Ice-binding proteins that aid the survival of freeze-avoiding, cold-adapted organismsby inhibiting the growth of endogenous ice crystals are called antifreeze proteins(AFPs). The binding of AFPs to ice causes a separation between the melting point andthe freezing point of the ice crystal . TH produced byhyperactive AFPs is an order of magnitude higher than that produced by a typical fishAFP. The basis for this difference in activity remains unclear. Here, we havecompared the time dependence of TH activity for both hyperactive and moderatelyactive AFPs using a custom-made nanolitre osmometer and a novel microfluidics system.We found that the TH activities of hyperactive AFPs were time-dependent, and that theTH activity of a moderate AFP was almost insensitive to time. Fluorescence microscopymeasurement revealed that despite their higher TH activity, hyperactive AFPs from twoinsects (moth and beetle) took far longer to accumulate on the ice surface than did amoderately active fish AFP. An ice-binding protein from a bacterium that functions asan ice adhesin rather than as an antifreeze had intermediate TH properties.Nevertheless, the accumulation of this ice adhesion protein and the two hyperactiveAFPs on the basal plane of ice is distinct and extensive, but not detectable formoderately active AFPs. Basal ice plane binding is the distinguishing feature ofantifreeze hyperactivity, which is not strictly needed in fish that require onlyapproximately 1°C of TH. Here, we found a correlation between the accumulationkinetics of the hyperactive AFP at the basal plane and the time sensitivity of themeasured TH. ModeratPseudopleuronectes americanus, could increase over time. The sameresearchers found that TH increased by a factor of 5 over time and reached a plateau60–180 min after exposure of an ice crystal to the AFP I solution, depending onthe concentration. They hypothesized that time-dependent TH resulted from theaccumulation of AFP molecules on the ice surfaces or the rearrangement of adsorbed AFPmolecules on the ice (or a combination of the two) [et al. observed the samephenomenon in a solution prepared from an AFPIII derived from Notched-fin eelpout. Theyfound that the TH activity increased by a factor of 2.5 after allowing for an annealingtime of 3 h [et al.[Although the dependence of TH activity on the AFP concentration has been extensivelystudied, the time dependence of this process remains unclear. A handful of experimentalstudies of the time-dependent TH activities of moderate AFPs have been reported ,19–21, ae of 3 h . This mo [et al. measured [et al..et al.have found evidence for these protein-bound ice-like waters [Various computational studies have suggested that AFPs form an ice-binding structure byconstraining water molecules present at the ice-binding sites into a structural orderingthat resembles ice ,25. Garne waters . Howevere waters , it is let al. [et al. suggested a mechanism by which AFGPs altered the dynamics ofwater molecules in the hydration shell in such a way that the solution freezing processwas inhibited [Dendroides canadensis,DcAFP) and identified direct short-range interactions between theAFP and the ice surface, in addition to long-range ice–water interactionsmediated by the AFP [et al.suggested that AFGPs interrupted the arrangement of the ice–water interfacethrough kinetic effects rather than through mediating binding between the ice surfaceand water molecules. Their final conclusion was that AFGPs could not be accuratelydescribed using the Gibbs–Thomson model [Sander & Tkachenko developed a ‘kinetic pinning’ mathematical modelthat described the AFP molecules as obstacles that induced surface pinning on thegrowing ice surface . The modet al.  examinednhibited , thereby the AFP . Solid-s the AFP . Zepada on model . Knight on model . These fStandard methods for measuring TH activity  include the use of ananolitre osmometer  in whichTenebrio molitor AFP (TmAFP) and spruce budworm AFP(sbwAFP) could be increased by a factor of 40 by increasing the exposure time. Weexamined the accumulation rates of both the moderate and hyperactive AFPs on thedifferent crystal planes, as well as the TH activity.Our objectives in this work were to characterize the effects of the exposure time on theTH properties of various AFPs and to identify the distinctions between the kineticmechanisms by which various AFPs function. We found that the TH activities ofThe findings reported here suggest a new approach to measuring and enhancing THactivity. Appropriate kinetic models of AFP adsorption to ice crystal planes can improveour understanding of the mechanisms by which these proteins act and could lead to aunified description of AFP activity.2.2.1.−1. TH measurements were as follows (Tm) was determined as describedelsewhere [Tm. The temperature was subsequently decreased at a fixedrate (0.15°C per min by reducing the temperature by 0.01°C every 4 s)until the crystal underwent sudden, uncontrolled growth (burst). The time between thecrystal formation (the end of melting) and the growth burst was defined as thecrystal ‘exposure time’. The output AVI files were analysed after theexperiments to accurately identify the melting point and the growth burst of thecrystal [We used a custom-designed nanolitre osmometer system that includes a 3040 Newporttemperature controller  and a previously described custom-madecooling stage ,33. The  follows : sub-miclsewhere . The bulcrystal . The exp2.2.Escherichia coli: TmAFP-GFP [MpAFP-GFPfrom Marinomonas primoryensis [2, 25 mMTris–HCl (pH 8); and isoform 501 of sbwAFP-GFP [Several proteins were tested in this study. Three hyperactive GFP-tagged AFPs wereproduced in mAFP-GFP  was stororyensis  was storwAFP-GFP  was storwAFP-GFP  was stor2.3.Previously, we developed and used microfluidic devices to understand the effects offree unbound AFPs on ice crystal growth and the binding mechanism of the AFPs to ice. With thAFP-free buffer solution in the microfluidic channel spontaneously nucleated to forma bulk ice crystal at approximately –20°C. The bulk ice crystal wassubsequently warmed to a temperature near the equilibrium melting temperature to forma single crystal. A 980 nm, 500 mW, diode laser  was used to heat and melt any unwanted ice to help obtain the singlecrystal in the designated cell. After ice formation, AFP solution was injected to theflow line using a glass syringe. By this method, no AFP was present in the core ofthe ice and the AFP molecules adsorbed to the crystals only at the surface .3.3.1.TmAFP is a 9 kDa protein with eight disulfide bonds [TmAFP. The positioning of the GFP tag in the fusion protein didnot interfere with the ability of the proteins during binding to ice and haveactually enhanced the TH activity as a result of the larger protein size (36 kDa)[a,b). The addition of 27 kDa to the AFP may, however,have slowed down the diffusion rate and influenced the adsorption rate. We tested thesignificance of the mass effects by comparing the dependence of TH on incubation timefor the TmAFP and TmAFP-GFP proteins under severalconditions. Indistinguishable kinetic behaviours were observed . de bonds . We used(36 kDa),40 . The TH activity increased by factors of between 10(for the 1 µM solution) and 3 (for the 40 µM solution). A long exposuretime (12 h) at a very low TmAFP concentration (1 µM)increased the TH activity to a level comparable to that obtained from incubating anice crystal in a 10 µM solution for a much shorter period of time (10min).The kinetics of the TH activity were measured using a custom-made nanolitreosmometer. The procedure is illustrated schematically in measured a. The THTmAFP solutions obviously depended on the concentration (a). A plot ofthe TH values obtained from the four TmAFP concentrations as afunction of the exposure time followed an approximately hyperbolic relationship(c). Theexposure time was calculated as the sum of the incubation time and the cooling timeuntil bursting. Higher TH values were obtained with extended crystal incubation times and under two supercooling conditions(incubation at 0.02°C or 0.1°C below the melting point). The results is a 38 kDa protein and is somewhatlarger than most AFPs (3–16 kDa). The adsorption kinetics ofMpAFP to ice were distinct from those of AFPIII, although theywere closer to the kinetics of AFPIII than to the kinetics of TmAFP.The TH activity was found to depend on the exposure time only at low concentrations(2.4 µM) of MpAFP-GFP  on its ice-binding site, similar tothose found in TmAFP and its orthologue from DcAFP[TmAFP, The TH of sbwAFPdepended profoundly on the exposure time (TmAFP (i.e. more time was needed for sbwAFP to depress thefreezing point) and was extremely slow compared with adsorption rate of AFPIII. TheTH activity increased by a factor of 40 as the sbwAFP exposure time was increasedfrom 8 s to 850 min. This TH ratio increase value was the highest value yet measuredamong the AFPs tested thus far .The accumulation of AFP molecules on the ice surfaces was measured by imaging the icecrystals in the presence of AFP molecules fused to GFP markers, AFP-GFP. Microfluidictechniques were used to prepare a large ice crystal in an AFP-free solution. AnAFP-GFP solution was then injected into the microfluidic channel, and the largecrystal was partially melted. Once melting had ceased, the AFP-GFP molecules adsorbedto the ice crystal surface under constant temperature conditions . The outer layer of the crystalthen became covered with AFP-GFPs, and no AFP-GFPs were observed within the crystal.Note that if the ice crystals had been initially nucleated in solutions containingAFP-GFP, the crystals that formed would have included AFP-GFPs within the ice, andnot only on the crystal surfaces. This procedure was performed for both theAFPIII-GFP and g. 3b in ). The foTmAFP-GFP. TmAFP, figure7a,b) displayed continuous accumulationof TmAFP-GFP molecules on the basal plane over an exposure time of 4h. We did not observe further accumulation during an additional 1.5 h of exposuretime suggesting that any subsequent change in fluorescence was too small to detectusing our instrumentation. The accumulation on the basal plane revealed that theadsorption rate (the time derivative of the fluorescence intensity) was high withinthe first few minutes after ice growth had ceased. After approximately 1000 s, theadsorption rate slowed and nulled after approximately 4 h. The accumulation kineticsmeasurements over periods of at least 1 h were repeated six times. Accumulationexperiments over shorter periods of time were repeated a greater number of times(more than 200 experiments). The accumulation experiments performed using AFPIII showed that the accumulation ofAFPIII-GFP at the prism plane reached saturation very rapidly (within 6 min) comparedto TmAFP-GFP (within 4 h). The solid line in figure 7d followed the equationI is the fluorescence intensity,Imax is the maximum fluorescence intensity parameter,t is the time from the ice formation andτ = 70 s is a time constant parameter that fit thedata. Our experiments revealed that 6 s ≥ τ ≥90 s for AFPIII-GFP depending on the solution concentration. We found a linearcorrelation between the inverted τ and the concentration. Assuming that1/τ = KonC, whereKon is the adsorption constant and Cis the AFP concentration in the solution, we found thatKon = 0.008 ± 0.001µM−1 s−1 . Nevertheless, single exponent formula did not fit the data forTmAFP-GFP for any parameter set. A model that included threeexponents to describe the process of TmAFP accumulation, however,provided a good description of the dataN = 3 andτi = s, respectively. The fluorescence signal was measured and extracted using the Nis-Elements program. An in-house Matlab program was used to analyse the fluorescencesignals and extract a maximum from each line profile of the fluorescence intensityperpendicular to the crystal plane that crossed the ice–solution interface.This procedure was used to measure the fluorescence signal from the ice surface overtime in the microfluidic channel. The intensity was interpreted as indicating theamount of bound AFP present on the ice surface and was used to monitor the binding ofboth AFPIII-GFP and a,b.The lack of binding between the moderate AFPs and the basal plane has been discussedpreviously  and has figure74.Tm, on the hysteresis activities of several AFPs. Thisinfluence was then found to be correlated with the accumulation of the tagged AFPs onthe surfaces of the ice crystals. Our study of moderate and hyperactive AFPs revealedvery different TH dynamics. This study showed that the TH of hyperactive AFPs increasedwith longer AFP exposure times. Although the TH activities of the TmAFPand sbwAFP solutions were sensitive to the exposure time , the TH activity of the AFPIII solution was relatively insensitive to theexposure time (the TH activity varied by only a factor of 1.5 in an hour). The THactivity of MpAFP, a bacterial