H2O2 activates G protein, α 12 to disrupt the junctional

H2O2activates G protein,α12to disrupt the junctional complex and enhance ischemia reperfusion injury

Wanfeng Yu a,Sarah Beaudry b,1,Hideyuki Negoro b,2,Ilene Boucher a,Mei Tran a,Tianqing Kong b,

and Bradley M.Denker a,3

a Renal Division,Beth Israel Deaconess Medical Center and Harvard Medical School,Boston,MA02215;and

b Renal Division,Brigham and Women’s Hospital and Harvard Medical School,Boston,MA02115

Edited*by Peter Agre,Johns Hopkins Malaria Research Institute,Baltimore,MD,and approved March12,2012(received for review October11,2011)

The epithelial cell tight junction separates apical and basolateral domains and is essential for barrier function.Disruption of the tight junction is a hallmark of epithelial cell damage and can lead to end organ damage including renal failure.Herein,we identify Gα12 activation by H2O2leading to tight junction disruption and demon-strate a critical role for Gα12activation during bilateral renal ische-mia/reperfusion injury.Madin–Darby canine kidney(MDCK)cells with inducible Gα12(Gα12-MDCK)and silenced Gα12(shGα12-MDCK)were subjected to ATP depletion/repletion and H2O2/cata-lase as models of tight junction disruption and recovery by moni-toring transepithelial resistance.In ATP depleted cells,barrier disruption and recovery was not affected by Gα12,but reassembly was accelerated by Gα12depletion.In contrast,silencing of Gα12 completely protected cells from H2O2-stimulated barrier disruption, a response that rapidly occurred in control cells.H2O2activated Src and Rho,and Src inhibition(by PP2),but not Rho(by Y27632), protected cells from H2O2-mediated barrier disruption.Immuno?u-orescent and biochemical analysis showed that H2O2led to increased tyrosine phosphorylation of numerous proteins and altered membrane localization of tight junction proteins through Gα12/Src signaling pathway.Gα12and Src were activated in vivo during ischemia/reperfusion injury,and transgenic mice with renal tubular QLα12(activated mutant)expression were delayed in re-covery and showed more extensive injury.Conversely,Gα12 knockout mice were nearly completely protected from ischemia/ reperfusion injury.Taken together,these studies reveal that ROS stimulates Gα12to activate injury pathways and identi?es a thera-peutic target for ameliorating ROS mediated injury.

kidney disease|acute kidney injury|signaling|tyrosine kinases

E pithelial cells lie at the boundary between two distinct com-

partments and function to maintain homeostasis in different milieus.Thus,epithelia provide a barrier to inappropriate mixing between compartments and maintain balance through regulated absorption and secretion.Effective epithelial function requires a cell-cell connection that modulates paracellular movement and separates transporters and ion channels into discrete apical and basolateral plasma membrane domains.The tight junction(TJ)is the most apical component of the junctional complex and is es-sential for these functions.TJs are linked to the actin cytoskeleton and composed of integral membrane proteins(claudins,occlu-din),scaffolding proteins(ZO-1,ZO-2,ZO-3),and numerous signaling molecules.TJs regulate steady-state paracellular prop-erties,and when disrupted with epithelial injury,lead to back-leak and major disturbances in organ function.The reassembly of the TJ is essential postinjury repair(1,2),and similar mechanisms are important during epithelial organ development.

Acute kidney injury(AKI)is common in hospitalized patients and contributes to increased mortality,morbidity,and cost(3). Accumulating evidence suggests that AKI is a risk factor for chronic progressive kidney disease and the development of end-stage renal disease requiring dialysis or transplantation(4).Toxic and ischemia/reperfusion injuries(IRIs)are the most common causes of AKI and result in disassembly of TJs,increased apo-ptosis and proliferation,and increased tubular cell detachment (reviewed in ref.5).Once injured,renal tubular epithelia may recover or progress to a?brotic phenotype,and recovery re-quires multiple integrated processes including reestablishment of the TJ.Studies of delayed graft function in human kidney allografts revealed that TJ disruption is an early and potentially reversible target of IRI(6).Currently,there are no therapeutic options to alter the natural history of AKI.

Cultured epithelial cells and animal models of injury have re-vealed that ATP depletion triggers an altered distribution of tight and adherens junction proteins with loss of barrier function and increased tyrosine phosphorylation(7–9).Reactive oxygen spe-cies are important mediators of injury,and H2O2stimulates TJ disruption through tyrosine kinase activation(10,11).Injury-mediated TJ disruption,and its subsequent reassembly,are reg-ulated by numerous signaling pathways,including Rho GTPases. Inhibition of Rho led to TJ disassembly in various epithelial cell lines,and basal Rho activity was required for normal TJ function (12).Protein kinase C(PKC)has multiple effects on the phos-phorylation of TJ proteins and diverse roles in TJ assembly(ref. 13and reviewed in ref.14).The ser/thre phosphatase PP2A localizes to the TJ and regulates the phosphorylation of ZO-1and occludin antagonizing aPKC phosphorylation of these proteins

(15).We have demonstrated an important role for heterotrimeric

G proteins in regulating the TJ(16).Several Gαsubunits localize to the TJ including Gαo,Gαi2,and Gαs(17,18)and stimulate TJ assembly.Conversely,Gα12is also found in the TJ(19)and interacts with ZO-1(20),but its activation inhibits TJ assembly and increases paracellular permeability by tyrosine phosphoryla-tion(via Src and Hsp90)of TJ proteins(21,22).Based on these ?ndings,we hypothesized that Gα12may be activated during is-chemia/reperfusion and contribute to kidney injury through dis-ruption of the junctional complex,delaying epithelial recovery. Herein,we identi?ed Gα12activation during renal IRI and examined these mechanisms in cell culture and animal models of https://www.sodocs.net/doc/5d11989217.html,ing MDCK cells in ATP depletion/repletion and H2O2/ catalase injury models,we found that H2O2,but not ATP de-pletion,activated Gα12to promote disruption of the junctional complex via Src phosphorylation of TJ proteins.Furthermore, bilateral renal artery IRI in transgenic mice with conditional renal tubular expression of QLα12(Q229L,constitutively acti-vated)and Gα12?/?mice con?rmed that Gα12has a pivotal role mediating injury.Taken together,these studies suggest that Gα12inhibition may provide a therapeutic strategy to block in-jury mechanisms.

Author contributions:B.M.D.designed research;W.Y.,S.B.,H.N.,I.B.,M.T.,and T.K.per-formed research;W.Y.,I.B.,and B.M.D.analyzed data;and B.M.D.wrote the paper. The authors declare no con?ict of interest.

*This Direct Submission article had a prearranged editor.

