythroid differentiation and erythroid gene expression.
Cyclin E-dependent protein kinase activity regulates niche retention of Drosophila ovarian follicle stem cells
Authors: Wang Zhu A. , Kalderon Daniel
Published: December 2009

Whether stem cells have unique cell cycle machineries and how they integrate with niche interactions remains largely unknown. We identified a hypomorphic
cyclin E
allele
WX
that strongly impairs the maintenance of follicle stem cells (FSCs) in the
Drosophila
ovary but does not reduce follicle cell proliferation or germline stem cell maintenance. CycE
WX
protein can still bind to the cyclin-dependent kinase catalytic subunit Cdk2, but forms complexes with reduced protein kinase activity measured in vitro. By creating additional CycE variants with different degrees of kinase dysfunction and expressing these and CycE
WX
at different levels, we found that higher CycE-Cdk2 kinase activity is required for FSC maintenance than to support follicle cell proliferation. Surprisingly,
cycE

WX

FSCs were lost from their niches rather than arresting proliferation. Furthermore, FSC function was substantially restored by expressing either excess DE-cadherin or excess E2F1/DP, the transcription factor normally activated by CycE-Cdk2 phosphorylation of retinoblastoma proteins. These results suggest that FSC maintenance through niche adhesion is regulated by inputs that normally control S phase entry, possibly as a quality control mechanism to ensure adequate stem cell proliferation. We speculate that a positive connection between central regulators of the cell cycle and niche retention may be a common feature of highly proliferative stem cells.
Myosin ATP turnover rate is a mechanism involved in thermogenesis in resting skeletal muscle fibers
Authors: Stewart Melanie A. , Franks-Skiba Kathleen , Chen Susan , Cooke Roger
Published: December 2009

Thermogenesis by resting muscle varies with conditions and plays an active role in homeostasis of body weight. The low metabolic rate of living resting muscles requires that ATP turnover by myosin be inhibited relative to the purified protein in vitro. This inhibition has not been previously seen in in vitro systems. We used quantitative epifluorescence microscopy of fluorescent nucleotides to measure single nucleotide turnovers in relaxed, permeable skeletal muscle fibers. We observed two lifetimes for nucleotide release by myosin: a fast component with a lifetime of ≈20 s, similar to that of purified myosin, and a slower component with a lifetime of 230 ± 24 s. We define the latter component to be the “super relaxed state.” The fraction of myosins in the super relaxed state was decreased at lower temperatures, by substituting GTP for ATP or by increased levels of myosin phosphorylation. All of these conditions have also been shown to cause increased disorder in the structure of the thick filament. We propose a model in which the structure of the thick filament modulates the nucleotide turnover rates of myosin in relaxed fibers. Modulation of the relative populations of the super relaxed and conventional relaxed states could have a profound effect on muscle thermogenesis, with the capacity to also significantly alter whole-body metabolic rate.
Evolution of the human lifespan and diseases of aging: Roles of infection, inflammation, and nutrition
Authors: Finch Caleb E.
Published: December 2009

Humans have evolved much longer lifespans than the great apes, which rarely exceed 50 years. Since 1800, lifespans have doubled again, largely due to improvements in environment, food, and medicine that minimized mortality at earlier ages. Infections cause most mortality in wild chimpanzees and in traditional forager-farmers with limited access to modern medicine. Although we know little of the diseases of aging under premodern conditions, in captivity, chimpanzees present a lower incidence of cancer, ischemic heart disease, and neurodegeneration than current human populations. These major differences in pathology of aging are discussed in terms of genes that mediate infection, inflammation, and nutrition. Apolipoprotein E alleles are proposed as a prototype of pleiotropic genes, which influence immune responses, arterial and Alzheimer’s disease, and brain development.
Structural dynamics of the myosin relay helix by time-resolved EPR and FRET
Authors: Agafonov Roman V. , Negrashov Igor V. , Tkachev Yaroslav V. , Blakely Sarah E. , Titus Margaret A. , Thomas David D. , Nesmelov Yuri E.
Published: December 2009

