Carbohydrate moiety of time-interval measuring enzyme regulates time measurement through Its interaction with time-holding peptide PIN

An ATPase called EA4 seems to measure time as a diapause-duration timer in the seasonal cycle of the silkworm, Bombyx mori. A peptide named PIN seems to regulate the time measurement of EA4. We characterize the EA4 as the first step to analyse its interaction with PIN. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry shows EA4 forms an equimolar complex with PIN. The binding affinity of EA4 for PIN is about 460 nM, as measured by surface plasmon resonance. Western blot analysis of EA4 with a variety of biotinylated lectins suggests that EA4 is a glycoprotein containing N-linked oligosaccharide. On enzymatic cleavage of the glycosyl chain, the carbohydrate is revealed to be essential for the regulation of EA4-time measurement through the interaction with PIN. PIN holds the timer by binding to EA4, and the dissociation of the complex could constitute the cue for the time measurement.     

家蚕滞育性卵盐酸处理的靶物质

酯酶A4（EA4）是家蚕卵的滞育生物钟蛋白质。从家蚕C108品种产下后48 h的滞育性卵和盐酸活化处理卵分离纯化出EA4酶蛋白,使用合成的EA4活性多肽抑制因子PIN（氨基酸结构：SIFMTKQHSQ DDIIQHPLDY VEQQIHQQKQ KLQKQTLN）,研究了PIN对EA4酶蛋白的作用机制。滞育性卵的EA4酶蛋白和PIN在25℃混合24h后,用矩阵辅助激光解吸离子质谱法,检测到了二者的结合体,该结合体在盐酸处理后消失;盐酸活化处理蚕卵的EA4酶蛋白和合成PIN之间没有出现这种结合体。体外25℃,滞育性蚕卵EA4的ATPase特征性活性峰在6.5 h后出现,而盐酸活化处理蚕卵的EA4在1.5 h后出现活性峰值。盐酸处理可能通过解除PIN对EA4的抑制作用,在短时间内激活EA4酶蛋白,从而活化滞育性蚕卵。     

Interactions between TIME and PIN could play a role in the system that controls the duration of diapause development and synchronization with seasonal cycles in the silkworm Bombyx mori*

The significance of winter cold in the termination of diapause was investigated with regards to TIME and PIN in eggs of the silkworm Bombyx mori. TIME (time interval measuring enzyme) is an ATPase that can measure time intervals by exhibiting a transitory burst of activation of the enzyme in accordance with diapause development, which requires cold for resumption of embryonic development in the silkworm. The possible timer function of TIME comprises a built‐in mechanism in the protein structure. TIME is a metallo‐glycoprotein consisting of 156 amino acid residues with a unique sequence in the N‐terminal region to which a sugar chain is attached. PIN (peptidyl inhibitory needle) inhibits the ATPase activity of TIME. PIN is not a simple enzyme inhibitor, but holds the timer by forming a time‐regulatory complex with TIME. The carbohydrate moiety of TIME is essential for the assembly of a high‐affinity PIN‐binding site within the timer motif of the TIME structure. The binding interaction between TIME and PIN was much tighter (nearly 1000 times) at 25°C than that at 4°C, as measured by fluorescence polarization. Because the logEC₅₀ at 4°C was approximately 7 nmol/L, PIN must dissociate from TIME at the physiological concentration of TIME in eggs in the winter cold. Based on the results of our study, we propose that the dissociation of the TIME–PIN complex in the winter cold cues a series of conformational changes of TIME, ultimately reaching the active form of ATPase which in turn causes the completion of diapause development and initiates new developmental programs.     

Time-measurement-regulating peptide PIN may alter a timer conformation of Time Interval Measuring Enzyme (TIME)

The TIME (Time Interval Measuring Enzyme) ATPase measures time intervals in accordance with diapause development, which indispensably requires cold for resumption of embryonic development in the silkworm (Bombyx mori). The PIN (Peptidyl Inhibitory Needle) peptide regulates the time measurement function of TIME. In the present study we investigated the interaction between TIME and PIN in order to address the mechanism of diapause development. When TIME was isolated from eggs later than 12 days after oviposition, transient bursts of ATPase activity occurred 18h after isolation of TIME, and the younger the eggs and pupal ovaries from which TIME was isolated, the earlier the bursts of ATPase activity appeared. However, no interval-timer activation of ATPase occurred in ovaries earlier than 6 days after pupation. Similar patterns of ATPase activity occurred in test tubes after mixing TIME with PIN. The shorter the time PIN was mixed with TIME, the earlier the ATPase activity appeared. The timer may be built into the protein conformation of TIME, and PIN (which is present in ovaries beginning 6 days after pupation) appears able to alter this timer conformation through pupal stages to laid eggs. We discuss the possible mechanism of diapause development in relation to the timer mechanism of TIME.     

Crystal structures of the clock protein EA4 from the silkworm Bombyx mori

Many insects pass the winter in an arrested developmental stage called diapause, either as eggs, as pupae, or even as adults. Exposure to the prolonged cold of winter is required to permit awakening from diapause in the spring. In the diapause eggs of the silkworm Bombyx mori, a metalloglycoprotein, esterase A4 (EA4), has been suggested to serve as a cold-duration clock because its characteristic ATPase activity is transiently elevated at the end of the necessary cold period. This timer property of EA4 is known to start with the dissociation of an inhibitory peptide (called “peptidyl inhibitory needle”) under cold conditions, but its time-measuring mechanism is completely unknown. Here we present the crystal structures and functional properties of EA4 with and without glycosylation. We show that EA4 is a homodimeric ATPase, with each subunit consisting of a copper-zinc superoxide dismutase fold. There is an additional short N-terminal region that is capable of binding one more copper ion, suggesting a timer mechanism in which this ion is involved. The sugar chain appears to reinforce the binding of peptidyl inhibitory needle, which may in turn stabilize the initial conformation of the N-terminal domain, explaining the requirement for glycosylation and for the peptide to set the clock.     

The peptide PIN changes the timing of transitory burst activation of timer-ATPase TIME in accordance with diapause development in eggs of the silkworm, Bombyx mori

TIME is an ATPase that measures a time interval by exhibiting transitory burst activation in eggs of the silkworm, Bombyx mori L. PIN is a peptide that regulates the time measurement of TIME. To address the mode of action of PIN, interactions between TIME and PIN were investigated. First, TIME was mixed with PIN for various periods (days) at 25 degrees C. The longer TIME was mixed with PIN, the later the transitory burst activation of TIME ATPase activity occurred, while no such delay occurred at 5 degrees C. Second, the capacity of PIN to bind with TIME was measured at the two temperatures by fluorescence polarization. The binding interaction was much tighter (nearly 1000 times) at 25 degrees C than that at 4 degrees C. Because the log EC50 (in nM) at 4 degrees C was about 7, PIN must dissociate from TIME at low temperatures at the physiological concentration of TIME in eggs. Thus, TIME appears to be restructured into a time-measuring conformation by PIN at the high temperatures of summer, whereas the TIME-PIN complex would dissociate at the low temperatures of winter. This dissociation acts as the preliminary cue for the ATPase activity burst of TIME, which in turn causes the completion of diapause development and initiates new developmental programs.     

Flavonol‐mediated stabilization of PIN efflux complexes regulates polar auxin transport

The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue‐native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo‐ and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1‐naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole‐3‐acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex.     

The Complexity and Reversibility of the Timer for the Onset of Aggregation in Dictyostelium

A simple conditional method is used to probe the complexity of the rate limiting pathway, or timer, for the onset of aggregation in the cellular slime mold Dictyostelium discoideum . The method depends upon a difference in the time required for the onset of aggregation under two sets of environmental conditions in which one parameter (ionic strength) is varied, and involves reciprocal shifts between the two sets of conditions at short time intervals. Evidence is presented that the timer for the onset of aggregation is composed of at least two components operating in sequence. In addition, a shift in one direction during the progress of the initial component of the timer causes the timer to be reset to zero. Our results also indicate that resetting the timer delays the acquisition of several aggregation associated changes, including chemotactic responsiveness, cAMP binding sites on the cell surface, and EDTA-resistant agglutination.     

Pinacyanol as effective probe of fibrillar beta-amyloid peptide: comparative study with Congo Red

The binding of pinacyanol (PIN), a cationic cyanine dye, to beta-amyloid fibrils (Abeta), which are associated with Alzheimer disease, was quantified by absorption spectrophotometry to measure the concentration of PIN bound to Abeta as a function of the Abeta concentration or by means of the separation of free PIN from bound PIN by centrifugation and subsequent analysis of the supernatant by visible-absorption spectrophotometry. Both methods gave equivalent results. The stoichiometry of PIN binding to Abeta was 1, and the curve representing the concentration effect of Abeta on the concentration of a dye-Abeta complex showed a biphasic curve instead of the hyperbolic curve that is characteristic of weak ligand-macromolecule interactions [e.g., as shown by Congo Red (CR)]). This and the fact that a Scatchard plot could not be fitted to the experimental data suggested that PIN binds tightly to Abeta. A comparison to the interaction of CR with Abeta led us to conclude that PIN is more sensitive than CR.     

High-grade prostatic intraepithelial neoplasia

High-grade prostatic intraepithelial neoplasia is considered the most likely precursor of prostatic carcinoma. The only method of detection is biopsy; prostatic intraepithelial neoplasia (PIN) does not significantly elevate serum prostate-specific antigen concentration and cannot be detected by ultra-sonography. The incidence of PIN in prostate biopsies averages 9% (range, 4%-16%), representing 115,000 new cases of PIN diagnosed each year in United States. PIN has a high predictive value as a marker for adenocarcinoma, and its identification warrants repeated biopsy for concurrent or subsequent invasive carcinoma. Carcinoma will develop in most patients with PIN within 10 years. PIN is associated with progressive abnormalities of phenotype and genotype that are intermediate between normal prostatic epithelium and cancer, indicating impairment of cell differentiation and regulatory control with advancing stages of prostatic carcinogenesis. Androgen deprivation therapy decreases the prevalence and extent of PIN, suggesting that this form of treatment may play a role in chemoprevention.     

A new method for examining the complexity and relationships of “timers” in developing systems

Simple methods are developed for analyzing the rate-limiting pathways, or “developmental timers,” for consecutive stages in a developing system. Two conditions are first defined for short and long timing to a developmental stage. Shifts are then performed at time intervals from short to long and long to short conditions. The total time to the stage (time under first condition plus time under second condition) is scored and plotted as a function of the time of shift, resulting in two plots, one for shifts from the short to long condition, and the other for shifts from the long to short condition. Each plot is then analyzed for the number of components, slopes of components, absolute times of origins and termini of components, and discontinuities between components. This information is then used (1) to distinguish between single- and multiple-component timers, (2) to assess the sensitivity of each timer component to the change in the environmental condition employed in the method, including reversibility, (3) to test for the addition of a new timer component under long conditions, and (4) to test for an identity change of a timer component between short and long conditions. These interpretations in turn provide a minimum estimate of the complexity of the rate-limiting pathway to a developmental stage, temporally define major transition points between timer components, and provide some insight into the nature of timer components. By characterizing the rate-limiting pathway from the origin of a developmental program for each consecutive stage in that program, distinctions can also be made between single, parallel, sequential, and branching timer relationships. From these interpretations, a detailed temporal “map” of the rate-limiting program can be generated for any developmental system in which consecutive stages can be reproducibly monitored with time.     

Photoperiodic control of reproduction in the male lizardAnolis carolinensis

Summary In contrast to the higher vertebrates the photoperiodic time measuring system in the male lizardAnolis carolinensis seems to rely on an hourglass timer which lacks endogenous rhythmicity. This timer appears to measure the absolute length of the light portion of light-dark (LD) cycles. The present study further characterized the nature of theAnolis photoperiodic timer and demonstrated: (1) The gonadal response is quite sensitive to photostimulation. Exposure to as few as three 16 h photoperiods (over a 3 week period) can maintain testicular function in summer anoles whereas exposure to as few as six 16 h photoperiods (over a 3 week period) can elicit maximal testicular development in the fall. (2) The photoperiodic timer does not have to be reset daily by a dark interruption. (3) The dark portion of LD cycles may be involved in a complex fashion in reversing a light-initiated reaction and (4) Comparisons of entrained circadian activity rhythms with testicular responses to various light cycles argue against the participation of a circadian clock in photoperiodic time measurement.Abbreviation CRPP circadian rhythm of photoperiodic photo-sensitivity     

Interaction of ethanolamine with alcohol dehydrogenase from the horse liver

The pattern of kinetic behaviour of ethanolamine (EA), an ethanol structural analog, in the alcohol dehydrogenase reaction has been studied. EA has been shown to manifest a mixed type inhibition versus ethanol and a noncompetitive behaviour towards the second substrate, NAD. A graphical analysis of the experimental results as well as the construction of secondary graphs provide evidence in favour of a mechanism, according to which the interaction between EA and the enzyme results in a dead-end complex formation (ESI). A direct conversion into reaction products can be achieved only after EA separation from the complex. The Ki value for the E-EA complex is 1.3 mM; that for EA release from the E-EA is 1.8 mM. An analysis of competitive interactions with NAD showed these constants to be equal in values (2 mM). Taking account of real concentrations of tissue EA and of experimental values of Ki, a conclusion is drawn on possible participation of EA in the alcohol dehydrogenase reaction control.     

Basic helix-loop-helix proteins and the timing of oligodendrocyte differentiation

An intracellular timer in oligodendrocyte precursor cells is thought to help control the timing of their differentiation. We show here that the expression of the Hes5 and Mash1 genes, which encode neural-specific bHLH proteins, decrease and increase, respectively, in these cells with a time course expected if the proteins are part of the timer. We show that enforced expression of Hes5 in purified precursor cells strongly inhibits the normal increase in the thyroid hormone receptor protein TR(&bgr;)1, which is thought to be part of the timing mechanism; it also strongly inhibits the differentiation induced by either mitogen withdrawal or thyroid hormone treatment. Enforced expression of Mash1, by contrast, somewhat accelerates the increase in TR(beta)1 protein. These findings suggest that Hes5 and Mash1 may be part of the cell-intrinsic timer in the precursor cells.     

Arabidopsis phosphatidylinositol monophosphate 5-kinase 2 is involved in root gravitropism through regulation of polar auxin transport by affecting the cycling of PIN proteins

Phosphatidylinositol monophosphate 5-kinase (PIP5K) catalyzes the synthesis of PI-4,5-bisphosphate (PtdIns(4,5)P(2)) by phosphorylation of PI-4-phosphate at the 5 position of the inositol ring, and is involved in regulating multiple developmental processes and stress responses. We here report on the functional characterization of Arabidopsis PIP5K2, which is expressed during lateral root initiation and elongation, and whose expression is enhanced by exogenous auxin. The knockout mutant pip5k2 shows reduced lateral root formation, which could be recovered with exogenous auxin, and interestingly, delayed root gravity response that could not be recovered with exogenous auxin. Crossing with the DR5-GUS marker line and measurement of free IAA content confirmed the reduced auxin accumulation in pip5k2. In addition, analysis using the membrane-selective dye FM4-64 revealed the decelerated vesicle trafficking caused by PtdIns(4,5)P(2) reduction, which hence results in suppressed cycling of PIN proteins (PIN2 and 3), and delayed redistribution of PIN2 and auxin under gravistimulation in pip5k2 roots. On the contrary, PtdIns(4,5)P(2) significantly enhanced the vesicle trafficking and cycling of PIN proteins. These results demonstrate that PIP5K2 is involved in regulating lateral root formation and root gravity response, and reveal a critical role of PIP5K2/PtdIns(4,5)P(2) in root development through regulation of PIN proteins, providing direct evidence of crosstalk between the phosphatidylinositol signaling pathway and auxin response, and new insights into the control of polar auxin transport.     

Selenium, selenoenzymes, oxidative stress and risk of neoplastic progression from Barrett's esophagus: results from biomarkers and genetic variants

Clinical trials have suggested a protective effect of selenium supplementation on the risk of esophageal cancer, which may be mediated through the antioxidant activity of selenoenzymes. We investigated whether serum selenium concentrations, selenoenzyme activity, oxidative stress and genetic variation in selenoenzymes were associated with the risk of neoplastic progression to esophageal adenocarcinoma (EA) and two intermediate endpoints, aneuploidy and tetraploidy. In this prospective cohort study, during an average follow-up of 7.3 years, 47 EA cases, 41 aneuploidy cases and 51 tetraploidy cases accrued among 361 participants from the Seattle Barrett's Esophagus Research Study who were free of EA at the time of blood draw and had at least one follow-up visit. Development to EA was assessed histologically and aneuploidy and tetraploidy by DNA content flow cytometry. Serum selenium concentrations were measured using atomic absorption spectrometry, activity of glutathione peroxidase (GPX) 1 and GPX3 by substrate-specific coupled test procedures, selenoprotein P (SEPP1) concentrations and protein carbonyl content by ELISA method and malondialdehyde concentrations by HPLC. Genetic variants in GPX1-4 and SEPP1 were genotyped. Serum selenium was not associated with the risk of neoplastic progression to EA, aneuploidy or tetraploidy (P for trend?=?0.25 to 0.85). SEPP1 concentrations were positively associated with the risk of EA [hazard ratio (HR)?=?3.95, 95% confidence intervals (CI)?=?1.42-10.97 comparing the third tertile with the first] and with aneuploidy (HR?=?6.53, 95% CI?=?1.31-32.58), but not selenoenzyme activity or oxidative stress markers. No genetic variants, overall, were associated with the risk of neoplastic progression to EA (global p?=?0.12-0.69). Our results do not support a protective effect of selenium on risk of neoplastic progression to EA. Our study is the first to report positive associations of plasma SEPP1 concentrations with the risk of EA and aneuploidy, which warrants further investigation.