hyperactive AFP, was only sensitive tothe exposure time at low concentrations (2.4 µM). Over short exposure times (afew seconds), all hyperactive AFPs showed very low TH activity. The differences betweenthe kinetics suggested that TmAFP and sbwAFP bound to ice at a muchslower rate than MpAFP, which bound slower than the moderately activeAFP. The rapid adsorption of AFPIII to the ice crystal could explain the observed THactivity. Evidence for rapid adsorption was obtained from fluorescence microscopymeasurements during ice formation in the presence of AFPIII-GFP. Significant amounts ofthe measured fluorescence signal on fast growing ice was observed the instant the icegrowth halted. In AFPIII accumulation experiments, in which the fluorescence intensitywas measured after the crystal had melted to a smaller size and then shaped slowly to abipyramidal structure , we measured singleexponent intensity growth. The fast increase in the fluorescence signal from the surfaceof the fast growing crystals might be due to high local AFP concentration that developedduring growth. Alternatively, this fast kinetics can attribute to modified ice surfacein comparison to the gentle growth within the TH gap. We note that in the presence ofthe moderate AFP, the ice grew along the c-axis, and the ice surfacedeveloped within the hysteresis gap. This behaviour contrasts with ice crystal shapingin the presence of hypAFP that occurs during ice melting and not within the TH gap[et al. [b). This unique feature both enhanced the ability to measurefluorescence on the surfaces and indicated that the AFP trapped inside of an ice crystaldid not influence the TH.This study examined the influence of the exposure time, at a temperature slightly belowthe e TH gap. The rap[et al. . Note thMpAFP, onthe other hand, includes the ice-binding domain of an ice adhesin is thought to beinvolved in docking its host bacterium to an ice crystal [MpAFP showed an almost sigmoidal increase in TH activity, but thenrapidly approached a plateau value slightly greater than 2°C. By contrast, theinsect AFPs approached TH values of 5–6°C at modest AFP concentrations[Tenebrio molitor beetle and spruce budworm, canendure much lower temperatures (–20°C) than are indicated by the THmeasured in the nanolitre osmometer [in vitro TH measurements and the survival temperatures of theinsects. Long exposure times to tiny ice crystals, as is allowed in the haemolymph of aninsect, can increase the apparent TH in the insect beyond the values that are typicallymeasured in the nanolitre osmometer.It was not surprising that the two insect AFPs displayed similar TH dynamics, becausethese two proteins have the same function of protecting their hosts from freezing insub-zero temperatures. The proteins also have very similar ice-binding sites within theprotein structure that comprise tandemly arrayed TXT motifs.  crystal . Althoug crystal .MpAFP strations. In natusmometer . This ansmometer . Anothersmometer .Crystalsmometer . Ourfinet al. [et al. [The binding kinetics of the AFPs to ice were characterized using fluorescence microscopytechniques to measure the accumulation of the AFPs on the different ice crystal planes.Takamichi et al.  measured [et al.  estimate [et al. . Their r [et al. .TmAFP. Subsequently, the hyperactiveTmAFP accumulated progressively on the basal plane over a few hours.The following section discusses these results provides an interpretation to place theresults into context.Our direct measurements of the AFP accumulation rates revealed that the initialadsorption of the AFPs to ice (over a few seconds) was very fast, and this process wassimilar for the AFPIII and σ is the surface coverage,σmax is the maximum possible coverage,t is the time from the ice formation and τis the time constant for the accumulation. This equation is a modified version of theoriginal model that takes into account the incubation time. Indeed, our measurements ofthe AFPIII-GFP fluorescence intensity over time were consistent with this model, asshown in figure 7d.Knight & DeVries suggested that the inhibition of ice growth by those AFPs thatcould not bind to the basal plane (moderate AFPs) was not time-dependent and was insteada function of the AFP concentration and the degree of supercooling [Kon = 0.008 ± 0.001µM−1 s−1), and the TH was found to beinsensitive (relative to the behaviour of hypAFP) to the exposure time. The modest timedependence occurred on a timescale that exceeded the accumulation kinetics of thefluorescence signal. The measured Kon value presented hereis the first experimental report of the adsorption constant of AFP molecules to ice.Several models have been proposed to describe the kinetics of AFP adsorption to ice,28. For rcooling . Thus, cTmAFP accumulated on the basal planein a slow and progressive manner. Our measurements of the accumulation ofTmAFP did not reveal any particular timescale(τ). A multi-exponential function with at least three timeconstants was needed to describe the fluorescence data. Processes that must be modelledacross many timescales or through the use of a stretched exponential are common inannealing processes, for example, in glass ageing [TmAFP-GFP to ice was influenced by both the number of binding sitesand by another process, such as the suitability of the available binding sites tointeractions with AFP. The secondary binding process proposed by Takamichi etal. [et al. [TmAFPand sbwAFP over long periods of time, as was observed in our TH measurements.Accordingly, the ability of the hypAFPs to bind to the basal plane and to inhibit icegrowth on this plane contributed most significantly to the time-dependent THactivity.On the other hand, the hyperactive s ageing . The Weis ageing  describei etal.  provides [et al.  suggesteet al. [et al. [c-direction and consequently decreases the basal plane surfacearea which leads to higher TH activities.The interpretations of our results may be summarized in terms of two kinetic processesthat contribute to TH. First, the AFPs blocked ice crystal growth in aconcentration-dependent manner, as described by Sander et al.  and Kniget al. . Accordiet al. . We foun [et al.  explaine [et al. . One supTmAFP. Therelationship between the number of AFP molecules accumulated onto an ice surface and themeasured TH will be investigated further in future studies.Our experiments using fluorescence microscopy techniques in conjunction withmicrofluidic devices demonstrated the binding of AFPs to ice crystals during the activeexchange of solutions around a crystal. This study revealed the complex dynamics ofprotein attachment to the ice crystals and the time-dependent effects of proteinattachment on the TH activity. The sensitivity of TH to the exposure time highlights thenecessity of reporting the exposure time parameter along with any reported AFP THactivity. This is especially important for the TH activities of proteins that are highlysensitive to the exposure time, such as sbwAFP and


This effect can be explained by inhibition of LARP6 phosphorylation and suggests that Akt inhibitors may be effective in treatment of various forms of fibrosis.La ribonucleoprotein domain family, member 6 (LARP6) is the RNA binding protein, which regulates translation of collagen mRNAs and synthesis of type I collagen. Posttranslational modifications of LARP6 and how they affect type I collagen synthesis have not been studied. We show that in lung fibroblasts LARP6 is phosphorylated at 8 serines, 6 of which are located within C-terminal domain. Phosphorylation of LARP6 follows a hierarchical order; S451 phosphorylation being a prerequisite for phosphorylations of other serines. Inhibition of PI3K/Akt pathway reduced the phosphorylation of LARP6, but had no effect on the S451A mutant, suggesting that PI3K/Akt pathway targets S451 and we have identified Akt as the responsible kinase. Overexpression of S451A mutant had dominant negative effect on collagen biosynthesis; drastically reduced secretion of collagen and induced hyper-modifications of collagen α2 (I) polypeptides. This indicates that LARP6 phosphorylation at S451 is critical for regulating translation and folding of collagen polypeptides. Akt inhibitor, GSK-2141795, which is in clinical trials for treatment of solid tumors, reduced collagen production by human lung fibroblasts with EC Type I collagen is triple helical protein composed of two α1 (I) polypeptides and one α2 (I) polypeptide. The constitutive rate of collagen synthesis is low, because the protein is very stable with half life of 60 daysThe biosynthesis of type I collagen is involving translation of the individual polypeptides, their posttranslational modifications, folding of three polypeptides into a triple helix, secretion of the triple helix into the extracellular space and processing of the secreted procollagen into mature collagen356756910111213147569Collagen polypeptides are cotranslationally inserted into the ER lumen. During this process nascent polypeptides acquire several modifications, including hydroxylations of selected lysines and prolines and glycosylation of hydroxyl-lysine residues. Then, two pro α1(I) and one pro α2(I) chains associate at the carboxyl terminus and initiate folding into triple helix, which is propagated towards the amino terminus in a zipper-like fashion to form a procollagen molecule422245There have been no reports on phosphorylation of LARP6 in the literature. The aim of this study was to determine if LARP6 is phosphorylated, to identify the phosphorylation sites, the kinase(s) involved and the role of LARP6 phosphorylation in type I collagen expression. We report that LARP6 is phosphorylated at multiple serines and that phosphorylation of S451 by Akt is critical for its regulation of type I collagen expression. We also propose a novel mechanism for the anti-fibrotic effect of Akt inhibition.To assess if LARP6 is phosphorylated, we analyzed LARP6 migration in SDS-PAGE gels. In one dimensional SDS-PAGE (1DGE) LARP6 from human lung fibroblasts (HLFs) was resolved as two bands, which were reduced to a single, faster migrating, band after calf intestinal alkaline phosphatase (CIP) treatment , suggestTo determine which amino acids are modified by phosphorylation, we analyzed the phospho-peptides by Mass Spectrometry. In three independent experiments 8 phosphorylation sites on LARP6 were identified; they are Ser56, Ser58, Ser348, Ser396, Ser409, Ser421, Ser447, and Ser451 . IdentifThe sequence comparison between LARP3 and LARP6 is shown in 1021To confirm that the serines identified by Mass Spectrometry are phosphorylated we individually mutated the 6 serines within the C-terminal domain into alanines and subjected the mutants to 2DGE analysis (isoelectric focusing (IEF) followed by SDS-PAGE and Western blotting). The rationale was that if a serine is phosphorylated, its mutation into alanine will result in a loss of a negative charge, which can be scored as a shift of the pI spectrum into the more basic region of the isoelectric focusing strip. Consequently, if there is a gain of negative charge by phosphorylation the pI values will be shifted to the more acidic region. Based on the importance of S451 phosphorylation, the next step was to identify the kinase involved. We surmised that, if S451 phosphorylation is required for acquisition of other phosphorylations, inhibition of the kinase involved in phosphorylation of S451 would result in drastic hypophosphorylation of LARP6, which could be detected in 1DGE see . Therefo3233To provide additional evidence that PI3K/Akt signaling pathway phosphorylates LARP6 we analyzed the pI of HA-LARP6 after treatment of Akt inhibitor, GSK-2141795. The inhibitor shifted the pI of HA-LARP6 molecules from 6.8–7.4 , panel 1When constitutive active Akt (CA Akt)Because most of the phosphorylation sites were identified in the carboxyl terminal domain of LARP6, predicted to be unstructured, we assessed if this domain can undergo phosphorylations independently of the other domains of LARP6. 2DGE showed that HA-CTER was resolved with pI spectrum of 6.6–9.0 with multiple phospho signals, consistent with multiple phosphorylations , panel 1In vitro kinase assay was used to corroborate that Akt is involved in phosphorylation of LARP6 at serine 451. As substrate, LARP6 had to be immunoprecipitated after expression in mammalian cells. In the immunoprecipitate containing HA-LARP6 radiolabeling of the protein was observed  polypeptide was not significantly changed, while the level of α2 (I) polypeptide was slightly reduced. The densitometric scans of Western blots are shown in the bottom panel of We also analyzed modifications of collagen α2(I) polypeptide by 2DGE in cells treated with GSK-2141795. Failure of the available antibody to recognize α1(I) polypeptide after isoelectric focusing prevented the analysis of this polypeptide. In control cells the pI of the collagen α2(I) polypeptide was distributed over a narrow pI range, centered around 8.8, indicating presence of uniformly modified molecules  and α2(I) polypeptide was similar in mock transfected cells and cells overexpressing wt or S451A HA-LARP6 . HoweverWe also analyzed modifications of collagen α2 (I) polypeptide in cells overexpressing S451A by 2DGE. Overexpression of S451A mutant induced appearance of α2(I) molecules with more acidic pI, between 7.9 and 8.1 , suggestIf the inhibition of Akt reduces secretion of type I collagen due to absence of S451 phosphorylation, then expression of the LARP6 S451D mutant should overcome this inhibitory effect. Therefore, we analyzed if the phospho-mimetic mutation, S451D, can rescue type I collagen secretion when Akt is inhibited. Inhibition of Akt by GSK-2141795 did not change the intracellular level of collagen α1(I) and α2(I) polypeptides in cells overexpressing either the S451D mutant or the wt HA-LARP6 . HoweverWhile we have demonstrated that phosphorylation of S451 is a prerequisite for additional phosphorylations and for participation of LARP6 in collagen biosynthesis, we have also observed that the S451 phosphorylation regulates half life of the protein. wt HA-LARP6 decayed with a half life of 15.5 hours, S451D mutant had a half life of 11.5 hours and S451A mutant had a half life of >18 hours . This inTo investigate if there is an association between the half life of LARP6 and collagen expression, we artificially prolonged the half life of endogenous LARP6 by treating HLFs with proteasome inhibitor, MG132. Between 3 hours and 6 hours of MG132 treatment we observed that LARP6 accumulated at the higher level in the cells, suggesting that its degradation has been retarded . When we50 for Akt1 of 180 nM, for Akt2 of 328 nM and for Akt3 of 38 nM3150 for Akt isoforms and may reflect nonspecific effects seen at high concentrations of most inhibitors. However, GSK-2141795 reduced secretion of type I collagen at concentrations of 160 nM which is lower than IC50 for Akt1 and Akt2 (50) was about 150 nM  LARP6 is phosphorylated at 8 serines, (ii) phosphorylation of LARP6 at S451 by Akt is required for phosphorylation of several other sites, (iii) the S451A mutant of LARP6 has dominant negative effect on type I collagen production by reducing secretion and causing hyper-modifications of α2 (I) polypeptide, (iv) similar effect can be induced by Akt inhibition, (v) Akt inhibitor GSK-2141795 reduces secretion of type I collagen at therapeutic concentrations. These results demonstrate that phosphorylation of LARP6 at S451 is an important step to activate LARP6 in collagen biosynthesis and to regulate translation and folding of type I collagen polypeptides.This is also the first report on functional significance of phosphorylation of LARP6. Large scale mapping of phosphoproteins in A498 and Hela cells identified phosphorylation of LARP6 at S56 and S5838101121Our results are consistent with hypothesis that phosphorylation of S451 in LARP6 is an early phosphorylation event that is required for priming of other phosphorylation sites. The reasoning is based on several experiments. First, when S451 was mutated into alanine, the mutant LARP6 showed absence of multiple phospho-isoforms and not only one phosphorylation, as determined by disappearance of at least 3 phospho signals from 2DGE , panel 6in vivo and in vitro approaches, but the evidence that Akt directly phosphorylates S451 is still lacking. Bacterially expressed LARP6 could not be phosphorylated by purified Akt; although this could be due to a misfolded protein or to absence of some accessory factors, as described for other Akt substrates4041Our efforts to identify the kinase involved in LARP6 phosphorylation pointed to the PI3K/Akt pathway . Identifin vitro when the protein is immunoprecipitated from cells. Immunoprecipitated LARP6 was phosphorylated in the absence of co-expressed active Akt or pure Akt added to the precipitate  and α2(I) polypeptides is synchronized to the rate of their posttranslational modifications and folding into a triple helixWe also found that S451D mutant has shorter half life than wt LARP6 or S451A mutant . As this50 of 150 nM  is due to the inhibition of LARP6 phosphorylation at the activating phosphorylation site, S451. Our finding has two practical implications: 1. at low doses, GSK-2141795 may be tried as treatment of fibrosis, 2. GSK-2141795 treatment of solid tumors may not only reduce the tumor growth, but also reduce the fibrous stroma surrounding the tumor, allowing easier penetration of other chemotherapeutics to the tumor cells.Fibrosis is characterized by excessive synthesis of type I collagen. Several studies pointed to the role of PI3K/Akt in development of fibrosis48495051f 150 nM , which iIn conclusion, we showed that phosphorylation of LARP6 plays an important role in regulating type I collagen biosynthesis. Primary phosphorylation of LARP6 at S451 requires Akt and Akt can be coimmunoprecipitated with LARP6. The phosphorylation of S451 is a prerequisite for phosphorylations of additional serines and for participation of LARP6 in regulating translation and subsequent folding and secretion of type I collagen. This findings support a novel mechanism for the anti-fibrotic effect of Akt inhibition through modulation of LARP6 phosphorylation.pcDNA3 vectors expressing human LARP6 tagged with HA tag at the amino terminus (HA-LARP6) and the HA-tagged carboxyl terminal domain (HA-CTER) of LARP6 have been described previouslyAdenoviruses were constructed by re-cloning the full-length wild type HA-LARP6 and HA-tagged LARP6 carrying single point mutations from pcDNA3 vectors into pAd-CMV-Track vector, followed by recombination with pAdEasy vector in BJ5183 E. coli cellsPDK1 inhibitor was from Calbiochem (124015). LY294002 was purchased from Cayman Chemical (154447-36-6). U0126 was from Calbiochem (662005). GSK-2141795 was from Activebiochem (A-1504). Pure active Akt enzyme was purchased from SignalChem (A16-10G). Calf intestinal alkaline phosphatase (CIP) was from New England Biolabs (M0290S). MG132 was from VWR International (89161566). Antibodies used were: anti LARP6 antibody from Abnova (H00055323-B01P), anti-HA antibody from Sigma-Aldrich (H9658), anti-collagen α1 (I) antibody from Rockland (600-401-103), anti-collagen α2 (I) antibody from Santa Cruz Biotechnology (sc-8786), anti-fibronectin antibody from BD Transduction Laboratories (610077), anti-β-actin antibody from abcam (ab8227), and anti-pan Akt antibody from Cell Signaling (4691).HEK293 cells and human lung fibroblasts (HLFs) immortalized by expression of telomerase reverse transcriptaseTransduction of human lung fibroblasts with adenoviruses was done at multiplicity of infection (MOI) of 500. With this MOI, 95-100% of the cells were transduced, as visualized by the expression of GFP. 48 hours after the transduction, cell extracts were made and analyzed by Western blotting or 2DGE. The inhibitors were added at the indicated concentrations for 12 hours prior to cell harvesting for analysis of collagen expression and for 6 hours when LARP6 phosphorylation was analyzed by 1DGE. For estimation of half life of LARP6, HLFs were transduced with adenovirus for 6 hours and treated with cycloheximide (100 μg/ml) for 0, 6, 12, and 18 hours and analyzed by Western blotting.RNA was extracted from cultured HLFs using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. 1 μg of RNA was used to prepare cDNA using the Superscript First Strand Synthesis System for RT-PCR (Invitrogen), according to the manufacturer’s instructions. 5 μl of 10-fold-diluted cDNA was used in SYBR Green qPCR assay (Applied Biosystems) on LightCycler 480 Real-Time RCR instrument (Roche Applied Sciences). The primers used for PCR amplification were shown in For one dimensional Western blotting analysis of LARP6, HLFs were treated with DMSO or kinase inhibitors, PDK1 inhibitor (4 μM), LY294002 (100 μM), U0126 (10 μM), and GSK-2141795 (0.1 μM) for 2 hours, adenovirus overexpressing wt HA-LARP6 or S451A LARP6 mutant was added and incubated for additional 4 hours. When needed, HLFs were treated with MG132 (8 μM) for 3 and 6 hours. The cells were lysed in Tris-HCl (50 mM), pH 7.5, NaCl (150 mM), 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, Dithiothreitol (1 mM) and protein concentrations were estimated with the Bradford assay , with bovine serum albumin (BSA) as the standard. 40 μg of total cellular protein was typically used for Western blotting analysis.For Western blotting analysis of cellular medium proteins, equal numbers of cells were seeded in 6-well plates and after 24–48 hours the cells were washed three times with serum free medium . 600 μl of serum free medium was then added to the cells and incubation continued for 3 hours. The medium was collected and an aliquot directly analyzed by Western blotting.Western blot signals were quantified using ImageJ software using the results of at least three independent experiments (n = 3). The level of LARP6 was normalized to the level of β-actin in each sample. The level of collagen α1 (I) (COL1A1) and collagen α2 (I) (COL1A2) polypeptides was normalized to the level of β-actin (intracellular) or fibronectin (cellular medium). The statistical significance of the LARP6 and collagen results was calculated using Student’s t test, with P values of <0.05 as significant. The results are presented as mean ± SD.Cells were lysed in 0.5% NP-40, Tris-HCl (50 mM), pH 7.5, NaCl (150 mM), phenylmethylsulfonyl fluoride (170 μg/ml), 1 × proteinase inhibitor, sodium fluoride (50 mM), β-glycerophosphate (5 mM) and sodium orthovanadate (1 mM) were added when phosphorylation of LARP6 was analyzed. Proteins were precipitated with 9 volumes of 100% ethanol and recovered by centrifugation at 2,284 × g for 10 minutes at 4 °C56Cells were lysed in 500 μl of Tris-HCl (50 mM), pH 7.5, NaCl (150 mM), 0.5% NP-40, Dithiothreitol (1 mM), phenylmethylsulfonyl fluoride (170 μg/ml), 1 × proteinase inhibitors and cleared lysate was incubated with 1 μg of antibody for 3 hours at 4 °C. 30 μl of equilibrated protein A/G-agarose beads (Santa Cruz Biotechnology) was added, and incubation continued for 1 hour. The beads were washed three times with lysis buffer and loaded onto SDS PAGE gels followed by Western blotting.Homo sapiens specific FASTA database and the Percolator peptide validator. Phosphorylation was detected by both SequestHT and Mascot and was verified by inbuilt phosphoRS node in proteome discoverer. Scaffold  was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm.HA-LARP6 was expressed and immunoprecipitated from HLFs in presence of phosphatase inhibitors. The immunoprecipitated protein was resolved on SDS PAGE gel and stained with GelCode Blue Stain Reagent . The HA-LARP6 band was excised and in-gel trypsin digest was done using ProteoExtract All-in-One Trypsin Digestion Kit  for 2 hours at 37 °C with shaking. Peptides were eluted in 50 μl 0.1% formic acid, separated on LCMS and the LC eluent was directly nano-sprayed into an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). The MS data were acquired using the following parameters: 10 data-dependent collisional-induced-dissociation (CID) MS/MS scans per full scan (400 to 2000 m/z) at a mass resolution for MS1 of 60000, minimum signal required to trigger MS2 was 500, MS mass range 0 to 1000000 and dynamic exclusion enabled with following parameters: Repeat count:1, Repeat Duration: 30.00, exclusion list size: 500, exclusion duration: 60.00, exclusion mass width relative to low and high mass: 10 ppm. All measurements were performed at room temperature and three technical replicates per sample were run to allow for statistical comparisons. The raw files were analyzed using Proteome Discoverer (version 1.4) software package with SequestHT and Mascot search nodes using 2 (10 mM), sodium fluoride (5 mM), β-glycerophosphate (5 mM), sodium orthovanadate (5 mM), ATP (50 μM), [γ-32P]ATP (0.5 μCi). GSK-2141795 at 0.1 μM (diluted in kinase reaction buffer) was preincubated with the immunoprecipitate for 30 minutes at 37 °C prior to addition of the kinase buffer. When the reaction was supplemented with exogenous Akt, 200 ng of pure, active Akt protein  was added to the kinase buffer. The kinase reactions were incubated at 37 °C for 1 hour, stopped by adding 6 × SDS protein loading buffer, heated and run on SDS polyacrylamide gel. The gel was dried and subjected to autoradiography.HLFs were transduced with adenovirus encoding wt HA-LARP6 and HA-LARP6 S451A mutant. After 48 hours the cell lysate was prepared and subjected to immunoprecipitation with anti-HA antibody. The immunoprecipitate was washed three times and resuspended in 30 μl of kinase reaction buffer (Τris-HCl (50 mΜ), pH 7.5, MgClHow to cite this article: Zhang, Y. and Stefanovic, B. Akt mediated phosphorylation of LARP6; critical step in biosynthesis of type I collagen. Sci. Rep.6, 22597; doi: 10.1038/srep22597 (2016).