We have used two complementary time-resolved spectroscopic techniques, dipolar electron–electron resonance and fluorescence resonance energy transfer to determine conformational changes in a single structural element of the myosin motor domain, the relay helix, before and after the recovery stroke. Two double-Cys mutants were labeled with optical probes or spin labels, and interprobe distances were determined. Both methods resolved two distinct structural states of myosin, corresponding to straight and bent conformations of the relay helix. The bent state was occupied only upon nucleotide addition, indicating that relay helix, like the entire myosin head, bends in the recovery stroke. However, saturation of myosin with nucleotide, producing a single biochemical state, did not produce a single structural state. Both straight and bent structural states of the relay helix were occupied when either ATP (ADP.BeF
x
) or ADP.P
i
(ADP.AlF
4
) analogs were bound at the active site. A greater population was found in the bent structural state when the posthydrolysis analog ADP.AlF
4
was bound. We conclude that the bending of the relay helix in the recovery stroke does not require ATP hydrolysis but is favored by it. A narrower interprobe distance distribution shows ordering of the relay helix, despite its bending, during the recovery stroke, providing further insight into the dynamics of this energy-transducing structural transition.
DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa
Authors: Tritschler Felix , Braun Joerg E. , Motz Carina , Igreja Catia , Haas Gabrielle , Truffault Vincent , Izaurralde Elisa , Weichenrieder Oliver
Published: December 2009

DCP1 stimulates the decapping enzyme DCP2, which removes the mRNA 5′ cap structure committing mRNAs to degradation. In multicellular eukaryotes, DCP1-DCP2 interaction is stabilized by additional proteins, including EDC4. However, most information on DCP2 activation stems from studies in
S. cerevisiae
, which lacks EDC4. Furthermore, DCP1 orthologs from multicellular eukaryotes have a C-terminal extension, absent in fungi. Here, we show that in metazoa, a conserved DCP1 C-terminal domain drives DCP1 trimerization. Crystal structures of the DCP1-trimerization domain reveal an antiparallel assembly comprised of three kinked α-helices. Trimerization is required for DCP1 to be incorporated into active decapping complexes and for efficient mRNA decapping in vivo. Our results reveal an unexpected connectivity and complexity of the mRNA decapping network in multicellular eukaryotes, which likely enhances opportunities for regulating mRNA degradation.
CD4
+
T cell regulation of CD25 expression controls development of short-lived effector CD8
+
T cells in primary and secondary responses
Authors: Obar Joshua J. , Molloy Michael J. , Jellison Evan R. , Stoklasek Thomas A. , Zhang Weijun , Usherwood Edward J. , Lefrançois Leo
Published: December 2009

Both CD4
+
T cell help and IL-2 have been postulated to “program” activated CD8
+
T cells for memory cell development. However, the linkage between these two signals has not been well elucidated. Here we have studied effector and memory CD8
+
T cell differentiation following infection with three pathogens (
Listeria monocytogenes
, vesicular stomatitis virus, and vaccinia virus) in the absence of both CD4
+
T cells and IL-2 signaling. We found that expression of CD25 on antigen-specific CD8
+
T cells peaked 3–4 days after initial priming and was dependent on CD4
+
T cell help, likely through a CD28:CD80/86 mediated pathway. CD4
+
T cell or CD25-deficiency led to normal early effector CD8
+
T cell differentiation, but a subsequent lack of accumulation of CD8
+
T cells resulting in overall decreased memory cell generation. Interestingly, in both primary and recall responses KLRG1
high
CD127
low
short-lived effector cells were drastically diminished in the absence of IL-2 signaling, although memory precursors remained intact. In contrast to previous reports, upon secondary antigen encounter CD25-deficient CD8
+
T cells were capable of undergoing robust expansion, but short-lived effector development was again impaired. Thus, these results demonstrated that CD4
+
T cell help and IL-2 signaling were linked via CD25 up-regulation, which controls the expansion and differentiation of antigen-specific effector CD8
+
T cells, rather than “programming” memory cell traits.
Binding of a potent small-molecule inhibitor of six-helix bundle formation requires interactions with both heptad-repeats of the RSV fusion protein
Authors: Roymans Dirk , De Bondt Hendrik L. , Arnoult Eric , Geluykens Peggy , Gevers Tom , Van Ginderen Marcia , Verheyen Nick , Kim Hidong , Willebrords Rudy , Bonfanti Jean-François , Bruinzeel Wouter , Cummings Maxwell D. , van Vlijmen Herman , Andries Koen
Published: December 2009