1Present address:Harvard Apparatus,Holliston,MA01746.

2Present address:University of Tokyo,Tokyo Medical and Dental University,Tokyo 113-8655,Japan.

3To whom correspondence should be addressed.E-mail:bdenker@https://www.sodocs.net/doc/5d11989217.html,. This article contains supporting information online at https://www.sodocs.net/doc/5d11989217.html,/lookup/suppl/doi:10. 1073/pnas.1116800109/-/DCSupplemental.

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Results

G α12Delays TJ Assembly in ATP Depletion/Repletion.Based on

previous studies (20–22),we hypothesized that G α12/Src signaling may be important during epithelial cell injury.We analyzed MDCK cells by using ATP depletion/repletion,an established model of epithelial injury (23).Baseline transepithelial resistance (TER)was determined in Tet-off inducible G α12(G α12-MDCK)and G α12-silenced MDCK cells (shG α12-MDCK)(20,24).As previously reported,G α12overexpression in MDCK cells -doxycycline (-dox)led to a decrease in baseline TER (Fig.1A ),and silencing G α12(shG α12-MDCK cells)led to a small increase in baseline TER (Fig.1B ).Next,the time course of TJ disruption and recovery in the ATP depletion/repletion model was examined (Fig.1C and D ).ATP depletion caused a rapid decrease of TER in all cell lines within an hour,and there were no differences in the kinetics of barrier disruption.Recovery of the barrier upon ATP repletion was not signi ?cantly different in G α12-MDCK cells (-dox)compared with the +dox controls (Fig.1C )but was accelerated in the shG α12-MDCK cells (Fig.1D ).

Activated G α12during ATP depletion/repletion was measured by GST-TPR pulldown.Activated G α12/13binds the PP5TPR domain (25),and Fig.1E con ?rms the pulldown of QL α12with GST-TPR from QL α12-MDCK cells,but not with GST alone (22).ATP-depleted G α12-MDCK cells (-dox)did not lead to any detectable G α12activation (Fig.1E ).However,during the ATP repletion phase,G α12was activated by 10min and was further increased at 30min.The activation of G α12during recovery (repletion),but not TJ disruption,was similar to the calcium switch (22),and these effects are summarized in Table S1.However,the signaling downstream of G α12was Src-dependent

in the calcium switch,but as shown in Fig.S1,was neither Src-nor Rho-dependent during recovery from ATP depletion.Silencing G α12Protects Cells from H 2O 2Induced TJ Injury.Reactive

oxygen species (ROS),including H 2O 2,are important in IRI and signal through numerous mechanisms (reviewed in ref.26).ROS were shown to directly activate G αi/o but not G αs through a re-ceptor-independent mechanism (27),although G α12was not examined.To determine whether G α12is activated by H 2O 2,G α12-MDCK cells with or without dox were treated with 5mM H 2O 2for 1h followed by the addition of catalase at t =0(initiates recovery;no signi ?cant effect on ATP levels;ref.11).Fig.2A shows rapid loss of barrier function in G α12-MDCK cells with or without dox and prompt recovery to values above baseline within 60min of catalase treatment in the controls (+dox).There was a signi ?cant delay in the recovery of G α12-MDCK cells (-dox),eventually reaching baseline at 300–420min.Likewise,shGFP-MDCK control cells dropped to 25%of baseline TER at 60min of H 2O 2,and with catalase,recovered over the next several hours.Surprisingly,shG α12-MDCK cells treated with H 2O 2were com-pletely protected from H 2O 2-stimulated TJ disruption (Fig.2B ).To con ?rm that H 2O 2activates G α12,two different methods were used —GST-TPR and [35S]GTP γS binding.Fig.2C shows GST-TPR pulldowns at various time points of H 2O 2exposure in G α12-MDCK cells.There was a signi ?cant increase in G α12ac-tivation by 10min H 2O 2exposure that peaked at 30min.This time course of activation is similar to that observed with G pro-tein-coupled receptors (GPCRs),and the duration of activation is determined by the GTPase activity of G α12.Next,ROS activation of G α12was con ?rmed by measuring [35S]GTP γS (non-hydrolyzable GTP analog)binding to membrane preparations of G α12-MDCK cells stimulated with H 2O 2.Fig.2D shows [35S]GTP γS-labeled G α12immunoprecipitated from lysed G α12-MDCK cells after vehicle,thrombin,or H 2O 2stimulation for 1h.Both thrombin and H 2O 2stimulated [35S]GTP γS binding to G α12.Taken together with previous studies,these studies show that H 2O 2activates G α12and is essential for barrier disruption.

H 2O 2Disrupts TJs Through G α12/Src.To distinguish between G α12activating Src and/or Rho,cells were analyzed with or without inhibitors.Preincubation of control cells with Y27632

(Rho

Fig.1.G α12inhibits TJ recovery after ATP depletion.Baseline TER in con-?uent G α12-MDCK cells with or without dox (A ),and shG α12-and shGFP-MDCK cells (B ).*,signi ?cance at P <0.05.TER time course in ATP depletion [antimycin (10μM)+2-deoxy-D-glucose (2mM)for 60min followed by ATP repletion injury model using G α12-MDCK cells with or without dox (C )and shG α12-and shGFP-MDCK cells (D )].The experiment was done four times for G α12-MDCK cells (n =3–5per time point)and three times with shG α12-MDCK cells.TER did not signi ?cantly differ among G α12-MDCK cells with or without dox,but was signi ?cantly different (P <0.05)in shG α12-MDCK cells at each time point during recovery (catalase)except for 300min in each experiment.(E )GST-TPR pulldown of activated G α12.QL α12(constitutively active G α12)expressing MDCK cells were used as controls (?rst two lanes).G α12expressing MDCK cells were analyzed in parallel at speci ?ed times of ATP depletion/repletion.Top shows precipitated G α12(activated)with total G α12from ≈75μg of total lysate shown in Middle .(Lower )GAPDH is shown as a loading

control.

Fig.2.G α12is activated by H 2O 2and required for barrier disruption.(A and B )TER was measured at speci ?ed times in G α12-MDCK cells with or without dox (A )and shG α12or control cells (B )during H 2O 2treatment for 1h followed by catalase added at t =0as described in Materials and Methods .(C )G α12activation was determined by GST-TPR pull down at indicated times of H 2O 2exposure and recovery.(D )[35S]GTP γS binding to G α12.G α12expressing MDCK cells were stimulated with thrombin (2U/mL),H 2O 2(5mM),or vehicle for 1h at 30°C.Lysates were prepared and G α12immu-noprecipitated as described in Materials and Methods .Samples were ana-lyzed by SDS/PAGE and autoradiography.Exposure time =14d.

