Rapid down-regulation of mammalian Period genes during behavioral resetting of the circadian clock

The pervasive role of circadian clocks in regulating physiology and behavior is widely recognized. Their adaptive value is their ability to be entrained by environmental cues such that the internal circadian phase is a reliable predictor of solar time. In mammals, both light and nonphotic behavioral cues can entrain the principal oscillator of the hypothalamic suprachiasmatic nuclei (SCN). However, although light can advance or delay the clock during circadian night, behavioral events trigger phase advances during the subjective day, when the clock is insensitive to light. The recent identification of Period (Per) genes in mammals, homologues of dperiod, which encodes a core element of the circadian clockwork in Drosophila, now provides the opportunity to explain circadian timing and entrainment at a molecular level. In mice, expression of mPer1 and mPer2 in the SCN is rhythmic and acutely up-regulated by light. Moreover, the temporal relations between mRNA and protein cycles are consistent with a clock based on a transcriptional/translational feedback loop. Here we describe circadian oscillations of Per1 and Per2 in the SCN of the Syrian hamster, showing that PER1 protein and mRNA cycles again behave in a manner consistent with a negative-feedback oscillator. Furthermore, we demonstrate that nonphotic resetting has the opposite effect to light: acutely down-regulating these genes. Their sensitivity to nonphotic resetting cues supports their proposed role as core elements of the circadian oscillator. Moreover, this study provides an explanation at the molecular level for the contrasting but convergent effects of photic and nonphotic cues on the clock.

Atomic structure of a thermostable subdomain of HIV-1 gp41

Infection by HIV-1 involves the fusion of viral and cellular membranes with subsequent transfer of viral genetic material into the cell. The HIV-1 envelope glycoprotein that mediates fusion consists of the surface subunit gp120 and the transmembrane subunit gp41. gp120 directs virion attachment to the cell–surface receptors, and gp41 then promotes viral–cell membrane fusion. A soluble, α-helical, trimeric complex within gp41 composed of N-terminal and C-terminal extraviral segments has been proposed to represent the core of the fusion-active conformation of the HIV-1 envelope. A thermostable subdomain denoted N34(L6)C28 can be formed by the N-34 and C-28 peptides connected by a flexible linker in place of the disulfide-bonded loop region. Three-dimensional structure of N34(L6)C28 reveals that three molecules fold into a six-stranded helical bundle. Three N-terminal helices within the bundle form a central, parallel, trimeric coiled coil, whereas three C-terminal helices pack in the reverse direction into three hydrophobic grooves on the surface of the N-terminal trimer. This thermostable subdomain displays the salient features of the core structure of the isolated gp41 subunit and thus provides a possible target for therapeutics designed selectively to block HIV-1 entry.

Identification of a novel vertebrate circadian clock-regulated gene encoding the protein nocturnin

Photoreceptors of the Xenopus laevis retina are the site of a circadian clock. As part of a differential display screen for rhythmic gene products in this system, we have identified a photoreceptor-specific mRNA expressed in peak abundance at night. cDNA cloning revealed an open reading frame encoding a putative 388 amino acid protein that we have named “nocturnin” (for night-factor). This protein has strong sequence similarity to the C-terminal domain of the yeast transcription factor, CCR4, as well as a leucine zipper-like dimerization motif. Nocturnin mRNA levels exhibit a high amplitude circadian rhythm and nuclear run-on analysis indicates that it is controlled by the retinal circadian clock at the level of transcription. Our observations suggest that nocturnin may function through protein–protein interaction either as a component of the circadian clock or as a downstream effector of clock function.

Phosphorylation of the Neurospora clock protein FREQUENCY determines its degradation rate and strongly influences the period length of the circadian clock

Under free running conditions, FREQUENCY (FRQ) protein, a central component of the Neurospora circadian clock, is progressively phosphorylated, becoming highly phosphorylated before its degradation late in the circadian day. To understand the biological function of FRQ phosphorylation, kinase inhibitors were used to block FRQ phosphorylation in vivo and the effects on FRQ and the clock observed. 6-dimethylaminopurine (a general kinase inhibitor) is able to block FRQ phosphorylation in vivo, reducing the rate of phosphorylation and the degradation of FRQ and lengthening the period of the clock in a dose-dependent manner. To confirm the role of FRQ phosphorylation in this clock effect, phosphorylation sites in FRQ were identified by systematic mutagenesis of the FRQ ORF. The mutation of one phosphorylation site at Ser-513 leads to a dramatic reduction of the rate of FRQ degradation and a very long period (>30 hr) of the clock. Taken together, these data strongly suggest that FRQ phosphorylation triggers its degradation, and the degradation rate of FRQ is a major determining factor for the period length of the Neurospora circadian clock.