Six-helix bundle (6HB) formation is an essential step for many viruses that rely on a class I fusion protein to enter a target cell and initiate replication. Because the binding modes of small molecule inhibitors of 6HB formation are largely unknown, precisely how they disrupt 6HB formation remains unclear, and structure-based design of improved inhibitors is thus seriously hampered. Here we present the high resolution crystal structure of TMC353121, a potent inhibitor of respiratory syncytial virus (RSV), bound at a hydrophobic pocket of the 6HB formed by amino acid residues from both HR1 and HR2 heptad-repeats. Binding of TMC353121 stabilizes the interaction of HR1 and HR2 in an alternate conformation of the 6HB, in which direct binding interactions are formed between TMC353121 and both HR1 and HR2. Rather than completely preventing 6HB formation, our data indicate that TMC353121 inhibits fusion by causing a local disturbance of the natural 6HB conformation.
Phosphorylation of aquaporin-2 regulates its endocytosis and protein–protein interactions
Authors: Moeller Hanne B. , Praetorius Jeppe , Rützler Michael R. , Fenton Robert A.
Published: December 2009

The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin–Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (
i
) apical plasma membrane abundance of AQP2, (
ii
) internalization of AQP2, (
iii
) AQP2 protein–protein interactions, and (
iv
) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life (
t
1/2
= 5.1 h and
t
1/2
= 4.4 h, respectively) compared to WT-AQP2 (
t
1/2
= 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein–protein interactions.
Strict coupling between CFTR’s catalytic cycle and gating of its Cl
−
ion pore revealed by distributions of open channel burst durations
Authors: Csanády László , Vergani Paola , Gadsby David C.
Published: December 2009

CFTR, the ABC protein defective in cystic fibrosis, functions as an anion channel. Once phosphorylated by protein kinase A, a CFTR channel is opened and closed by events at its two cytosolic nucleotide binding domains (NBDs). Formation of a head-to-tail NBD1/NBD2 heterodimer, by ATP binding in two interfacial composite sites between conserved Walker A and B motifs of one NBD and the ABC-specific signature sequence of the other, has been proposed to trigger channel opening. ATP hydrolysis at the only catalytically competent interfacial site is suggested to then destabilize the NBD dimer and prompt channel closure. But this gating mechanism, and how tightly CFTR channel opening and closing are coupled to its catalytic cycle, remains controversial. Here we determine the distributions of open burst durations of individual CFTR channels, and use maximum likelihood to evaluate fits to equilibrium and nonequilibrium mechanisms and estimate the rate constants that govern channel closure. We examine partially and fully phosphorylated wild-type CFTR channels, and two mutant CFTR channels, each bearing a deleterious mutation in one or other composite ATP binding site. We show that the wild-type CFTR channel gating cycle is essentially irreversible and tightly coupled to the ATPase cycle, and that this coupling is completely destroyed by the NBD2 Walker B mutation D1370N but only partially disrupted by the NBD1 Walker A mutation K464A.
PINK1-dependent recruitment of Parkin to mitochondria in mitophagy
Authors: Vives-Bauza Cristofol , Zhou Chun , Huang Yong , Cui Mei , de Vries Rosa L.A. , Kim Jiho , May Jessica , Tocilescu Maja Aleksandra , Liu Wencheng , Ko Han Seok , Magrané Jordi , Moore Darren J. , Dawson Valina L. , Grailhe Regis , Dawson Ted M. , Li Chenjian , Tieu Kim , Przedborski Serge
Published: December 2009