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kinase inhibitor)failed to prevent barrier disruption (Fig.3A ),although the magnitude of the decrease in TER was slightly less than that seen in the untreated controls.shG α12-MDCK cells were protected from H 2O 2stimulated barrier disruption,and the addition of Y27632had no effect.The rate of recovery in shGFP-MDCK cells with or without Y27632was similar (Fig.3A ).Rho activity in control and shG α12-MDCK cells with or without H 2O 2shows rapid activation of Rho in control cells with H 2O 2(Fig.3B ).In contrast,shG α12-MDCK cells showed no Rho activation with H 2O 2,and a partial decrease was observed.The lack of an effect of Y27632in protecting MDCK cells from H 2O 2stimulated loss of TER suggests that although Rho is ac-tivated through G α12,this pathway is not the primary signal mediating barrier disruption.shGFP-MDCK cells were in-cubated with Src inhibitor PP2with H 2O 2for 1h and were completely protected from barrier disruption (Fig.3C ).Src ac-tivity was assessed by p419Western blot,and dramatic activation was seen in shGFP-MDCK cells at 30min (Fig.3D )with only minimal activation of Src seen in H 2O 2treated shG α12-MDCK cells.These ?ndings were con ?rmed in inner medullary collect-ing duct (IMCD)cells by using lentivirus G α12shRNA (Fig.3E ).IMCD cells do not develop measurable TER,so paracellular ?ux of 3-kDa FITC dextran was measured with or without H 2O 2treatment.Paracellular ?ux signi ?cantly increased (1.77±0.18fold)with H 2O 2stimulation (Fig.3E )in control IMCD cells,whereas shG α12silenced IMCD cells were completely protected from barrier disruption.Src inhibition with PP2in control IMCD cells also prevented barrier disruption.We next asked whether the RGS domain of p115RhoGEF (a dominant negative for both G α12/13)inhibited H 2O 2stimulated barrier disruption.Fig.3F shows a slower rate of barrier disruption and accelerated re-covery in the RGS expressing cells.

H 2O 2Stimulates Disruption of Junctional Complex Proteins Through G α12/Src.Fig.S2A and B shows that TJ proteins ZO-1and

occludin were displaced from the lateral membrane with H 2O 2

treatment in control cells.The normal linear staining at the cell membrane was disrupted and punctuate,whereas pretreatment with PP2largely prevented these H 2O 2induced changes.shG α12cells treated with H 2O 2were nearly identical to the baseline condition.E-cadherin,localized within the adherens junction,is also required for TJ assembly (reviewed in ref.28)and interacts with G α12(29).Injury leads to E-cadherin degradation (30),and H 2O 2stimulated loss of E-cadherin could be prevented by treatment with PP2or silencing G α12(Fig.S2C ).Stress ?bers were nearly completely absent from H 2O 2-stimulated control cells (Fig.S2D ),and there was partial restoration in PP2treated,H 2O 2stimulated control cells.There were no signi ?cant differ-ences observed in shG α12-MDCK cells,indicating that G α12was required for formation of stress ?bers triggered by H 2O 2.

H 2O 2Stimulates Tyrosine Phosphorylation of ZO-1and Occludin Through G α12/Src.H 2O 2stimulated pTyr phosphorylation of nu-

merous cellular proteins,and much of the increased phosphory-lation could be inhibited with PP2(Fig.S3A ).Immunoprecipitated ZO-1was more heavily phosphorylated with H 2O 2stimulation in shGFP-MDCK cells,with little change in shG α12-MDCK cells (Fig.S3B ).Next,ZO-1and occludin were immunoprecipitated from control cells and phosphorylation examined by pTyr Western blot.Fig.S3C shows that H 2O 2stimulated increase in tyrosine phosphorylation was inhibited by PP2.Finally,the integrity of the TJ complex was assessed by determining the amount of ZO-1coprecipitated with ZO-2in these cells.Consistent with partial disruption of the complex by H 2O 2,less ZO-1was immunopreci-pitated,and this ?nding was partially reversed with Src inhibition (Fig.S3D ,Bottom ).

G α12and Src Are Activated in Vivo with Ischemic Injury.Unilateral renal artery ischemia reperfusion followed by GST-TPR pull-down shows activated G α12that was not seen in the nonischemic contralateral control kidney (Fig.4A ).Normally,G α12is not detectable by Western blot in kidney.Several time points

of

Fig.3.H 2O 2/G α12stimulated barrier disruption is mediated by Src and not Rho.(A )Time course of H 2O 2-stimulated barrier disruption in shG α12-and shGFP control MDCK cells with or without Rho kinase inhibitor Y27632(10μM)added 1h before the addition of H 2O 2.Catalase was added at t =0.(B )Rho activity in shG α12and control MDCK cells stimulated with H 2O 2at t =0.Values are normal-ized to baseline Rho A activity before H 2O 2treat-ment.(C )Time course of H 2O 2-stimulated barrier disruption in shG α12and shGFP control MDCK cells with or without Src kinase inhibitor PP2(10μM)added 1h before the addition of H 2O 2.The experiment was repeated three times with similar results.(D )Src activity in H 2O 2treated shG α12-and shGFP-MDCK cells.Western blot to pY419-Src (ac-tive Src)and total Src is shown at baseline and after 30or 60min of H 2O 2treatment.Samples were on the same gel,and one exposure time is shown.(E )Paracellular ?ux of 3-kDa FITC dextrans as described in Materials and Methods in con ?u-ent IMCD cells with G α12shRNA or control cells treated with H 2O 2with or without PP2(10μM).*indicates no overlap of 95%con ?dence intervals.(F )Time course of H 2O 2-stimulated barrier dis-ruption of MDCK cells with retroviral infected p115RhoGEF RGS domain or control.

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reperfusion were also examined but did not consistently show any G α12activation.Kidney lysates from ischemic and non-ischemic kidneys were also examined for Src activation by using pY419antibodies,and Fig.4B shows Src activation after 30min of ischemia and after 30min of reperfusion.

Transgenic Mice Reveal a Critical Role for G α12in Enhancing IRI.Two

different transgenic mice were used to examine the role of G α12in renal injury:conditional expression of mutationally activated G α12(QL α12)in renal tubules and G α12knockout mice (normal phenotype;ref.31).The expression of activated G proteins has been an invaluable tool for probing G protein signaling in-dependent of receptor activation.Fundamental insights into oncogenes (e.g.,Ras)and signaling pathways have been identi ?ed with this approach,which has been widely used in cell culture

studies,although less so in vivo.Epitope tagged (EE),?oxed LacZ human QL α12mouse was established (32),and Fig.S4shows a minority of tubules express LacZ (estimated to be 10–20%based on LacZ staining before and after Cre)due to mosaic expression (33).QL α12LacZ+mice were crossed with γGT-Cre mice for conditional proximal tubule expression and,EE-tagged QL α12could be detected by immuno ?uorescence (Fig.S4)in some tu-bular cells.These mice had no phenotype,but were more sus-ceptible to IRI.Fig.5A –D shows that QL α12Cre+mice were delayed in renal recovery after bilateral IRI compared with the controls.Baseline creatinine in these mice was 0.2±0.1mg/dL.One day after IR,plasma creatinine was increased to a similar degree in both groups (Fig.5A ),and control mice partially re-covered on day 2,whereas renal function in transgenic mice did not recover.Histologic analysis on day 2con ?rmed the persistence of more severe injury in the QL α12Cre+mice (Fig.5B –D ).There was more severe tubular injury with extensive necrosis,loss of brush border,more vacuolation,and desquamation in epithelial cells than was seen in the control mice (quanti ?ed in Fig.5B ).A longer time course of recovery after IRI was performed with these mice.Fig.S5shows that QL α12Cre+mice show recovery of serum creatinine to levels similar to controls by day 4after IRI.However,histology at day 6shows signi ?cantly more injury in QL α12Cre+mice.There was no signi ?cant difference in kidney/body weight ratios although QL α12Cre+have more weight loss after in-jury than the controls.There was no difference in recovery between males and females.These ?ndings are consistent with other studies in which injury to a subset of epithelial cells leads to more wide-spread organ damage (34).Finally,we hypothesized that if ROS activated G α12led to more severe injury in IR,then G α12knockout mice might be protected.G α12knockout mice are phenotypically normal (31).Fig.5E –H shows that G α12?/?mice were highly re-sistant to bilateral IRI and showed much less histologic injury.Body and kidney weight were not different in G α12?/?and control mice at baseline or at 48h after injury,and no signi ?cant differences in glomerular integrity were observed in these experiments.

Discussion

Oxidative stress disturbs the permeability barrier of epithelial cells,and loss of barrier function is an early and reversible event in epithelial injury.We present cell culture and animal data in two transgenic mouse models that highlight a pivotal role for G α12in promoting barrier disruption with injury and

suggest

Fig.4.G α12and Src are activated in vivo in IRI.(A )Wild-type male mice were subjected to 30min of unilateral renal ischemia and compared with the contralateral kidney (cont).Kidney lysates were analyzed by GST-TPR pull-down prepared as described in Materials and Methods .Ten percent of total kidney lysate was set aside,and the remainder was used for GST-TPR pull-down.Results from two mice analyzed on the same gel are shown in com-parison with the contralateral nonischemic kidney analyzed in parallel.(B )Src Western blots of kidneys obtained from unilateral IR and contralateral kidney from the same mouse analyzed at speci ?ed times of ischemia and reperfu-sion.Two percent of cell lysate was analyzed for these Western

blots.

Fig.5.Targeting constitutively activated G α12to renal tubules delays recovery in IRI and G α12knockout mice are protected from injury.(A )Serum creatinine on day 1and day 2after bilateral IR in QL α12Cre+(n =9)and QL α12Cre-(control;n =7)mice.*P =0.009.(B )Tubular injury score for QL α12Cre+and QL α12Cre-mice.*P =0.006.Scoring criteria are de ?ned in Materials and Methods .(C and D )Representative histology of QL α12Cre+and QL α12Cre-mice at 2d after bilateral IR.(E )Serum creatinine in G α12?/?(n =6)and littermate age matched wild-type controls (n =7)on day 1and day 2after bilateral IRI.*P =0.002;#P =0.001.(F )Tubular injury score from G α12?/?and controls on day 2.(G and H )Representative histology of G α12+/+and G α12?/?mice at 2d after bilateral IR.(Scale bar:50μm.)

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inhibiting Gα12may prevent subsequent damage.We?nd that H2O2activates Gα12and stimulates Src mediated physiological, biochemical,and immunocytochemical changes in the tight and adherens junctions.H2O2,but not ATP depletion,activates Gα12/Src to disrupt the epithelial cell barrier,and our results are consistent with direct activation of Gα12by H2O2(as shown for Gαi/o;ref.27).The delayed recovery from IRI in QLα12Cre+ mice implicates renal tubular Gα12in promoting renal epithelial injury,and the protection from injury in Gα12knockout mice supports a critical role for Gα12in early injury signaling(al-though likely through multiple mechanisms).Thus,ROS-acti-vated Gα12may be an effective target to inhibit activation of downstream signaling pathways that promote injury.

G proteins are molecular switches linking extracellular stimuli to cellular responses,yet because of the links to multiple signaling pathways,they have not been viewed as attractive therapeutic targets.Many drugs modulate cellular responses through GPCRs (number in the thousands),and the relatively speci?c therapeutic effects are achieved through binding to unique receptor subtypes. There are only16Gαsubunits(divided among the four major families),and downstream signaling occurs through either the activated Gαor dissociated Gβγsubunits(reviewed in ref.35). Therefore,the inhibition of a speci?c Gαsubunit would be pre-dicted to affect many pathways and have numerous off-target effects.However,there may be unique pathophysiologic con-ditions where blocking all of the signaling downstream of an ac-tivated Gαsubunit would be bene?cial.We suggest that targeting Gα12for inhibition during oxidative injury is one of these cir-cumstances,and is based on(i)Gα12is activated by H2O2and leads to barrier disruption,an early injury event;(ii)Gα12si-lenced cells and knockout mice are highly protected from injury; (iii)Gα12knockout mice are normal,indicating that adult cells do not require Gα12for survival;(iv)targeting only the activated conformation of Gα12would limit potential drug effects only to cells with activated Gα12(i.e.,at the site of injury);and(v)tar-geting activated Gα12for inhibition obviates the need to identify and inhibit numerous receptors capable of activating Gα12.We suggest that the results described herein provide a strong ratio-nale for pursuing further studies on identifying molecules that inhibit activated Gα12.

ROS are composed of several major species,target multiple cell components,and are major signaling molecules.ROS can directly oxidize target proteins through thiol ester formation of amino acids(usually cysteine;reviewed in ref.26).Oxidation of a target protein could lead to degradation,inactivation,or acti-vation.It was shown that Gαi and Gαo(both pertussis toxin substrates),but not Gαs,were directly activated by H2O2leading to subunit dissociation and ERK activation(27).We report a similar?nding of Gα12activation by H2O2.Alignment of Gαs, Gαi,Gαo,and Gα12does not identify a conserved cysteine(s) that could account for this speci?city.Gαcrystal structures reveal that with receptor activation,the C-terminalα5helix is displaced to uncover the bound GDP and,thus,lower the af?nity for the bound nucleotide(36).The selectivity for H2O2activation of Gαi/o,but not Gαs,may reside in the conserved cysteine residue four amino acids from the C terminus(pertussis toxin modi?-cation site).ADP ribosylation by pertussis toxin prevents Gαand Gβγdissociation and blocks subsequent signaling.Thiol modi?-cation of cysteine-350could promote displacement of theα5 helix and permit Gαi/o activation,because deletion of C-terminal 10amino acids of Gαo(345–354)led to an activated confor-mation via reduced GDP af?nity(37).Gα12has no cysteine in the C-terminal region,but the N terminus of Gα12is distinct from all other Gαsubunits and contains a unique cysteine at amino acid11.The N terminus is required for binding Gβγ,and modi?cation in this domain could result in Gβγdisplacement, a required event for Gαactivation.Site-directed mutagenesis and measurement of Gαactivation by ROS will be required to determine the speci?c amino acids targeted for H2O2-thiol modi?cation necessary for activation of speci?c Gαsubunits.

Multiple signaling molecules regulate loss of barrier integrity with injury and during recovery.G proteins,kinases,and phos-phatases all have a role,and cell culture models permit these mechanisms to be analyzed in ways that are dif?cult in vivo.Table S1summarizes the consequences of activating Gα12,Src,receptor tyrosine kinases,PKC,and phosphatases in three different models of TJ disruption and assembly.Although each model of barrier disruption and reassembly uses similar signaling molecules,the consequences of activation vary depending on the model(see Table S1for details).Our results indicate that observations obtained from cell culture models of injury are a valid and im-portant approach to understanding the complex role of speci?c signaling molecules in vivo.The?ndings in both ATP depletion and H2O2models of injury revealed important functions for Gα12 in maintenance and assembly of the barrier during and after injury. These?ndings provided the rationale for the transgenic animal experiments and help to interpret the respective phenotypes. Some of the bene?cial effects of various compounds reported in AKI may be mediated through inhibition of Gα12/Src sig-naling.For example,activation of the Gα12/13coupled LPA-3 receptor with oleoyl-me-thoxyphosphothionate,enhanced IRI, whereas an LPA1/LPA3-receptor antagonist,VPC-12249,re-duced IRI(38).Adenosine is also protective in AKI(39)and activates Gαs and Gαi,and we showed that Gαs and Gαi2 accelerates TJ reassembly in the calcium switch(17,18).This ?nding suggests that a balance of G protein signals normally regulates TJs.Interestingly,in a chronic?brosis model of uni-lateral ureteral obstruction,LPA-1receptor activation promoted tubulointerstitial?brosis that could be inhibited with an LPA-1 receptor antagonist(40).Thus,signaling through Gα12/13may also mediate chronic?brotic pathways and raises the possibility that inhibiting Gα12in AKI may have bene?ts in preventing chronic damage.Gα12has multiple roles important to numerous cellular functions including proliferation,apoptosis,cell migra-tion(reviewed in ref.41),and the protection from IRI afforded to the Gα12knockout mice could result from additional mech-anisms involving loss of Gα12from in?ammatory cells,pericytes, or vascular endothelial cells.Conditional knockout of Gα12will be necessary to determine its role in these other cell types.

In conclusion,these?ndings identify Gα12as a pivotal mediator of epithelial injury and provides a link from ROS to Src activation. Taken together,these?ndings suggest that targeting ROS acti-vated Gα12for inhibition has the potential to prevent disruption of the junctional complex and prevent subsequent organ damage. Materials and Methods

Cell Lines and Culture.MDCK cell lines were cultured at37°C in5%(vol/vol)CO2 and maintained in DMEM(Cellgro)containing5%(vol/vol)FBS(Clontech) (DMEM)and100μg/mL G418.Tet-off MDCK cells were maintained in40ng/mL dox and Gα12expression induced by dox removal for48–72h as described(20).

TER.Con?uent MDCK cells on polycarbonate?lters(Transwell;Costar)were analyzed for TER at speci?ed times(n=3–5)during H2O2/catalase or ATP depletion/repletion treatment with a Millipore electrical resistance system. Measurements are expressed as a mean±SEM of the original readings after subtraction of background values.

ATP Depletion/Repletion.Intracellular ATP was depleted by adding the gly-colytic inhibitor2-deoxy-D-glucose and antimycin as described(11).Con?u-ent MDCK monolayers were serum starved for24h,washed with PBS,and incubated with PBS containing2mM2-deoxy-D-glucose and10μM antimycin for1h.Repletion of intracellular ATP levels was achieved by changing to DMEM at time=0.

H2O2/Catalase.Con?uent monolayers were serum starved for24h and then incubated with5mM H2O2in serum-free DMEM for1h h as described(11). Recovery at T=0was induced by the addition of the ROS scavenger catalase (5,000U/mL)and cells analyzed at various times.

Silencing Gα12in IMCD Cells.Lentiviral vector transfer plasmids containing mouse Gα12shRNA were obtained from Fisher and Gα12silenced according to the manufacturer’s protocol.See SI Materials and Methods for additional details.

6684|https://www.sodocs.net/doc/5d11989217.html,/cgi/doi/10.1073/pnas.1116800109Yu et al.

Paracellular Flux.IMCD cells were cultured on Transwell Filters,FITC-tagged 3-kDa anionic dextran(Molecular Probes)was added to the apical chamber (0.25mg/mL),and paracellular?ux measured as described in SI Materials and Methods.

GST-TPR Pulldown.MDCK cells(≈1mg of total protein)or kidney lysates were incubated with GST-TPR coupled glutathione-agarose beads(Amersham Pharmacia)(≈1μg)overnight at4°C.Samples were centrifuged,washed with PBS+0.1%Triton X-100,and eluted samples were analyzed by SDS/ PAGE and Gα12Western blot.

GTPγS Binding.Gα12-MDCK cells were cultured in-dox for72h.Membranes were prepared from con?uent10-cm plates and incubated with5mM H2O2 in1μM[35S]GTPγS(PerkinElmer;1,250Ci/mM),thrombin(2U/mL),or vehicle for1h.Gα12was immunoprecipitated from membranes with rabbit Gα12-antisera(10μL)or protein-A beads only(control)overnight at4°C.Samples were centrifuged,washed,and analyzed by SDS/PAGE,Coommassie staining and EnHance(Dupont).Autoradiogram was exposed for14d.

RhoA Activation.RhoA activation was determined with G-LISA RhoA Acti-vation Assays(Cytoskeleton)according to the manufacturer’s protocol.See SI Materials and Methods for additional details.

Immuno?uorescent Microscopy.MDCK cells were cultured on coverslip chambers and analyzed after H2O2treatment for1h.See SI Materials and Methods for additional details.

Western Blotting and Immunoprecipitations.Gα12,Src and GAPDH Western blot,and immunoprecipitation of TJ proteins were done as described(22). See SI Materials and Methods for additional details.Transgenic Mice.Human Gα12(Q229L)EE tagged was cloned in to the CMV ?oxed LacZ cassette(provided by Larry Holzman,University of Pennsylvania, Philadelphia,PA).C57/B6mice were injected at the Brigham and Women’s Hospital Transgenic Mouse Facility and were then crossed withγGTCre mice (provided by Joe Bonventre,Brigham and Women’s Hospital,Boston,MA) on the same C57/B6background.Gα12?/?mice were obtained from Mel Si-mon(California Institute of Technology,Pasadena,CA),and age and sex matched wild-type mice were used as controls.

Animal Preparation and IRI.Experiments were performed in2-to5-mo-old C57/B6mice and age matched±2wk for each experiment.The renal arteries were accessed through the retroperitoneum,and ischemia was induced by bilateral clamping for30min for male,33min for female,or only the left kidney for30min.See SI Materials and Methods for additional details.

Renal Function and Histology.The plasma creatinine concentration was de-termined by the picric acid method.Kidney histology was examined on paraf?n sections stained with PAS.Tubule injury were determined by PAS stained paraf?n sections using a blinded scoring method.See SI Materials and Methods for additional details.

Statistics.Data are expressed as medians or means±SEM as indicated. Statistical analysis was performed by using Prism4for Macintosh(GraphPad) using the two-tailed t test.Statistical signi?cance was identi?ed at P<0.05.

ACKNOWLEDGMENTS.We thank Dr.Ted Meigs for his constructive reading of the manuscript and Qingyi Wang for help with IMCD cell knockdown studies.This work was supported by GM55223(to B.M.D.),and DK080179 (to T.K.).

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蛋白质的生理功能

蛋白质的生理功能 1、构造人的身体：蛋白质是一切生命的物质基础，是肌体细胞的重要组成部分，是人体组织更新和修补的主要原料。人体的每个组织：毛发、皮肤、肌肉、骨骼、内脏、大脑、血液、神经、内分泌等都是由蛋白质组成，所以说饮食造就人本身。蛋白质对人的生长发育非常重要。比如大脑发育的特点是一次性完成细胞增殖，人的大脑细胞的增长有二个高峰期。第一个是胎儿三个月的时候；第二个是出生后到一岁，特别是0---6个月的婴儿是大脑细胞猛烈增长的时期。到一岁大脑细胞增殖基本完成，其数量已达成人的9/10。所以0到1岁儿童对蛋白质的摄入要求很有特色，对儿童的智力发展尤关重要。 2、修补人体组织：人的身体由百兆亿个细胞组成，细胞可以说是生命的最小单位，它们处于永不停息的衰老、死亡、新生的新陈代谢过程中。例如年轻人的表皮28天更新一次，而胃黏膜两三天就要全部更新。所以一个人如果蛋白质的摄入、吸收、利用都很好，那么皮肤就是光泽而又有弹性的。反之，人则经常处于亚健康状态。组织受损后，包括外伤，不能得到及时和高质量的修补，便会加速机体衰退。 3、维持肌体正常的新陈代谢和各类物质在体内的输送。载体蛋白对维持人体的正常生命活动是至关重要的。可以在体内运载各种物质。比如血红蛋白—输送氧（红血球更新速率250万/秒）、脂蛋白—输送脂肪、细胞膜上的受体还有转运蛋白等。 4、白蛋白：维持机体内的渗透压的平衡及体液平衡。 5、维持体液的酸碱平衡。 6、免疫细胞和免疫蛋白：有白细胞、淋巴细胞、巨噬细胞、抗体（免疫球蛋白）、补体、干扰素等。七天更新一次。当蛋白质充足时，这个部队就很强，在需要时，数小时内可以增加100倍。 7、构成人体必需的催化和调节功能的各种酶。我们身体有数千种酶，每一种只能参与一种生化反应。人体细胞里每分钟要进行一百多次生化反应。酶有促进食物的消化、吸收、利用的作用。相应的酶充足，反应就会顺利、快捷的进行，我们就会精力充沛，不易生病。否则，反应就变慢或者被阻断。 8、激素的主要原料。具有调节体内各器官的生理活性。胰岛素是由51个氨基酸分子合成。生长素是由191个氨基酸分子合成。 9、提供热能。蛋白质和健康蛋白质是荷兰科学家格里特在1838年发现的。他观察到有生命的东西离开了蛋白质就不能生存。蛋白质是生物体内一种极重要的高分子有机物，占人体干重的54%。蛋白质主要由氨基酸组成，因氨基酸的组合排列不同而组成各种类型的蛋白质。人体中估计有10万种以上的蛋白质。生命是物质运动的高级形式，这种运动方式是通过蛋白质来实现的，所以蛋白质有极其重要的生物学意义。人体的生长、发育、运动、遗传、繁殖等一切生命活动都离不开蛋白质。生命运动需要蛋白质，也离不开蛋白质。人体内的一些生理活性物质如胺类、神经递质、多肽类激素、抗体、酶、核蛋白以及细胞膜上、血液中起“载体”作用的蛋白都离不开蛋白质，它对调节生理功能，维持新陈代谢起着极其重要的作用。人体运动系统中肌肉的成分以及肌肉在收缩、作功、完成动作过程中的代谢无不与蛋白质有关，离开了蛋白质，体育锻炼就无从谈起。在生物学中，蛋白质被解释为是由氨基酸借肽键联接起来形成的多肽，然后由多肽连接起来形成的物质。通俗易懂些说，它就是构成人体组织器官的支架和主要物质。 蛋白质能供给能量。这不是蛋白质的主要功能，我们不能拿“肉”当“柴”烧。但在能量缺乏时，蛋白质也必须用于产生能量。另外，从食物中摄取的蛋白质，有些不符合人体需要，或者摄取数量过多，也会被氧化分解，释放能量。

蛋白质的主要生理功能和作用

蛋白质的主要生理功能和作用 张世林外语学院日语14.1 学号：201407030120 摘要本文阐述了蛋白质的定义概念、组成特点、结构性质、生理功能以及作用。 关键词历史定义组成特点结构性质功能 正文： 在18世纪，安东尼奥·弗朗索瓦（Antoine Fourcroy）和其他一些研究者发现蛋白质是一类独特的生物分子，他们发现用酸处理一些分子能够使其凝结或絮凝。当时他们注意到的例子有来自蛋清、血液、血清白蛋白、纤维素和小麦面筋里的蛋白质。荷兰化学家格利特·马尔德（Gerhardus Johannes Mulder）对一般的蛋白质进行元素分析发现几乎所有的蛋白质都有相同的实验公式。用“蛋白质”这一名词来描述这类分子是由Mulder的合作者永斯·贝采利乌斯于1838年提出。Mulder随后鉴定出蛋白质的降解产物，并发现其中含有为氨基酸的亮氨酸，并且得到它（非常接近正确值）的分子量为131Da。 对于早期的生物化学家来说，研究蛋白质的困难在于难以纯化大量的蛋白质以用于研究。因此，早期的研究工作集中于能够容易地纯化的蛋白质，如血液、蛋清、各种毒素中的蛋白质以及消化性和代谢酶（获取自屠宰场）。1950年代后期，Armour Hot Dog Co.公司纯化了一公斤纯的牛胰腺中的核糖核酸酶A，并免费提供给全世界科学家使用。

这一构想最早是由威廉·阿斯特伯里于1933年提出。随后，Walter Kauzman在总结自己对变性的研究成果和之前Kaj Linderstrom-Lang的研究工作的基础上，提出了蛋白质折叠是由疏水相互作用所介导的。1949年，弗雷德里克·桑格首次正确地测定了胰岛素的氨基酸序列，并验证了蛋白质是由氨基酸所形成的线性（不具有分叉或其他形式）多聚体。原子分辨率的蛋白质结构首先在1960年代通过X射线晶体学获得解析；到了1980年代，NMR也被应用于蛋白质结构的解析；近年来，冷冻电子显微学被广泛用于对于超大分子复合体的结构进行解析。截至到2008年2月，蛋白质数据库中已存有接近50,000个原子分辨率的蛋白质及其相关复合物的三维结构的坐标。 蛋白质是一种复杂的有机化合物，旧称“朊（ruǎn)”。氨基酸是组成蛋白质的基本单位，氨基酸通过脱水缩合连成肽链。蛋白质是由一条或多条多肽链组成的生物大分子，每一条多肽链有二十至数百个氨基酸残基（-R）不等；各种氨基酸残基按一定的顺序排列。蛋白质的氨基酸序列是由对应基因所编码。除了遗传密码所编码的20种基本氨基酸，在蛋白质中，某些氨基酸残基还可以被翻译后修饰而发生化学结构的变化，从而对蛋白质进行激活或调控。多个蛋白质可以一起，往往是通过结合在一起形成稳定的蛋白质复合物，折叠或螺旋构成一定的空间结构，从而发挥某一特定功能。合成多肽的细胞器是细胞质中

蛋白质对人体的六大作用

蛋白质对人体的六大作用 2008-3-4 13:34:3 在人体中，蛋白质的主要生理作用表现在六个方面： 1)构成和修复身体各种组织细胞的材料 人的神经、肌肉、内脏、血液、骨骼等，甚至包括体外的头皮、指甲都含有蛋白质，这些组织细胞每天都在不断地更新。因此，人体必须每天摄入一定量的蛋白质，作为构成和修复组织的材料。 2)构成酶、激素和抗体 人体的新陈代谢实际上是通过化学反应来实现的，在人体化学反应的过程中，离不开酶的催化作用，如果没有酶，生命活动就无法进行，这些各具特殊功能的酶，均是由蛋白质构成。此外，一些调节生理功能的激素和胰岛素，以及提高肌体抵抗能力儿保护肌体免受致病微生物侵害的抗体，也是以蛋白质为主要原料构成的。 3)维持正常的血浆渗透压，是血浆和组织之间的物质交换保持平衡 如果膳食中长期缺乏蛋白质，血浆蛋白特别是白蛋白的含量就会降低，血液内的水分便会过多地渗入周围组织，造成临床上的营养不良性水肿。 4)供给肌体能量 在正常膳食情况下，肌体可将完成主要功能而剩余的蛋白质，氧化分解转化为能量。不过，从整个肌体而言，蛋白质的这方面功能是微不足道的。 5)维持肌体的酸碱平衡 肌体内组织细胞必须处于合适的酸碱度范围内，才能完成其正常的生理活动。肌体的这种维持酸碱平衡的能力是通过肺、肾脏以及血液缓冲系统来实现的。蛋白质缓冲体系是血液缓冲系统的重要组成部分，因此说蛋白质在维持肌体酸碱平衡方面起着十分重要的作用。 6)运输氧气及营养物质 血红蛋白可以携带氧气到身体的各个部分，供组织细胞代谢使用。体内有许多营养素必须与某种特异的蛋白质结合，将其作为载体才能运转，例如运铁蛋白、钙结合蛋白、视黄醇蛋白等都属于此类。 蛋白质是化学结构复杂的一类有机化合物，是人体的必须营养素。蛋白质的英文是protein，源于希腊文的proteios，是“头等重要”意思，表明蛋白质是生命活动中头等重要物质。蛋白质是细胞组分中含量最为丰富、功能最多的高分子物质，在生命活动过程中起着各种生命功能执行者的作用，几乎没有一种生命活动能离 开蛋白质，多以没有蛋白质就没有生命。 发现历史 人们对蛋白质重要性的认识经历了一个漫长的历程。1742年Beccari将面粉团不断用水洗去淀粉，分离出 麦麸，实际上就是谷蛋白之一。1841年Liebig发表了分析蛋白质的文章。此后于1883年John Kjedahl 发明了一个准确测定氮进而测定蛋白质含量的分析方法，至今仍被广为应用。随后，氨基酸也被发现。1902 年E.Fischer测定了氨基酸的化学结构，还测定了肽键的性质。大约在1927年，J.B.Summer证明了酶是

蛋白质的生理作用.