Nucleotide binding and autophosphorylation of the clock protein KaiC as a circadian timing process of cyanobacteria

A negative feedback control of kaiC expression by KaiC protein has been proposed to generate a basic oscillation of the circadian clock in the cyanobacterium Synechococcus sp. PCC 7942. KaiC has two P loops or Walker's motif As, that are potential ATP-/GTP-binding motifs and DXXG motifs conserved in various GTP-binding proteins. Herein, we demonstrate that in vitro KaiC binds ATP and, with lower affinity, GTP. Point mutation by site-directed mutagenesis of P loop 1 completely nullified the circadian rhythm of kaiBC expression and markedly reduced ATP-binding activity. Moreover, KaiC can be autophosphorylated in vitro. These results suggest that the nucleotide-binding activity of KaiC plays important roles in the generation of circadian oscillation in cyanobacteria.

The biological clock of very premature primate infants is responsive to light

Each year more than 250,000 infants in the United States are exposed to artificial lighting in hospital nurseries with little consideration given to environmental lighting cycles. Essential in determining whether environmental lighting cycles need to be considered in hospital nurseries is identifying when the infant’s endogenous circadian clock becomes responsive to light. Using a non-human primate model of the developing human, we examined when the circadian clock, located in the hypothalamic suprachiasmatic nuclei (SCN), becomes responsive to light. Preterm infant baboons of different ages were exposed to light (5,000 lux) at night, and then changes in SCN metabolic activity and gene expression were assessed. After exposure to bright light at night, robust increases in SCN metabolic activity and gene expression were seen at ages that were equivalent to human infants at 24 weeks after conception. These data provide direct evidence that the biological clock of very premature primate infants is responsive to light.

Unbinding process of adsorbed proteins under external stress studied by atomic force microscopy spectroscopy

We report the study of the dynamics of the unbinding process under a force load f of adsorbed proteins (fibrinogen) on a solid surface (hydrophilic silica) by means of atomic force microscopy spectroscopy. By varying the loading rate rf, defined by f = rf t, t being the time, we find that, as for specific interactions, the mean rupture force increases with rf. This unbinding process is analyzed in the framework of the widely used Bell model. The typical dissociation rate at zero force entering in the model lies between 0.02 and 0.6 s−1. Each measured rupture is characterized by a force f0, which appears to be quantized in integer multiples of 180–200 pN.

Vitamin B2-based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting the circadian clock in mammals

In mammals the retina contains photoactive molecules responsible for both vision and circadian photoresponse systems. Opsins, which are located in rods and cones, are the pigments for vision but it is not known whether they play a role in circadian regulation. A subset of retinal ganglion cells with direct projections to the suprachiasmatic nucleus (SCN) are at the origin of the retinohypothalamic tract that transmits the light signal to the master circadian clock in the SCN. However, the ganglion cells are not known to contain rhodopsin or other opsins that may function as photoreceptors. We have found that the two blue-light photoreceptors, cryptochromes 1 and 2 (CRY1 and CRY2), recently discovered in mammals are specifically expressed in the ganglion cell and inner nuclear layers of the mouse retina. In addition, CRY1 is expressed at high level in the SCN and oscillates in this tissue in a circadian manner. These data, in conjunction with the established role of CRY2 in photoperiodism in plants, lead us to propose that mammals have a vitamin A-based photopigment (opsin) for vision and a vitamin B2-based pigment (cryptochrome) for entrainment of the circadian clock.