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (
PINK1
) and
PARK2/Parkin
mutations cause autosomal recessive forms of Parkinson's disease. Upon a loss of mitochondrial membrane potential (ΔΨ
m
) in human cells, cytosolic Parkin has been reported to be recruited to mitochondria, which is followed by a stimulation of mitochondrial autophagy. Here, we show that the relocation of Parkin to mitochondria induced by a collapse of ΔΨ
m
relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal ΔΨ
m
. We also show that once at the mitochondria, Parkin is in close proximity to PINK1, but we find no evidence that Parkin catalyzes PINK1 ubiquitination or that PINK1 phosphorylates Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into mitochondrial aggregates and/or large perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin, together with PINK1, modulates mitochondrial trafficking, especially to the perinuclear region, a subcellular area associated with autophagy. Thus by impairing this process, mutations in either
Parkin
or
PINK1
may alter mitochondrial turnover which, in turn, may cause the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinson's disease.
Characterization and temporal development of cores in a mouse model of malignant hyperthermia
Authors: Boncompagni Simona , Rossi Ann E. , Micaroni Massimo , Hamilton Susan L. , Dirksen Robert T. , Franzini-Armstrong Clara , Protasi Feliciano
Published: December 2009

Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the Ca
2+
release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice (RYR1
Y522S/WT
), the mutation causes SR Ca
2+
leak and MHS. Here, we identified mitochondrial-deficient core regions in skeletal muscle fibers from RYR1
Y522S/WT
knock-in mice and characterized the structural and temporal aspects involved in their formation. Mitochondrial swelling/disruption, the initial detectable structural change observed in young-adult RYR1
Y522S/WT
mice (2 months), does not occur randomly but rather is confined to discrete areas termed presumptive cores. This localized mitochondrial damage is followed by local disruption/loss of nearby SR and transverse tubules, resulting in early cores (2–4 months) and small contracture cores characterized by extreme sarcomere shortening and lack of mitochondria. At later stages (1 year), contracture cores are extended, frequent, and accompanied by areas in which contractile elements are also severely compromised (unstructured cores). Based on these observations, we propose a possible series of events leading to core formation in skeletal muscle fibers of RYR1
Y522S/WT
mice: Initial mitochondrial/SR disruption in confined areas causes significant loss of local Ca
2+
sequestration that eventually results in the formation of contractures and progressive degradation of the contractile elements.
The shape of a long leaf
Authors: Liang Haiyi , Mahadevan L.
Published: December 2009

Long leaves in terrestrial plants and their submarine counterparts, algal blades, have a typical, saddle-like midsurface and rippled edges. To understand the origin of these morphologies, we dissect leaves and differentially stretch foam ribbons to show that these shapes arise from a simple cause, the elastic relaxation via bending that follows either differential growth (in leaves) or differential stretching past the yield point (in ribbons). We quantify these different modalities in terms of a mathematical model for the shape of an initially flat elastic sheet with lateral gradients in longitudinal growth. By using a combination of scaling concepts, stability analysis, and numerical simulations, we map out the shape space for these growing ribbons and find that as the relative growth strain is increased, a long flat lamina deforms to a saddle shape and/or develops undulations that may lead to strongly localized ripples as the growth strain is localized to the edge of the leaf. Our theory delineates the geometric and growth control parameters that determine the shape space of finite laminae and thus allows for a comparative study of elongated leaf morphology.
Chemical modifier screen identifies HDAC inhibitors as suppressors of PKD models
Authors: Cao Ying , Semanchik Nicole , Lee Seung Hun , Somlo Stefan , Barbano Paolo Emilio , Coifman Ronald , Sun Zhaoxia
Published: December 2009

Polycystic kidney disease (PKD) is a common human genetic disease with severe medical consequences. Although it is appreciated that the cilium plays a central role in PKD, the underlying mechanism for PKD remains poorly understood and no effective treatment is available. In zebrafish, kidney cyst formation is closely associated with laterality defects and body curvature. To discover potential drug candidates and dissect signaling pathways that interact with ciliary signals, we performed a chemical modifier screen for the two phenotypes using zebrafish

pkd2
hi4166

and

ift172
hi2211

models.
pkd2
is a causal gene for autosomal dominant PKD and
ift172
is essential for building and maintaining the cilium. We identified trichostatin A (TSA), a pan-HDAC (histone deacetylase) inhibitor, as a compound that affected both body curvature and laterality. Further analysis verified that TSA inhibited cyst formation in
pkd2
knockdown animals. Moreover, we demonstrated that inhibiting class I HDACs, either by valproic acid (VPA), a class I specific HDAC inhibitor structurally unrelated to TSA, or by knocking down
hdac1
, suppressed kidney cyst formation and body curvature caused by
pkd2
deficiency. Finally, we show that VPA was able to reduce the progression of cyst formation and slow the decline of kidney function in a mouse ADPKD model. Together, these data suggest body curvature may be used as a surrogate marker for kidney cyst formation in large-scale high-throughput screens in zebrafish. More importantly, our results also reveal a critical role for HDACs in PKD pathogenesis and point to HDAC inhibitors as drug candidates for PKD treatment.
A protein secretion system linked to bacteroidete gliding motility and pathogenesis
Authors: Sato Keiko , Naito Mariko , Yukitake Hideharu , Hirakawa Hideki , Shoji Mikio , McBride Mark J. , Rhodes Ryan G. , Nakayama Koji
Published: December 2009

Porphyromonas gingivalis
secretes strong proteases called gingipains that are implicated in periodontal pathogenesis. Protein secretion systems common to other Gram-negative bacteria are lacking in
P. gingivalis
, but several proteins, including PorT, have been linked to gingipain secretion. Comparative genome analysis and genetic experiments revealed 11 additional proteins involved in gingipain secretion. Six of these (PorK, PorL, PorM, PorN, PorW, and Sov) were similar in sequence to
Flavobacterium johnsoniae
gliding motility proteins, and two others (PorX and PorY) were putative two-component system regulatory proteins. Real-time RT-PCR analysis revealed that
porK
,
porL
,
porM
,
porN
,
porP
,
porT
, and
sov
were down-regulated in
P. gingivalis porX
and
porY
mutants. Disruption of the
F. johnsoniae porT
ortholog resulted in defects in motility, chitinase secretion, and translocation of a gliding motility protein, SprB adhesin, to the cell surface, providing a link between a unique protein translocation system and a motility apparatus in members of the
Bacteroidetes
phylum.
Expression of human cytokines dramatically improves reconstitution of specific human-blood lineage cells in humanized mice
Authors: Chen Qingfeng , Khoury Maroun , Chen Jianzhu
Published: December 2009

Adoptive transfer of human hematopoietic stem cells (HSCs) into mice lacking T, B and natural killer (NK) cells leads to development of human-blood lineage cells in the recipient mice (humanized mice). Although human B cell reconstitution is robust and T cell reconstitution is reasonable in the recipient mice, reconstitution of NK cells and myeloid cells is generally poor or undetectable. Here, we show that the poor reconstitution is mainly the result of a deficiency of appropriate human cytokines that are necessary for the development and maintenance of these cell lineages. When plasmid DNA encoding human IL-15 and Flt-3/Flk-2 ligand were delivered into humanized mice by hydrodynamic tail-vein injection, the expression of the human cytokine lasted for 2 to 3 weeks and elevated levels of NK cells were induced for more than a month. The cytokine-induced NK cells expressed both activation and inhibitory receptors, killed target cells in vitro, and responded robustly to a virus infection in vivo. Similarly, expression of human GM-CSF and IL-4, macrophage colony stimulating factor, or erythropoietin and IL-3 resulted in significantly enhanced reconstitution of dendritic cells, monocytes/macrophages, or erythrocytes, respectively. Thus, human cytokine gene expression by hydrodynamic delivery is a simple and efficient method to improve reconstitution of specific human-blood cell lineages in humanized mice, providing an important tool for studying human immune responses and disease progression in a small animal model.
A β-catenin/TCF-coordinated chromatin loop at
MYC
integrates 5′ and 3′ Wnt responsive enhancers
Authors: Yochum Gregory S. , Sherrick Colette M. , MacPartlin Mary , Goodman Richard H.
Published: December 2009

Aberrant
MYC
gene expression by the Wnt/β-catenin pathway is implicated in colorectal carcinogenesis. Wnt/β-catenin signaling stimulates association of the β-catenin coactivator complex with two Wnt responsive enhancers (WREs) located in close proximity to
MYC
gene boundaries. Each enhancer directly binds members of the TCF/Lef family of transcription factors that, in turn, recruit β-catenin. In a previous report, we showed that the downstream
MYC
enhancer (
MYC
3′ WRE) cooperated with the upstream enhancer (
MYC
5′ WRE) to activate expression of a heterologous reporter gene in response to Wnt/β-catenin and mitogen signaling. Here we use chromatin conformation capture (3C) to show that the
MYC
5′ and 3′ WREs are juxtaposed at the genomic
MYC
locus during active transcription. This
MYC
5′3′ chromatin loop is present in HCT116 human colorectal cancer cells that contain high levels of nuclear β-catenin and is absent in HEK293 cells that contain trace amounts of nuclear β-catenin. Depletion of functional β-catenin/TCF complexes blocks formation of the
MYC
5′3 chromatin loop. Furthermore, we find that the chromatin loop is absent in quiescent cells, but is rapidly and transiently induced by serum mitogens in a β-catenin-dependent manner. Thus, we propose that a distinct chromatin architecture coordinated by β-catenin/TCF-bound WREs accompanies transcriptional activation of
MYC
gene expression.
Functions of the unique N-terminal region of glycoprotein E in the pathogenesis of varicella-zoster virus infection
Authors: Berarducci Barbara , Rajamani Jaya , Zerboni Leigh , Che Xibing , Sommer Marvin , Arvin Ann M.
Published: December 2009

Varicella-zoster virus (VZV) is an alphaherpesvirus that infects skin, lymphocytes, and sensory ganglia. VZV glycoprotein E (gE) has a unique N-terminal region (aa1-188), which is required for replication and includes domains involved in secondary envelopment, efficient cell-cell spread, and skin infection in vivo. The nonconserved N-terminal region also mediates binding to the insulin-degrading enzyme (IDE), which is proposed to be a VZV receptor. Using viral mutagenesis to make the recombinant rOka-ΔP27-G90, we showed that amino acids in this region are required for gE/IDE binding in infected cells; this deletion reduced cell-cell spread in vitro and skin infection in vivo. However, a gE point mutation, linker insertions, and partial deletions in the aa27-90 region, and deletion of a large portion of the unique N-terminal region, aa52-187, had similar or more severe effects on VZV replication in vitro and in vivo without disrupting the gE/IDE interaction. VZV replication in T cells in vivo was not impaired by deletion of gE aa27-90, suggesting that these gE residues are not essential for VZV T cell tropism. However, the rOka-ΔY51-P187 mutant failed to replicate in T cell xenografts as well as skin in vivo. VZV tropism for T cells and skin, which is necessary for its life cycle in the human host, requires this nonconserved region of the N-terminal region of VZV gE.
Sequencing newly replicated DNA reveals widespread plasticity in human replication timing
Authors: Hansen R. Scott , Thomas Sean , Sandstrom Richard , Canfield Theresa K. , Thurman Robert E. , Weaver Molly , Dorschner Michael O. , Gartler Stanley M. , Stamatoyannopoulos John A.
Published: December 2009

Faithful transmission of genetic material to daughter cells involves a characteristic temporal order of DNA replication, which may play a significant role in the inheritance of epigenetic states. We developed a genome-scale approach—Repli Seq—to map temporally ordered replicating DNA using massively parallel sequencing and applied it to study regional variation in human DNA replication time across multiple human cell types. The method requires as few as 8,000 cytometry-fractionated cells for a single analysis, and provides high-resolution DNA replication patterns with respect to both cell-cycle time and genomic position. We find that different cell types exhibit characteristic replication signatures that reveal striking plasticity in regional replication time patterns covering at least 50% of the human genome. We also identified autosomal regions with marked biphasic replication timing that include known regions of monoallelic expression as well as many previously uncharacterized domains. Comparison with high-resolution genome-wide profiles of DNaseI sensitivity revealed that DNA replication typically initiates within foci of accessible chromatin comprising clustered DNaseI hypersensitive sites, and that replication time is better correlated with chromatin accessibility than with gene expression. The data collectively provide a unique, genome-wide picture of the epigenetic compartmentalization of the human genome and suggest that cell-lineage specification involves extensive reprogramming of replication timing patterns.