Additionally, a significant interaction between the type of inhibition (D1 vs D2) and the type of motor task (FT vs PT)  indicated that D1 inhibition was associated with a significant reduction in PSI levels from TF to TP , whereas no significant difference between the tasks was observed for D2 inhibition . These results suggest that D1 and D2 inhibitions of the soleus H-reflex are differentially modulated during the performance of plantarflexion FT and PT. The reduced level of ongoing PSI during PT suggests that, in comparison to FT, there is a larger reliance on inputs from muscle spindles primary afferents when the neuromuscular system is required to maintain position-controlled plantarflexion contractions.Presynaptic inhibition (PSI) has been shown to modulate several neuronal pathways of functional relevance by selectively gating the connections between sensory inputs and spinal motoneurons, thereby regulating the contribution of the stretch reflex circuitry to the ongoing motor activity. In this study, we investigated whether a differential regulation of Ia afferent inflow by PSI may be associated with the performance of two types of plantarflexion sensoriomotor tasks. The subjects (in a seated position) controlled either: 1) the force level exerted by the foot against a rigid restraint ; or 2) the angular position of the ankle when sustaining inertial loads  that required the same level of muscle activation observed in FT. Subjects were instructed to maintain their force/position at target levels set at ~10% of maximum isometric voluntary contraction for FT and 90° for PT, while visual feedback of the corresponding force/position signals were provided. Unconditioned H-reflexes (i.e. control reflexes) and H-reflexes conditioned by electrical pulses applied to the common peroneal nerve with conditioning-to-test intervals of 21 ms and 100 ms  were evoked in a random fashion. A significant main effect for the type of the motor task (FT  Presynaptic inhibition (PSI) is ubiquitous in the vertebrate spinal cord  and has More specifically, two types of motor tasks  have been used in laboratory settings to investigate several mechanisms underlying motor control and performance –15. The vs PTs when compared to the upper extremity muscles investigated by Baudry and colleagues  was used to detect main effects and interactions among experimental conditions. As a significant interaction was detected, simple effects tests (with Bonferroni’s correction) were conducted for the contrasts of theoretical interest. Additionally, confidence intervals (CIs) in conjunction with graphical presentation were used to allow appropriate data interpretation [In order to evaluate the effect of task type (FT and PT) on both D1 and D2 inhibitions, the amount of inhibition was measured for each conditioning protocol ,42. Therretation ,44. MoreAs the measurements of amount of inhibition described above may eventually “hide” information on difference among control parameters (i.e. unconditioned reflexes), a t-test for paired samples was used to compare the amplitude of the control (unconditioned) H-reflexes measured during FT and PT trials. Similarly, paired t-tests were used to compare the levels of background soleus EMG  between FT and PT trials.2p) indices [Statistical analysis were performed using the statistical package SPSS 15.0 for Windows , with significance level set at p < 0.05. Effect sizes are reported using partial eta-squared  positioned over the femoral triangle and the reference electrode  over the posterior and upper aspect of the thigh. The interval between conditioning and test stimuli was chosen based on a previous exploration of the time course variation of the heteronymous Ia facilitation for each subject (not recorded) and ranged between -3 and -4 ms.The protocol employed in the additional experiments replicated the previous one described above, except that the trials (in FT and PT conditions) consisted in evoking control H-reflexes (unconditioned), and H-reflexes conditioned by a stimulus  applied to the femoral nerve. Conditioning stimuli were delivered with the active electrode , with the amplitude of the reflexes expressed as a percentage of the mean amplitude observed in the control condition. = 13.64, p = 0.005, η2p = 0.603), indicating higher inhibition for the FT as compared to the PT condition. A significant main effect was also obtained for the factor “duration of conditioning-to-test interval”  = 11.909, p = 0.007, η2p = 0.570), with higher inhibition observed for D2 as compared to D1. ANOVA also revealed a significant interaction between the factors “type of contraction” and “duration of conditioning-to-test interval”  = 5.868, p = 0.038, η2p = 0.395). Simple effects tests indicated that the amount of inhibition was higher for D2 inhibition (as compared to D1) in both TF  and TP  conditions. Comparisons between the “type of contraction” showed that the amount of inhibition in FT was higher than in PT for D1 inhibition , whereas no significant difference between FT and PT was found for D2 inhibition . The panels in The two-way ANOVA test performed on amount of inhibition measures revealed a significant main effect for the “type of contraction”  = 0.29, p = 0.77, η2p = 0.010 and T(9) = 1.37, p = 0.20, η2p = 0.172, for soleus EMG and control H-reflexes, respectively). These results indicate that the background levels of soleus EMG and the amplitude of control reflexes did not change considerably between FT and PT. Lastly, the t-tests performed on the levels of soleus EMG activation  and on the amplitude of the control (unconditioned) H-reflexes revealed no significant differences between FT and PT trials  . While tDifferent methods have been proposed to estimate changes in presynaptic inhibition of Ia terminals during voluntary contraction in humans. However, none of these methods can provide by itself unequivocal evidence for a change in presynaptic inhibition of Ia terminals . For exaIn this study, we chose to control H-reflex amplitude by keeping unconditioned H-reflexes at ~20%Mmax see , so as tIn an investigation on the upper limbs of human subjects, Aimonetti et al.  showed tThe present experiments were performed with matched levels of soleus EMG between FT and PT trials . AdditioIles  showed tIt is also unlikely that Ib afferent input from Golgi tendon organs have contributed to the present results because muscle tension (and consequently Ib activation) was probably similar in FT and PT, as suggested by the lack of activation of tibialis anterior and thigh muscles and the matched levels of soleus EMG activation in both tasks . On the Different levels of PSI converging onto the Ia afferents acting on the soleus motoneuron pool must be counterbalanced by appropriate levels of descending drive to yield the similar levels of soleus EMG imposed for the FT and PT. However, whether an augmented descending drive is needed when the control system acts with higher levels of PSI (i.e. during FT), as suggested by Baudry et. al , may be A recent study  suggesteStudies with a biologically plausible computational model –63 have A malfunction in some spinal pathways responsible for controlling the excitability of the stretch reflex has been associated with the development of spasticity in conditions such as following stroke and spinal cord injury. For instance, reduced PSI of Ia terminals –67 has bThe present study addressed the modulation of PSI as a function of the type of the plantarflexion task (force or position-controlled contractions). In comparison to FTs, PTs are undertaken with lower levels of Ia PSI converging onto the soleus motoneuron pool. Furthermore, it is suggested that D1 and D2 inhibitions of the soleus H-reflex are differentially modulated during the performance of plantarflexion force and position tasks. The results enhance our knowledge of the spinal physiology of plantarflexion neuromuscular control and may provide relevant implications for the practice of rehabilitation and training.


The SWAT  programme, established by the All-Ireland Hub for Trials Methodology Research in collaboration with the Medical Research Council Network of Hubs in the UK and others, is developing methods to resolve uncertainties about trial conduct through embedded research. SWAT-1 provides an initial example.To evaluate the effects of a site visit on recruitment rates in a multi-centre randomised trial.Using the SWAT-1 design, a before-and-after comparison used the date of the site visit as the time point for the intervention. Site A received the site visit. Sites B and C did not receive it and acted as the controls. The primary outcomes were the difference in recruitment in each site from 1 and 3 months pre-intervention to 1 and 3 months post-intervention.Recruitment rates increased in Site A post-intervention . No differences in recruitment occurred in Site B or in Site C. At 3 months post-intervention, a significant difference was detected in favour of higher recruitment in A compared to B+C . These findings suggest that recruitment might increase from 2 participants per week before the site visit to 4-5 per week after it.This initial example of a SWAT provides evidence that a site visit increases recruitment rates in a trial. Further SWAT-1s are required to substantiate these findings and to examine the effects in different trials in different settings.


LITAF/SIMPLE gene shared a functional link to the PMP22 gene, whose duplication or deletion results in CMT1A and HNPP, respectively. By studying a large cohort of CMT1A/HNPP-affected patients, we found that the LITAF I92V sequence variant predisposes patients to an earlier age of onset of both the CMT1A and HNPP diseases. Using cell transfection experiments, we showed that the LITAF I92V sequence variant partially mislocalizes to the mitochondria in contrast to wild-type LITAF which localizes to the late endosome/lysosomes and is associated with a tendency for PMP22 to accumulate in the cells. Overall, this study shows that the I92V LITAF sequence variant would be a good candidate for a biomarker in the case of the CMT1A/HNPP disorders.Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) represent the most common heritable neuromuscular disorders. Molecular diagnostics of CMT1A/HNPP diseases confirm clinical diagnosis, but their value is limited to the clinical course and prognosis. However, no biomarkers of CMT1A/HNPP have been identified. We decided to explore if the  Charcot-Marie-tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) belong to the group of most common heritable neuromuscular disorders caused by submicroscopic duplication or deletion of the 17p11.2-p12 region, respectively , 10, 3, It has been suggested that lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF) may play a significant role in CMT. While patients who initially lacked mutations to known genes were designated to the CMT1C subtype, the molecular basis of the latter has recently been identified as a mutation in LITAF . Such LIPMP22) gene seems to be a critical factor where the clinical course of CMT1A/HNPP is concerned. Thus, the number of copies of the PMP22 gene has been shown to correspond with the clinical course of neuropathy in laboratory transgenic animals [PMP22 gene resulted in an improved clinical course of CMT in transgenic animals (mice/rats) [PMP22 regulatory sequences in the gene-dosage effect, we decided to analyze an extended region of the 5′UTR sequence encompassing 5000 kb and the coding sequence of the PMP22 gene in patients with CMT1A/HNPP (duplication/deletion of the PMP22 gene). Unfortunately, we failed to detect any significant variants within the PMP22 regulatory sequence that could be responsible for the gene-dosage effect and the observed clinical variability in the CMT1A/HNPP diseases [The dosage of the peripheral myelin protein 22  . Given tdiseases .Baby green monkey kidney (BGMK) was obtained from the American Type Culture Collection . BGMK cells were cultured in Dulbecco-modified Eagle’s medium  with 7 % fetal bovine serum , 2-mM L-glutamine, 100-U/mL penicillin, and 100-μg/mL streptomycin, at 37 °C with 5 % CO2. The following antibodies/probes were used during immunofluorescence: Lysotracker®DND-99 from Molecular Probes , the 9E10 mouse and mouse myc monoclonal antibody from Roche , rabbit anti-FLAG , and FITC/Cy3-conjugated goat anti-mouse or anti-rabbit immunoglobulin G (IgG) from Jackson ImmunoResearch Inc. ; and MitoTRACKER Red FM from Molecular Probes . LITAF WT was synthesized by GenScript . Myc-tag was added to the N-terminus of the protein to facilitate imaging. PMP22 was synthesized by Sino Biological Inc. . The gene was pCMV/hygro and contained a flag-tag.PMP22 gene was confirmed using Real-Time PCR (Q-PCR) [PMP22 gene) and VIC . The Q-PCR reaction was performed on the ABI-7500 Real-Time PCR system (Applied Biosystems). The relative dosage (RQ) of the PMP22 gene ranges from 0.700 to 1.090 in normal individuals (two copies of the PMP22 gene), from 0.359 to 0.595 in HNPP patients (one copy of the PMP22 gene), and from 1.176 to 2.324 in CMT1A patients (three copy of the PMP22 gene) [One hundred six patients affected with CMT were examined by neurologists at the Neuromuscular Unit and Warsaw Department of Neurology. Family trees of at least three generations were constructed for all the patients. The clinical diagnosis of CMT/HNPP was then confirmed in all patients by means of electromyography examination (EMG). Patients displaying symmetrical, generalized neuropathy involving upper and lower limbs were included in this study. All members of the CMT1A/HNPP-affected families gave their signed informed consent for the study, which gained the approval of the local Ethics Committee at Warsaw Medical University . The control group consists of 50 unaffected individuals. Genomic DNA was extracted from peripheral blood lymphocytes using the salting-out procedure. The duplication/deletion of the LITAF gene were amplified by PCR . The LITAF gene sequence was analyzed by comparison with reference sequence NM_004862.3 (transcript variant 1).The three coding exons 2–4 of the ribed by ). The PCTotal RNA was isolated from peripheral blood lymphocytes using the TRIzol Reagent, in line with the instructions from the manufacturer (Invitrogen).RT-PCR reactions were carried out using a First Strand cDNA Synthesis Kit (Fermentas), again in line with the manufacturer’s instructions.U test. The p < 0.05 level was considered statistically significant, data being analyzed using the StatSoft statistical software package version 5.The data was presented as means ± SD, medians, and ranges of values Table . DiffereLITAF WT and the I92V LITAF mutation were amplified using a 50-μL PCR mixture containing 1× PCR buffer (Invitrogen), 3.0-mM MgCl2 (Invitrogen), 0.2-mM forward and reverse primers, and 2.5-U Taq DNA polymerase . LITAF WT and mutations were used as template DNA for the following primers: 5′-ATGGAGCAGAAACTGATTAGT-3′ (LITAF-forward), 5′-TACCAGTCT TTGTAAGTTCC-3′ (LITAF-reverse). The cycling conditions used were 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 90 s for 30 cycles. The resulting PCR product was cloned into pTARGET .In order to confirm that transformation into cloning vectors was performed correctly, and to identify potential LITAF mutations, LITAF samples were prepared and sent for sequencing at the Robarts Institute . Once sequencing was completed, data was analyzed using Codon Aligner 4.0.4 .BGMK cells were grown to 70 % confluency and transfected using a polyethylenimine (PEI) reagent. DNA (5 μl) was diluted with 400 μl of serum-free DMEM. PEI was added in a 4:1 ratio of PEI:DNA and left to incubate at room temperature for 15 min. The transfection mix was then added to cells along with fresh DMEM.In 24-h post-transfection, the cells were probed with MitoTRACKER Red FM or LysoTRACKER then fixed using 3.7 % paraformaldehyde solution in phosphate buffer solution (PBS) and permeabilized using a 0.1 % Triton X-100 solution in PBS. Cells were blocked for 2 h at room temperature in block buffer  (w/v), 50-mM Tris HCl (pH 7.4), 150-mM NaCl, 0.5 % NP-40 (v/v)) followed by several 5-min washes in wash buffer  (w/v), 50-mM Tris HCl (pH 7.4), 150-mM NaCl, 0.5 % NP- 40 (v/v)). Primary antibody diluted in wash buffer was incubated on cells for 1 h at room temperature. The primary antibody was removed following several 5-min washes in wash buffer, and secondary antibody was diluted in wash buffer and applied to cells. The secondary antibody was left on cells for 1 h at room temperature in darkness before removal with several 5-min washes in wash buffer. Cells were mounted using VECTASHIELD , and fluorescence was detected using a Leica DM SP2 confocal microscope . Images were assembled using Adobe Photoshop CS4 . This process was repeated at least three times, and results were analyzed using ImageJ by using the method described by Burgess et al. [LITAF gene is associated with an earlier age at onset of CMT1A and HNPP diseases in the group of patients manifesting with CMT1A/HNPP after 10 years of age. In this group of patients, we found a statistically significant association (p = 0.005 and p = 0.023) for CMT1A and HNPP, respectively [Table We found that the presence of I92V heterozygous substitution in the In general, the CMT1A and HNPP-affected patients harboring the I92V mutation manifest with CMT1A/HNPP 13 years earlier than the patients without I92V sequence variant.The statistically significant association was not detected for the CMT1A/HNPP patients with early age at onset (before 10 years of age) harboring Ile92Val mutation.LITAF mutation.We have also taken into consideration other features of the CMT1A/HNPP phenotype . No statistically significant associations have been found for clinical parameters of CMT1A/HNPP and the presence of I92V To conclude, within numerous clinical parameters of CMT1A/HNPP phenotypes, only age at onset has been shown to be associated with the presence of I92V mutation in the group of older patients (after 10 years of age).Since we observed differences in age of onset of CMT1A and HNPP in patients depending on the occurrence of the LITAF I92V sequence variant, we were interested to see if the I92V LITAF mutant is found in the same subcellular location as the WT LITAF. To further explore the relocation of LITAF, we transfected into BGMK cells either wild-type LITAF or I92V. We observed that WT LITAF co-localizes with the lysosomal marker CD63, suggesting that WT LITAF is normally present in the lysosomes Fig. . Next, wLITAF has been implicated in protein degradation , 23 whilIn the present study, we show that the I92V sequence variant in addition to PMP22 mutations predispose patients to an earlier age of onset of both CMT1A/HNPP diseases. The I92V LITAF sequence variant has not been considered a biomarker of CMT1A/HNPP. Nevertheless, in a single CMT1A family study, the I92V sequence variant has been shown to contribute to a more severe CMT1A phenotype . SimilarLITAF gene was again found to be present [The I92V sequence variant occurs in different ethnic groups with a relatively high frequency in the healthy individuals. In our study, the I92V mutation was found in 47 % of CMT1A-affected patients and 54 % HNPP patients [Table  present . Thus, CPMP22 gene dosage regulated by LITAF activity is not the single, exclusive molecular mechanism influencing the severity of CMT1A. While our study also analyzed other parameters of CMT1A, the only statistically significant result obtained was the correlation between age at onset and the I92V mutation within the LITAF gene.Interestingly, a statistically significant effect of the I92V sequence variant on CMT1A/HNPP phenotype has only been found in the group of patients manifesting CMT1A/HNPP disease after 10 years of age. We therefore cannot preclude the action on the part of other modifying genetic factors in the case of a group of younger patients with a more severe, early age at onset clinical course for CMT1A/HNPP. In other words, PMP22, the mislocalization of LITAF from lysosomes to mitochondria may be associated with a less effective degradation of PMP22 protein. Since gene dosage is considered crucial to CMT1A severity, the decrease of LITAF within lysosomes may be a key factor in molecular pathogenesis of CMT1A. We therefore hypothesized that the LITAF gene might play a role in the regulation of PMP22 gene dosage. In this study, we explored the effect of I92V LITAF on PMP22 level within the cell and found through immunofluorescence that when BGMK cells are co-transfected with PMP22 and I92V, there is a tendency for PMP22 to accumulate in the cell, suggesting that LITAF plays a role on PMP22 regulation.Given the primary role of LITAF protein in the degradation of In summary, our results indicate that the LITAF I92V sequence variant is associated with an earlier age of onset of the CMT1A/HNPP diseases and may be considered a genetic modifier of clinical variability. To conclude with respect to the Polish population at least, the I92V LITAF sequence variant may be considered a genetic modifying factor.


There is increasing evidence of a pivotal role for regulated mRNA translation in control of developmental cell fate transitions. Physiological and pathological stem and progenitor cell self-renewal is maintained by the mRNA-binding protein, Musashi1 through repression of translation of key mRNAs encoding cell cycle inhibitory proteins. The mechanism by which Musashi1 function is modified to allow translation of these target mRNAs under conditions that require inhibition of cell cycle progression, is unknown.Xenopus oocytes. S337 phosphorylation in mammalian NSPCs and human SH-SY5Y cells correlates with the de-repression and translation of a Musashi reporter mRNA and with accumulation of protein from the endogenous Musashi target mRNA, p21WAF1/CIP1. Inhibition of Musashi regulatory phosphorylation, through expression of a phospho-inhibitory mutant Musashi1 S337A or over-expression of the wild-type Musashi, blocked differentiation of both NSPCs and SH-SY5Y cells. Musashi1 was similarly phosphorylated in NSPCs and SH-SY5Y cells under conditions of nutrient deprivation-induced cell cycle arrest. Expression of the Musashi1 S337A mutant protein attenuated nutrient deprivation-induced NSPC and SH-SY5Y cell death.In this study, we demonstrate that differentiation of primary embryonic rat neural stem/progenitor cells (NSPCs) or human neuroblastoma SH-SY5Y cells results in the rapid phosphorylation of Musashi1 on the evolutionarily conserved site serine 337 (S337). Phosphorylation of this site has been shown to be required for cell cycle control during the maturation of Our data suggest that in response to environmental cues that oppose cell cycle progression, regulation of Musashi function is required to promote target mRNA translation and cell fate transition. Forced modulation of Musashi1 function may present a novel therapeutic strategy to oppose pathological stem cell self-renewal. WAF1/CIP1, Numb and others [The extensive efforts directed towards identification of the gene transcription mechanisms controlling stem and progenitor cell differentiation have yielded a wealth of relevant information  in primary embryonic rat NSPCs using a phospho-specific antiserum that recognizes mammalian S337 [We have recently demonstrated that regulatory phosphorylation of Musashi1 is required to allow the target mRNA translation that mediates cell cycle control during maturation of  oocytes . To deteian S337 . A basalWAF1/CIP1 mRNA is a target of Musashi1 [WAF1/CIP1 protein levels, consistent with de-repression and translation of the p21WAF1/CIP1 mRNA . The mutant Musashi S337A was fused to eGFP for visualization and utilized to assess the role of regulatory phosphorylation in differentiation of NSPCs and SH-SY5Y cells. Control, eGFP-transfected NSPCs exhibited a high level of neurite extension after culture in differentiation conditions,  and pcDNA Fluc β-globin (Fluc-con) respectively [The mouse Musashi-1 protein sequence (GenBank NM_008629) was amplified by PCR from the IMAGE OC29D08 clone. The product was cloned in-frame with the downstream Enhanced Green Flourescent Protein (EGFP) open reading frame using the Xho I and BamH I sites of the pEGFP-N1 plasmid (Clontech) to generate pEGFPN-Msi1. Phosphorylation mutant Musashi protein was generated from pEGFPN-Msi1 as a starting template using site directed mutagenesis  to create the S337A mutant. The pcDNAFluc2 reporter plasmid was created through insertion of a Hind III restriction site 128 bp downstream of the BGH poly(A) site in pcDNA3.1(−) (Life Technologies) using the QuikChange kit, followed by plasmid digestion with Hind III and religation to form a modified vector. The Firefly luc2 ORF (Promega) was isolated with Xho I and Eco RI from pCR-Blunt (Life Technologies) and ligated into the Xho I and Eco RI-digested modified pcDNA3.1(−). For luciferase mRNA translation reporter assays we utilized the previously characterized Musashi binding element (MBE) from the A 3′ UTR . This MBPrimary neural stem/progenitor cells (NSPCs) were cultured from embryonic rat hippocampal/cortical tissue (Genlantis) and were cultured on low-adhesion plates (Corning) in Neurobasal medium  with B-27 (−vitamin A), 0.5 mM glutamine and 10 μg/ml gentamycin, supplemented with 10 ng/ml bFGF (PeproTech) and 10 ng/ml EGF (PeproTech) to promote proliferation and formation of neurospheres . Prior tTotal cell lysates were prepared in NP-40 lysis buffer supplemented with 1 mM DTT, 0.1 mM PMSF and protease inhibitors . ProteinSH-SY5Y cells were co-transfected with the Firefly Luciferase (Fluc) reporter constructs and control Renilla Luciferase plasmid  and cultured under proliferation or differentiation conditions, as described previously . Lucifert*SEM, where t is the 97.5 percentile from the appropriate t-distribution. Student’s two tailed, paired t-tests were used to analyze the data, where p < 0.05 was adopted for statistical significance. Where indicated, statistical significance was also assessed by one way ANOVA with Bonferroni multiple comparison test and in each case a probability of p < 0.05 was adopted for statistical significance.Quantitated data are presented either as mean +/− SEM, or as mean and 95% confidence interval (CI). The 95% CI is simply calculated as the mean +/−


Recent evidence indicates that inhibition of histone deacetylase (HDAC) protects the heart against myocardial injury and stimulates endogenous angiomyogenesis. However, it remains unknown whether HDAC inhibition produces the protective effect in the diabetic heart. We sought to determine whether HDAC inhibition preserves cardiac performance and suppresses cardiac remodeling in diabetic cardiomyopathy.Control, Control + Sodium butyrate (NaBu), STZ and STZ + Sodium butyrate (NaBu), respectively. Myocardial function was serially assessed at 7, 14, 21 weeks following treatments.Adult ICR mice received an intraperitoneal injection of either streptozotocin  to establish the diabetic model or vehicle to serve as control. Once hyperglycemia was confirmed, diabetic mice received sodium butyrate (1%), a specific HDAC inhibitor, in drinking water on a daily basis to inhibit HDAC activity. Mice were randomly divided into following groups, which includes Echocardiography demonstrated that cardiac function was depressed in diabetic mice, but HDAC inhibition resulted in a significant functional improvement in STZ-injected mice. Likewise, HDAC inhibition attenuates cardiac hypertrophy, as evidenced by a reduced heart/tibia ratio and areas of cardiomyocytes, which is associated with reduced interstitial fibrosis and decreases in active caspase-3 and apoptotic stainings, but also increased angiogenesis in diabetic myocardium. Notably, glucose transporters (GLUT) 1 and 4 were up-regulated following HDAC inhibition, which was accompanied with increases of GLUT1 acetylation and p38 phosphorylation. Furthermore, myocardial superoxide dismutase, an important antioxidant, was elevated following HDAC inhibition in the diabetic mice.HDAC inhibition plays a critical role in improving cardiac function and suppressing myocardial remodeling in diabetic heart.The online version of this article (doi:10.1186/s12933-015-0262-8) contains supplementary material, which is available to authorized users. Cardiovascular complications are the leading cause of diabetes-related morbidity and mortality worldwide . Diabete]. Inhibition of HDACs using small molecules is regarded to be one of the most promising approaches for many pathological disorders.Histone acetyltransferases (HAT) and HDAC have recently garnered attention because they have emerged as important mechanisms in the regulation of a variety of cellular responses. Histone acetylation is mediated by HAT, which results in the modification of the structure of chromatin leading to nucleosomal relaxation, and altered transcriptional activation. In contrast, the reverse reaction is mediated by HDACs which induce deacetylation, chromatin condensation, and transcriptional repression. The acetylation status of histone tails is determined by the interplay between HATs and HDACs –15. Rece22Our studies have demonstrated that inhibition of HDACs plays a critical role in the prevention of myocardial damages, mediation of cardiogenesis, and stimulation of myocardial repair –27. In aTwo-month old ICR male mice were purchased from Charles River Laboratories . All animal experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee of Institute, which conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .Control group: mice only received an injection of vehicle (citrate buffer); Control + NaBu group: mice received sodium butyrate (1%), a specific HDAC inhibitor, in drinking water on a daily basis; STZ-treated group: mice received intraperitoneal injection of STZ; STZ + NaBu group: mice received intraperitoneal injection of STZ injection followed by sodium butyrate (1%), in drinking water on a daily basis. Tail vein blood glucose samples were measured using One Touch II Glucometer  to confirm the induction of diabetes. All animals were euthanized at 21 weeks after injection of STZ.Two month-old mice were made diabetic by intraperitoneal injection of a single dose of freshly prepared STZ solution  following overnight fasting . DiabetiEchocardiographic parameters were accessed before and 7, 14, and 21 weeks after the sodium butyrate treatments. Echocardiography was performed to evaluate left ventricular (LV) functions using an Acuson Sequoia C512 system  equipped with a 15L8 linear array transducer. Mice were placed in supine position on a heating pad after being anesthetized with 1.5% isoflurane mixed with oxygen. Pre-warmed ultrasound gel was applied on the chest throughout the measurements. At the signal depth of 25 mm, 2-D B-mode and M-mode images were recorded on short axis views at the level of the papillary muscles. The following parameters were measured on the M-mode tracings and averaged from 3 to 6 cardiac cycles: left ventricular internal dimension-diastole , left ventricular internal dimension-systole , ejection fraction (EF), fraction shortening (FS), left ventricular posterior wall thickness in end-diastole , and end-systole . Data were calculated with accompanying software.Tissue sections were de-paraffinized for 30 min at 70°C and subsequently immersed in xylene and ethanol at decreasing concentration. Immunostaining was performed as described previously . All de-® TdT In Situ Apoptosis Detection Kits  following the manufacturer’s instructions. TUNEL positive cells were observed using confocal laser scanning microscopy LSM 700 (Carl Zeiss). The numbers of TUNEL positive cells were determined and were normalized to the tissue area. DAPI was used to counter stain for nuclei.De-paraffinized sections were processed for a TUNEL assay using a TACSHeart tissues were homogenized on ice in RIPA buffer with Halt protease and phosphatase inhibitor cocktail. Proteins (50 µg/lane) were resolved by SDS gel and transferred onto PVDF membranes. The membranes were then blocked with 5% powdered milk in TBST for 1 h and were subsequently probed with primary antibodies overnight at 4°C. The following primary antibodies were used in this study: HDAC4, acetylated-Lysine, phosphorylated p38, and active casepase-3 from Cell Signaling ; GLUT 1, GLUT4, p38, SOD1, and β-actin were from Santa Cruz Biotechnology . Horseradish peroxidase-conjugated monoclonal antibodies  were used for chemiluminescence detection.For immunoprecipitation, samples were pre-cleared prior to immunoprecipitation to reduce the amount of non-specific contaminants. The EZView red protein A affinity gel  was incubated with myocardial lysates for 60 min at 4°C. Samples were centrifuged, and supernatants were obtained. Proteins were incubated with the indicated primary antibodies at 4°C overnight. Beads were added to lysate plus antibody mix, and proteins were further incubated for 2 h at 4°C. In addition, IgG was also used as immunoprecipitation control and non-immunoprecipitated lysate was used as a control for detected molecular weight. After incubation, samples were washed with RIPA buffer five times, and proteins were eluted with 4× loading buffer by boiling and subjected to SDS-PAGE.Myocardial HDAC activity was measured as described preciously in detail .p < 0.05 was considered to be a significant difference.All data are expressed as mean ± SEM. Differences among multigroups were analyzed by one-way analysis of variance (ANOVA), followed by Bonferroni correction. A probability of A single injection of STZ (200 mg/kg) was used to induce diabetes in ICR mice. At 1 week after the injection, the fasting blood glucose levels of >450 mg/dl were detected in all mice that received STZ injection. Approximately 20% mice did not survive to sustain the toxicity of STZ by day 14. The surviving mice had blood glucose levels of >450 mg/dl throughout the study period. As shown in Fig. Serial echocardiography was performed immediately before and up to 21 weeks after treatments. As shown in Fig. STZ-induced diabetic mice WGA staining was performed to access the cross sectional cardiomyocyte size. As shown in Figs. The ratio of heart weight to tibia length was used to evaluate the hypertrophic response. STZ injection resulted in an increase in the heart weight/tibia length ratio as compared to control hearts. Treatment of STZ mice with sodium butyrate significantly reduced the heart weight/tibia length ratio Fig. a. In addWhen we measured HDAC activity in the diabetic myocardium, HDAC activity demonstrated an increase in STZ-induced diabetic heart, but HDAC activity was significantly reduced following the treatment of animals with sodium butyrate Fig. a. In addThe apoptotic marker active caspase-3 was examined in the myocardium. As shown in Fig. Superoxide dismutase has been implicated in protective effects against myocardial ischemic injury. The levels of SOD1 in the myocardium were examined. As shown in Fig. As shown in Fig. Our study is the first to document that HDAC inhibition preserves cardiac performance and suppresses cardiac remodeling in diabetic cardiomyopathy. Specifically, (1) Serial echocardiographic evaluation indicates that HDAC inhibition resulted in the preservation of ventricular function in STZ-induced diabetic heart; (2) HDAC inhibition plays a profound effect in suppressing interstitial fibrosis and attenuating myocyte hypertrophy in the diabetic myocardium; (3) HDAC inhibition mitigated the frequencies of apoptosis in the diabetic myocardium by decreasing active caspase 3 and TUNEL positive signals; (4) HDAC inhibition resulted in the increase of SOD1, GLUT1, and GLUT4 protein levels in diabetic hearts, and acetylation of GLUT1 was elevated following HDAC inhibition; (5) Diabetic hearts exhibited significant decreases in CD31 and α-SMA positive microvessels, which was prevented following HDAC inhibition.Recent evidence has indicated a genetic association between diabetes and HDACs. HDAC inhibitors promote β-cell development and function that positively affect diabetic microvascular complications . VentricClinical studies demonstrate that diabetes mellitus increased the susceptibility of the myocardium to ischemic injury , 36. In Myocardial fibrosis is another important hallmark of diabetic cardiomyopathy, and it is also featured by the accumulation of interstitial collagens in the hearts . The preWe reported that HDAC inhibition protects the heart against ischemia/reperfusion through blocking of reactive oxidant species . OxidatiIt is known that pathological cardiac hypertrophy with reduced contractility is accompanied by impaired coronary angiogenesis . The redTwo glucose transporter proteins (GLUT1 and GLUT4) were reported to be reduced in the diabetic myocardium . We haveOur study is the first to document that HDAC inhibition attenuated diabetic cardiomyopathy by improving ventricular function, attenuating remodeling, and suppressing cardiac hypertrophy. The protective effects of HDAC inhibition in the diabetic myocardium are closely associated with decreased apoptosis, stimulation of endogenous angiogenesis, an increase of anti-oxidant SOD1, and activations of GLUT1 acetylation and p38 phosphorylation. HDAC inhibition provides a novel approach to the treatment of patients with diabetic cardiomyopathy. Our study indicated that inhibition of HDACs effectively prevented cardiac dysfunction and cardiac remodeling in the diabetic heart, which is related to the promotion of angiogenesis, anti-apoptosis, and acetylation of GLUT1.Diabetes mellitus threatens to become a global health crisis; treatment of diabetes and its implications constitutes a major health care expenditure. A significant proportion of diabetic patients are known to develop diabetic cardiomyopathy, which is one of the leading causes of increased morbidity and mortality in patients with diabetes mellitus. HDAC regulates transcriptional changes and plays a critical role in mediating myocardial development and disease. From both a mechanistic and a translational point of view, the pathway identified in the present study will provide a strong foundation on which HDAC inhibition may serve as the novel therapeutic target to prevent the progression of diabetes-induced cardiac dysfunction. HDAC inhibitors are well tolerated in humans, and clinical trials investigating their efficacy in anti-cancer therapy are currently underway. Therefore, the therapeutic merit of HDAC inhibition in treating diabetic cardiomyopathy is potentially important. Exploration of the functional role of HDAC inhibition and evaluation of its clinical outcome will provide direct evidence to support a potential clinical implication.


Taenia solium cystatin, one of the cestode cystatins, demonstrated that the N-terminus of T. solium cystatin formed a five turn α-helix, a five stranded β-pleated sheet and a hydrophobic edge, similar to the structure of chicken cystatin. Although no conserved disulfide bond was found in T. solium cystatin, the models of T. solium cystatin and chicken cystatin corresponded at the site of the first disulfide bridge of the chicken cystatin. However, the two models were not similar regarding the location of the second disulfide bridge of chicken cystatin. These results showed that T. solium cystatin and chicken cystatin had similarities and differences, suggesting that the biochemistry of T. solium cystatin could be similar to chicken cystatin in its inhibitory function and that it may have further functional roles. The same results were obtained for other cestode cystatins. Phylogenetic analysis showed that cestode cystatins constituted an independent clade and implied that cestode cystatins should be considered to have formed a new clade during evolution.The cystatin superfamily is comprised of cysteine proteinase inhibitors and encompasses at least 3 subfamilies: stefins, cystatins and kininogens. In this study, the platyhelminth cystatin superfamily was identified and grouped into stefin and cystatin subfamilies. The conserved domain of stefins  was observed in all members of platyhelminth stefins. The three characteristics of cystatins, the cystatin-like domain , a signal peptide, and one or two conserved disulfide bonds, were observed in platyhelminths, with the exception of cestodes, which lacked the conserved disulfide bond. However, it is noteworthy that cestode cystatins had two tandem repeated domains, although the second tandem repeated domain did not contain a cystatin-like domain, which has not been previously reported. Tertiary structure analysis of  Chicken cystatin was first described by Barrett, who discovered that it inhibited various cysteine proteases. Subsequently, many similar molecules present in metazoans and plants have been identified –3. The cRecently, some studies have described the capacity of nematode cystatins to regulate not only the activity of parasite proteases but also that of host proteases . AdditioCysteine proteases have been described in cestodes and trematodes –23. The Schmidtea mediterranea, Schistosoma japonicum, Schistosoma mansoni, Echinococcus multilocularis, Echinococcus granulosus, Hymenolepis microstoma, T. solium, T. saginata and T. asiatica. With the advantage of having T. solium, T. saginata and T. asiatica genomic data and samples, their corresponding cystatin superfamily was cloned and analyzed, which may provide a comprehensive insight into its abundance, diversity and evolution in platyhelminths.In the present study, the cystatin superfamily was characterized and explored for their phylogeny in nine flatworms including S. japonicum and S. mansoni and the cestodes T. solium, E. multilocularis, E. granulosus and H. microstoma can be found at http://www.genedb.org/homepage. The genome of the planarian S. mediterranea is available at http://smedgd.neuro.utah.edu. Each genome was investigated using the word “cystatin” as the query parameter. The expressed sequence tags (ESTs) or RNA-Seq data of each species were searched for the details of their transcription. For full-scale analysis, the nr and EST databases of NCBI (http://blast.ncbi.nlm.nih.gov/) were also included in the searches.The annotated genomes of 6 platyhelminths are available for public study. Those of the trematodes T. saginata and T. asiatica were used for the identification of the cystatin family. A combined strategy was employed using both the motifs-based method and the sequence similarity-based method to search for proteins or molecules with conserved cystatin-like domains in the genomes of the two tapeworms. Putative members of the cystatin superfamily in T. saginata and T. asiatica were characterized using the TBlastN program, with the annotated T. solium cystatin superfamily gene sequences (TsM_000671000 and TsM_000687900) as query sequences, to search the two genome databases with the cut-off e-value of 10−5 [http://merops.sanger.ac.uk/) and those belonging to proteinase inhibitor I25 [The unpublished genome sequences of  of 10−5 . In addiT. solium, T. saginata and T. asiatica are available in our lab [The DNA and cDNA samples of  our lab , 26. In www.cbs.dtu.dk/services/SignalP/) [http://smart.embl-heidelberg.de) [http://swissmodel.expasy.org/) [Signal peptides were predicted using SignalP 4.0  . DisulfiignalP/) , 29 and berg.de) , respectsy.org/) . The temsy.org/) –36. VMD Protein sequences were aligned using ClustalW2  and manuT. solium and T. saginata, whereas one cystatin and two stefins were found in the T. asiatica genome. The expression of the stefins and the cystatins in the above mentioned three species was then confirmed by PCR using their corresponding cDNA as template   Figs  and S2. T. solium, T. saginata and T. asiatica by PCR and sequencing   . Similarity site . These rF. hepatica [It was interesting to note that the size of cystatins in cestodes ranged from 274 to 295 amino acids , substanhepatica . Cestodehepatica . Howeverhepatica . The secThere are two or three cystatin-like proteins in each planarian, nematode and trematode species . HoweverThe other typical characteristic of cystatins is that they possess one or two conserved disulfide bridges. The analysis of cystatins in vertebrates, trematodes, nematodes, and planarians demonstrated that they had one or two conserved disulfide bonds . HoweverAbove results of comparison of amino acid sequences of cystatin superfamily revealed that the cystatin sequences were poorly conserved in platyhelminth species, except for the conserved cystatin-like domain . HoweverS. japonicum cystatin1 was annotated to have a cystatin-like domain, no signal peptide and cystatin-like domain was predicted using SMART. Thus, it was discarded when the tree was constructed. For S. mansoni, one of three splicing forms of cystatin, with an intact cystatin-like domain was chosen to construct the tree. The phylogenetic analysis clearly showed that the members of platyhelminth cystatin superfamily were divided into two independent groups, the stefin and cystatin subfamilies. The stefins in trematodes, nematodes and cestodes branched together in a clade discrete from the planarian one. All platyhelminth species had a single copy for the stefin except for T. asiatica with two stefins (stefin1 and stefin2). All stefin genes in T. solium, T. saginata and T. asiatica (cestodes) were confirmed by PCR using their respective cDNA as template  almost overlaps Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigThe 3D structures for chicken cystain are shown in yellow, cestode cystains in other colors. Conserved cysteines (in green stick view) are displayed. The three parts of the papain binding domain (in stick view) are surrounded by ellipses.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


In vivo, phosphorylated Odf2 was evaluated in mouse sperm using a specific phospho-Ser-76 Odf2 antibody and LC-MS/MS. These findings are the first to demonstrate the phosphorylation site in Odf2 by Tssk4, providing essential clues regarding the function of Tssk4 in regulating sperm motility and/or structure and thus male fertility.As a member of the testis-specific serine/threonine protein kinase (TSSK) family, Tssk4 is exclusively expressed in the testis and plays an essential role in male fertility. We previously reported that Tssk4 can associate with and phosphorylate Odf2, but the phosphorylation site is still unknown. Here we confirm that the C-terminal region (amino acids 214-638) of Odf2 is required for association with Tssk4. Furthermore, to identify the site at which Tssk4 phosphorylates Odf2, we generated several Odf2 point mutants (Ser/Thr/Lys to Ala) and identified serine 76 of Odf2 as one of the phosphorylation sites.  Reversible protein phosphorylation is critically significant in biology because of its influence on almost every important cellular process throughout phylogeny, including cell growth, cell differentiation, cell cycle, and cell migrationDEx6,7/DEx6,7 mice display uncoordinated ciliary beating and a coughing/sneezing phenotype because of basal bodies lacking basal feetThe outer dense fibers (ODFs) are prominent sperm tail-specific cytoskeletal structures. They function in forming “9+9+2” structures, together with nine outer doublet microtubules and a central pair of microtubules45648513testis-specific serine/threonine protein kinase (TSSK) family, whose members play an important role in spermatogenesis and/or spermiogenesis. Thus far, five members of this family have been reported, including Tssk1, Tssk2, Tssk3, Tssk4 , and Tssk6. Due to their specific expression patterns during the processes of spermatogenesis and/or spermiogenesis, their physiological functions were explored by establishing gene knockout mouse models. The Tssk1 and Tssk2 double-deletion mouse was haploinsufficient and could not transmit the null genotypeTssk4 belongs to the We previously reported that Tssk4 is expressed exclusively in the testis and can maintain its kinase activity through autophosphorylation at Thr-197in vitro and in vivo. The confirmation of the fragment required for the cooperation of two proteins and the phosphorylation site of Odf2 provide crucial clues that deepen our understanding of the roles of Tssk4 and Odf2 in sperm structure and motility and, thus, male fertilityIn the present study, we defined the C-terminal fragment of Odf2, which is essential for the modification of Tssk4, and we then identified Ser-76 as a Tssk4 phosphorylation site in Odf2 both Tssk4 knockout mouse modelIt has been previously reported that Tssk4 is exclusively expressed in the testis and that male Tssk4 deficiency mice exhibit a subfertile phenotype due to decreased sperm motility and impaired sperm structureOn the one hand, the presence of an Odf2 band with a slower migration rate appeared only when Odf2 was co-transfected with wild-type Tssk4 but not the dead mutant kinase K54M  and or t21To identify the essential fragment of Odf2 that is required for altering the phosphorylation state of Tssk4, we generated several truncated constructs of murine Odf2 (GenBank number: NM013615) according to its various functional domains predicted by SMART software (Simple Modular Architecture Research Tool). The computational results revealed 3 major functional domains : a leuciThe six truncated constructs were subsequently transfected either alone or together with Tssk4/Tssk4 (K54M). The results revealed that the Odf2-C1 and Odf2-C2 constructs altered the phosphorylation state of Tssk4 , suggestin vitro  or threonine (Thr) or tyrosine (Tyr) sites in Odf2 were mutated to alanine, either separately or in combination . All of in vitro , implyinTo further confirm that the Odf2 Ser-76 phosphorylation site was modified by Tssk4, we generated an anti-phospho-Ser76 Odf2 polyclonal antibody to evaluate the phosphorylation state. The antibody can specifically recognize the phosphorylated Odf2 (Ser-76) protein band . When coin vivo, we performed Western blot analysis using the phospho-Ser76 Odf2 antibody to assess the phosphorylation state in mouse sperm (C57BL-6 strain). As shown in To determine whether the phosphorylated Ser-76 of Odf2 is present SSEK-80. The MS/MS spectrum of this phosphorylated peptide is presented in The identification of the Ser-76 site phosphorylated by Tssk4 was further verified using LC-MS/MS. A mouse sperm lysate was separated via SDS-PAGE, and the corresponding band of Odf2 was excised and digested with trypsin. LC-MS/MS confirmed the identification of the Odf2 protein (data not shown) and indicated the phosphorylation of the peptide sequence 64-NPPHCLEITPPIn conclusion, as a kinase exclusively expressed in testis, Tssk4 could phosphorylate Odf2 at Ser-76, depending on both its kinase activity and autophosphorylation modification. Meanwhile, Odf2 phosphorylation is required for maintaining the autophosphorylation activity at Thr-197. in vitro and in vivo. However, as the electrophoretic migration rate of the Odf2 (S76A) mutant when it was co-expressed with Tssk4 was slower compared with singly expressed Odf2 (S76A)  , Ser-76 Conversely, the electrophoretic migration rate of Tssk4 changed when it was co-expressed with full-length Odf2 or the Odf2-C1 and Odf2-C2 fragments, indicating that RPT and the filament domain were essential for the modification of Tssk4 and that the type of modification was phosphorylation catalyzed by Tssk4 itself, which was previously reported as auto-phosphorylation activityTssk4 and Odf2 can cooperate to regulate sperm structure and motility, and the deficiency of Tssk4 in a mouse model can lead to disrupted sperm function, thus reducing male fertilityPrevious studies indicate that ODF functions in maintaining the elastic recoil and passive elastic structures of the sperm flagellum and protecting the sperm flagellum against shear forces during epididymal transport and ejaculation27in vivo, our discoveries that Tssk4 could phosphorylate Odf2 at Ser-76 and, conversely, that Odf2 phosphorylation is required for the autophosphorylation activity at Thr-197 serve as relevant novel findings that will be useful for further characterization of the role of Tssk4 and phosphorylated Odf2 in sperm. The mutual modification through phosphorylation between Tssk4 and Odf2 and the identification of a functional interaction between these proteins could also provide further insight into signaling pathways that lead to sperm motility and/or structure.In light of the fact that Odf2 is known to be highly phosphorylated The pCMV-HA-Odf2 and pCMV-Myc-Tssk4 vectors and the dead mutant Myc-Tssk4 kinase (K54 M) were previously describedFull-length Odf2 and 6 truncated Odf2 sequences were amplified from murine testis cDNA via PCR and ligated into the pCMV-HA vector in frame. All of the constructs were verified through DNA sequencing. The sequences of full-length Odf2 and the truncated constructs were amplified with primer pairs as showed in Each primer contains a restriction enzyme recognition sequence, including a 5′ EcoRI sequence and a 3′ Xhol sequence, and the PCR products were cloned into the EcoRI/Xhol-digested pCMV-HA vector. Mutants of Odf2  were generated through PCR mutagenesis using the TaKaRa MutanBEST Kit according to the manufacturer’s instructions.For transfection, HEK-293T cells were cultured in Dulbecco’s Modified Eagle’s Medium containing 10% fetal calf serum (FCS). Transient transfection was performed using Lipofectamine 2000 according to the manufacturer’s instructions. The subsequent Western blot assay was described previously21232EDTA; 1 mM EGTA; 1% Triton; 2.5 mM sodium pyrophosphate; 1 mM Na3VO4; 1 mg/mL leupeptin) for 30 min at 4 °C with rocking. Following centrifugation  for 20 min at 4 °C, supernatants were collected, and the protein content was measured using a bicinchoninic acid (BCA) protein assay. The samples were mixed with loading buffer with 2-mercaptoethanol before being heated to 95 °C for 10 min.Cells were harvested and incubated in cell lysis buffer  and incubated for 30 min at 37 °C. Then, the sperm in the supernatant were centrifuged at 1000 g for 5 min. The collected sperm were then resuspended in PBS and passed through an 80-mm pore size filter for purification. Sperm were directly mixed with 1× loading buffer and boiled at 95 °C for 10 min.Cell or sperm extracts were subjected to 10% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% non-fat milk in Tris-buffered saline (TBS) for 1 h at room temperature and then incubated with corresponding primary antibodies overnight at 4 °C with rocking, followed by washing three times in TBST (0.1% tween-20) and incubation of the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1.5 h. After washing three times in TBST, the signals were developed using ECL chemiluminescence reagents on X-film.Mice (C57BL-6 strain) were handled following the guidelines for animal care and use in research of the National Institutes of Health, and all animal-related procedures were approved by the State Key Laboratory of Genetic Engineering and Institutional Animal Care and Use Committee (IACUC) of Fudan University. Mouse breeding and animal-related protocols were performed following the general guidelines published by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).2 MicroTrap, followed by washing with 80% ACN and 5% TFA and subsequent elution with 2% ACN and 1% NH4OH. The eluted phosphopeptides were desalted with a MicroTrap C8  and lyophilized using a SpeedVac , then resuspended in 20 μl of 0.1% formic acid/2% acetonitrile. All mass spectrometric experiments were performed on a LTQ orbitrap “XL” mass spectrometer connected to a Paradigm MDLC nanoflow LC system via an ADVANCE Spray Source LC-MS interface . The peptide mixture was loaded onto a 15 cm column with a 0.1 mm inner diameter packed with Magic C18AQ 3-um Reversed Phase resins and separated through a 60 minute linear gradient from 100% solvent A (0.1% formic acid/5% acetonitrile/95% water) to 35% solvent B (0.1% formic acid/100% acetonitrile) at a flow rate of 500 nl/min. The spray voltage was set to 1.5 KV, and the temperature of the ion transfer capillary was 160 °C. The mass spectrometer was operated in positive ion mode and used in data-dependent mode to automatically switch between MS and MS/MS using the Tune and Xcalibur 2.5.5 software package. One full MS scan from 350 to 1800 m/z was acquired at a high resolution of R = 100,000 (defined at m/z = 400), followed by 10 data = dependent MS/MS spectra in the linear ion trap from the 10 most abundant ions. All MS/MS ion spectra were analyzed using Sequest1  and were incorporated into the Sorcerer engine version 4.0.4 build (Sage-N Research). Sequest was set up to search the ipi.MOUSE.v3.65 database (56775 entries) (ftp.ebi.ac.uk/pub/databases/IPI/current) assuming semi-enzyme tryptic digestion allowing 3 missed tryptic cleavages, with a full mass from 600 to 4600. The precursor ion tolerance was set to ±10 p.p.m, and the MS2 ion tolerance was set to 1 Da. The search parameters for the total peptides allowed a static modification of Carbamidomethyl C (+57.021465) on cysteine and a dynamic modification of oxidized M (+15.99492) on methionine. Additional searches for phosphopeptides allowed dynamic modifications of phosphorylation STY (+79.966331), on serine, threonine, and tyrosine, with up to 6 total dynamic modifications and up to 4 of a particular dynamic modification. The cutoff values of the filter threshold were set at XCorr 2+, 3+, 4+: >2.5, >3.0, >3.5 and DeltaCN > 0.08, and two peptides were required for total peptide to protein identification, with an estimated false-discovery rate (FDR) < 1%. Finally, the results were presented using the Scaffold viewer.Gel slices containing the Odf2 protein were de-stained with 50% acetonitrile (ACN) in 50 mM ammonium bicarbonate. The dried gel slices were sequentially reduced with 10 mM DTT and alkylated with 55-mM iodoacetamide. The proteins were then digested overnight at 37 °C with sequencing-grade modified trypsin at a protein-to-enzyme ratio of 50:1. The peptides were extracted from the gel slices with 60% ACN and 1% trifluoroacetic acid (TFA) and were then desalted with a MicroTrap C8. Phosphopeptides were subsequently enriched with a MicroTrap Titanium Dioxide Phosphopeptide Trap , as described in the manual. Briefly, the reaction products dissolved in equilibration buffer  were loaded into the pre-equilibrated TiOHow to cite this article: Wang, X. et al. Testis-specific serine/threonine protein kinase 4 (Tssk4) phosphorylates Odf2 at Ser-76. Sci. Rep.6, 22861; doi: 10.1038/srep22861 (2016).