Protein kinase CK2 interacts with and phosphorylates the Arabidopsis circadian clock-associated 1 protein

The circadian clock-associated 1 (CCA1) gene encodes a Myb-related transcription factor that has been shown to be involved in the phytochrome regulation of Lhcb1*3 gene expression and in the function of the circadian oscillator in Arabidopsis thaliana. By using a yeast interaction screen to identify proteins that interact with CCA1, we have isolated a cDNA clone encoding a regulatory (β) subunit of the protein kinase CK2 and have designated it as CKB3. CKB3 is the only reported example of a third β-subunit of CK2 found in any organism. CKB3 interacts specifically with CCA1 both in a yeast two-hybrid system and in an in vitro interaction assay. Other subunits of CK2 also show an interaction with CCA1 in vitro. CK2 β-subunits stimulate binding of CCA1 to the CCA1 binding site on the Lhcb1*3 gene promoter, and recombinant CK2 is able to phosphorylate CCA1 in vitro. Furthermore, Arabidopsis plant extracts contain a CK2-like activity that affects the formation of a DNA–protein complex containing CCA1. These results suggest that CK2 can modulate CCA1 activity both by direct interaction and by phosphorylation of the CCA1 protein and that CK2 may play a role in the function of CCA1 in vivo.

Structural activity of a cloned potassium channel (ROMK1) monitored with the atomic force microscope: The “molecular-sandwich” technique

The atomic force microscope (AFM) was used to continuously follow height changes of individual protein molecules exposed to physiological stimuli. A AFM tip was coated with ROMK1 (a cloned renal epithelial potassium channel known to be highly pH sensitive) and lowered onto atomically flat mica surface until the protein was sandwiched between AFM tip and mica. Because the AFM tip was an integral part of a highly flexible cantilever, any structural alterations of the sandwiched molecule were transmitted to the cantilever. This resulted in a distortion of the cantilever that was monitored by means of a laser beam. With this system it was possible to resolve vertical height changes in the ROMK1 protein of ≥0.2 nm (approximately 5% of the molecule’s height) with a time resolution of ≥1 msec. When bathed in electrolyte solution that contained the catalytic subunit of protein kinase A and 0.1 mM ATP (conditions that activate the native ion channel), we found stochastically occurring height fluctuations in the ROMK1 molecule. These changes in height were pH-dependent, being greatest at pH 7.6, and lowering the pH (either by titration or by the application of CO2) reduced their magnitude. The data show that overall changes in shape of proteins occur stochastically and increase in size and frequency when the proteins are active. This AFM “molecular-sandwich” technique, called MOST, measures structural activity of proteins in real time and could prove useful for studies on the relationship between structure and function of proteins at the molecular level.

Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail

Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a prime target for drugs and herbicides. We have identified the interaction of EPSP synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography. The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain cleft. Glyphosate appears to occupy the binding site of the second substrate of EPSP synthase (phosphoenol pyruvate), mimicking an intermediate state of the ternary enzyme⋅substrates complex. The elucidation of the active site of EPSP synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs.

Protein folding from a highly disordered denatured state: The folding pathway of chymotrypsin inhibitor 2 at atomic resolution

Previous experimental and theoretical studies have produced high-resolution descriptions of the native and folding transition states of chymotrypsin inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation–condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding/unfolding pathway of CI2 at atomic resolution.

Visualization of unwinding activity of duplex RNA by DbpA, a DEAD box helicase, at single-molecule resolution by atomic force microscopy

The Escherichia coli protein DbpA is unique in its subclass of DEAD box RNA helicases, because it possesses ATPase-specific activity toward the peptidyl transferase center in 23S rRNA. Although its remarkable ATPase activity had been well defined toward various substrates, its RNA helicase activity remained to be characterized. Herein, we show by using biochemical assays and atomic force microscopy that DbpA exhibits ATP-stimulated unwinding activity of RNA duplex regardless of its primary sequence. This work presents an attempt to investigate the action of DEAD box proteins by a single-molecule visualization methodology. Our atomic force microscopy images enabled us to observe directly the unwinding reaction of a DEAD box helicase on long stretches of double-stranded RNA. Specifically, we could differentiate between the binding of DbpA to RNA in the absence of ATP and the formation of a Y-shaped intermediate after its progression through double-stranded RNA in the presence of ATP. Recent studies have questioned the designation of DbpA, in particular, and DEAD box proteins in general as RNA helicases. However, accumulated evidence and the results reported herein suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicases.