Highly Sensitive Detection of Individual HEAT and ARM Repeats with HHpred and COACH

BACKGROUND:HEAT and ARM repeats occur in a large number of eukaryotic proteins. As these repeats are often highly diverged, the prediction of HEAT or ARM domains can be challenging. Except for the most clear-cut cases, identification at the individual repeat level is indispensable, in particular for determining domain boundaries. However, methods using single sequence queries do not have the sensitivity required to deal with more divergent repeats and, when applied to proteins with known structures, in some cases failed to detect a single repeat. METHODOLOGY AND PRINCIPAL FINDINGS:Testing algorithms which use multiple sequence alignments as queries, we found two of them, HHpred and COACH, to detect HEAT and ARM repeats with greatly enhanced sensitivity. Calibration against experimentally determined structures suggests the use of three score classes with increasing confidence in the prediction, and prediction thresholds for each method. When we applied a new protocol using both HHpred and COACH to these structures, it detected 82% of HEAT repeats and 90% of ARM repeats, with the minimum for a given protein of 57% for HEAT repeats and 60% for ARM repeats. Application to bona fide HEAT and ARM proteins or domains indicated that similar numbers can be expected for the full complement of HEAT/ARM proteins. A systematic screen of the Protein Data Bank for false positive hits revealed their number to be low, in particular for ARM repeats. Double false positive hits for a given protein were rare for HEAT and not at all observed for ARM repeats. In combination with fold prediction and consistency checking (multiple sequence alignments, secondary structure prediction, and position analysis), repeat prediction with the new HHpred/COACH protocol dramatically improves prediction in the twilight zone of fold prediction methods, as well as the delineation of HEAT/ARM domain boundaries. SIGNIFICANCE:A protocol is presented for the identification of individual HEAT or ARM repeats which is straightforward to implement. It provides high sensitivity at a low false positive rate and will therefore greatly enhance the accuracy of predictions of HEAT and ARM domains

Myelin Proteomics: Molecular Anatomy of an Insulating Sheath

Fast-transmitting vertebrate axons are electrically insulated with multiple layers of nonconductive plasma membrane of glial cell origin, termed myelin. The myelin membrane is dominated by lipids, and its protein composition has historically been viewed to be of very low complexity. In this review, we discuss an updated reference compendium of 342 proteins associated with central nervous system myelin that represents a valuable resource for analyzing myelin biogenesis and white matter homeostasis. Cataloging the myelin proteome has been made possible by technical advances in the separation and mass spectrometric detection of proteins, also referred to as proteomics. This led to the identification of a large number of novel myelin-associated proteins, many of which represent low abundant components involved in catalytic activities, the cytoskeleton, vesicular trafficking, or cell adhesion. By mass spectrometry-based quantification, proteolipid protein and myelin basic protein constitute 17% and 8% of total myelin protein, respectively, suggesting that their abundance was previously overestimated. As the biochemical profile of myelin-associated proteins is highly reproducible, differential proteome analyses can be applied to material isolated from patients or animal models of myelin-related diseases such as multiple sclerosis and leukodystrophies

A temperature-compensated ultradian clock ticks in Schizosaccharomyces pombe.

An ultradian oscillation is described for Schizosaccharomyces pombe which meets the criteria for a cellular clock, i.e. timekeeping device. The rhythm can be induced by transfer from circadian conditions (stationary phase or very slow growth) to ultradian conditions (rapid growth). It can also be synchronized by ultradian temperature cycles of 6 degrees C difference. Released to constant temperature, the rhythm persists for 20 h without damping. The period of the free-running rhythm is temperature-compensated and in no experiment did period length fall outside the narrow range between 40 and 44 min. The parameter observed is the septum index, i.e. the percentage of cells occupying the last stage of the cell cycle in wild-type cells before final division. The results suggest control of the cell division processes by the ultradian clock

The aniline blue fluorochrome specifically stains the septum of both live and fixed Schizosaccharomyces pombe cells

A novel method is described which uses aniline blue for the specific fluorescent staining of the septa of dividing cells of the fission yeast, Schizosaccharomyces pombe. It gives the same results with live and fixed cells. In fixed or, more generally, dead cells there is no staining of the cytoplasm: this renders aniline blue superior to other dyes previously used to stain the septum of S. pombe. This feature allows quantitative analysis of the septum index for fixed samples and, therefore, makes aniline blue the stain of choice for cell cycle kinetic studies

A temperature-compensated ultradian clock explains temperature-dependent quantal cell cycle times

The effects of sublethal heat pulses on cell division have provided insights into possible molecular mechanisms. Thus Zeuthen's findings of 'set-backs' up to a transition point provides the basis for the idea that the continuous accumulation of a compound needed for cell division spans a major portion of the cell cycle. The accumulating substance is a 'division protein' which forms part of a structure which is unstable until completely assembled at the transition point. Experiments showing phase resetting of mammalian cells by temperature perturbation indicate limit-cycle oscillator control of the cell cycle with a phase-response curve with a repeat interval equal to the period of the clock. As well as providing a method for establishing synchronized cultures these observations have found application in the selective effects of hyperthermia as an antitumour agent. Circadian rhythms display several unique features distinguishing them from other periodic processes. Only recently has it been recognized that some of these characteristics may be properties of ultradian rhythms as well. The probably most striking feature of circadian timekeeping, i.e. independence of ambient temperature, was found for ultradian rhythmicity even at the level of the unicellular organization. Synchronous cultures of some lower eukaryotes were prepared by centrifugal size selection methods. Experiments with asynchronous control cultures substantiated the view that the conditions employed were such as to minimize any perturbative effects: most importantly the organisms were never removed from their culture medium. Whereas the control cultures showed smoothly increasing respiration rates, total RNA, total protein, enzyme activities and enzyme protein (e.g. for cytochrome aa3, ATPase, catalase), in synchronous cultures all these parameters showed oscillatory behaviour. Different periods were observed in different organisms: thus in Acanthamoeba castellanii the period was about 70 min, in Tetrahymena pyriformis strain ST it was about 50 min, in T. pyriformis AII it was 30 min, and in Candida utilis it was about 30 min (all measurements at 30 degrees C). In A. castellanii the periods of both the oscillations in rate of respiration and the total cell protein were hardly affected by the temperature of growth over the range 20 to 30 degrees C. The oscillations show no damping during experiments lasting 12 h: these properties suggest that we are observing temperature-compensated endogenous rhythms which presumably serve a timekeeping function in cells undergoing growth and division.(ABSTRACT TRUNCATED AT 400 WORDS