《食品化学与健康》电子教材 蛋白质的生理作用 一、是人体最重要的组成成分 人体中所有重要组织都有蛋白质参与如神经、肌肉、内脏、血液等都含有蛋白质。蛋白质是构成细胞和组织的“建筑材料”，在人体细胞中的含量仅次于水，占细胞干重的50%以上。一切生物膜，如细胞膜、细胞内各种细胞器的膜，几乎都是由蛋白质和脂类等物质组成。蛋白质是生命活动的重要物质基础。在体内多种重要生理活性物质的成分是蛋白质，蛋白质参与调节生理功能，如构成细胞核的核蛋白能影响细胞功能；促进食物消化、吸收和利用作用的是酶蛋白；维持机体免疫功能作用的是免疫蛋白；具有调节肌肉收缩的功能的是肌球蛋白；具有运送营养素的作用的是血液中的脂蛋白、运铁蛋白、视黄醇结合蛋白质；具有携带、运送氧气功能的是血红蛋白；具有调节渗透压、维持体液平衡的作用(肝癌) 是白蛋白；由蛋白质或蛋白质衍生物构成的某些激素，如垂体激素、甲状腺激素、胰岛素及肾上腺素等等都是机体的重要调节物质。蛋白质能向机体提供能量，大约占总热能的14%，每克蛋白质在体内代谢，能产生4千卡左右的能量。 二、蛋白质的生理作用表现为 1.参与生理活动和劳动做功 心脏跳动、呼吸运动、胃肠蠕动以及日常各种劳动做功等，都离不开肌肉的收缩，而骨肉的收缩又离不开具有骨肉收缩功能的蛋白质。 2.参与氧和二氧化碳的运输 在生命活动中，将氧气供给全身组织，同时将新陈代谢所产生的二氧化碳排出体外的运输工具就是血红蛋白。血红蛋白是红细胞的主要成分，也是红细胞行使其功能的物质基础。 3.参与维持人体的渗透压

血浆中有多种蛋白质，对维持血液的渗透压、维持细胞内外的压力平衡起着重要作用。 4.具有防御功能 血浆中含有的抗体，主要是丙种球蛋白，这是一种具有防御功能的蛋白质。 5.参与调节人体内物质的代谢 在物质代谢中，都需要酶系统的催化或调节，而酶的本质就是蛋白质。在调节代谢过程中，蛋白质以酶和激素的形式出现，发挥了生命活动中“指挥员”的作用。

蛋白质的营养生理作用

“蛋白质”一词，源于希腊字“Proteios”，其意是“最初的”、“第一重要的”；蛋白质是细胞的重要组成成份，在生命过程中起着重要的作用, 涉及动物代谢的大部分与生命攸关的化学反应。不同种类动物都有自己特定的、多种不同的蛋白质。在器官、体液和其它组织中，没有两种蛋白质的生理功能是完全一样的。这些差异是由于组成蛋白质的氨基酸种类、数量和结合方式不同的必然结果。 动物在组织器官的生长和更新过程中，必须从食物中不断获取蛋白质等含氮物质。因此，把食物中的含氮化合物转变为机体蛋白质是一个重要的营养过程。 蛋白质在动物的生命活动中的重要营养作用： (一)蛋白质是构建机体组织细胞的主要原料 动物的肌肉、神经、结缔组织、腺体、精液、皮肤、血液、毛发、角、喙等都以蛋白质为主要成份，起着传导、运输、支持、保护、连接、运动等多种功能。肌肉、肝、脾等组织器官的干物质含蛋白质80%以上。蛋白质也是乳、蛋、毛的主要组成成份。除反刍动物外，食物蛋白质几乎是唯一可用以形成动物体蛋白质的氮来源。 (二)蛋白质是机体内功能物质的主要成份 在动物的生命和代谢活动中起催化作用的酶、某些起调节作用的激素、具有免疫和防御机能的抗体（免疫球蛋白）都是以蛋白质为主要成分。另外，蛋白质对维持体内的渗透压和水分的正常分布，也起着重要的作用。 (三) 蛋白质是组织更新、修补的主要原料 在动物的新陈代谢过程中，组织和器官的蛋白质的更新、损伤组织的修补都需要蛋白质。据同位素测定，全身蛋白质6-7个月可更新一半。 (四)蛋白质可供能和转化为糖、脂肪 在机体能量供应不足时，蛋白质也可分解供能，维持机体的代谢活动。当摄入蛋白质过多或氨基酸不平衡时，多余的部分也可能转化成糖、脂肪或分解产热。正常条件下，鱼等水生动物体内亦有相当数量的蛋白质参与供能作用。 “蛋白质”一词，源于希腊字“Proteios”，其意是“最初的”、“第一重要的”；蛋白质是细胞的重要组成成份，在生命过程中起着重要的作用, 涉及动物代谢的大部分与生命攸关的化学反应。不同种类动物都有自己特定的、多种不同的蛋白质。在器官、体液和其它组织中，没有两种蛋白质的生理功能是完全一样的。这些差异是由于组成蛋白质的氨基酸种类、数量和结合方式不同的必然结果。 动物在组织器官的生长和更新过程中，必须从食物中不断获取蛋白质等含氮物质。因此，把食物中的含氮化合物转变为机体蛋白质是一个重要的营养过程。 蛋白质在动物的生命活动中的重要营养作用： (一)蛋白质是构建机体组织细胞的主要原料 动物的肌肉、神经、结缔组织、腺体、精液、皮肤、血液、毛发、角、喙等都以蛋白质为主要成份，起着传导、运输、支持、保护、连接、运动等多种功能。肌肉、肝、脾等组织器官的干物质含蛋白质80%以上。蛋白质也是乳、蛋、毛的主要组成成份。除反刍动物外，食物蛋白质几乎是唯一可用以形成动物体蛋白质的氮来源。 (二)蛋白质是机体内功能物质的主要成份 在动物的生命和代谢活动中起催化作用的酶、某些起调节作用的激素、具有免疫和防御机能的抗体（免疫球蛋白）都是以蛋白质为主要成分。另外，蛋白质对维持体内的渗透压和水分的正常分布，也起着重要的作用。 (三) 蛋白质是组织更新、修补的主要原料 在动物的新陈代谢过程中，组织和器官的蛋白质的更新、损伤组织的修补都需要蛋白质。据同位素测定，全身蛋白质6-7个月可更新一半。

蛋白质的作用及功能

（1）氨基酸、蛋白质的生理功能 蛋白质是人体必需的主要营养物质。蛋白质的分解产物是氨基酸；氨基酸的重要生理功能之一是作为蛋白质、多肽合成的原料，是蛋白质或多肽的基本组成单位。 蛋白质的生理功能： ①维持组织的生长、更新和修复：膳食中必须提供足够质和量的蛋白质，才能维持组织、细胞的生长、更新和修复。 ②参与多种重要的生理功能：如催化功能、调节功能、运输功能、储存功能、保护功能和维持体液胶体渗透压（如清蛋白）等。 ③氧化供能：体内蛋白质、多肽分解成氨基酸后，产生（17.19kJ/g）能量，成人每日约有18%的能量来自蛋白质。 ④转变为糖类和脂肪。 （2）营养必需氨基酸的概念和种类 体内需要而不能自身合成、或合成量不能满足机体需要，必须由食物供应的氨基酸称为营养必需氨基酸。营养必需氨基酸包括赖氨酸、色氨酸、苯丙氨酸、甲硫氨酸、苏氨酸、亮氨酸、异亮氨酸和缬氨酸。 蛋白质的功能和对人体的作用 人体的所有组织器官都会有蛋白质，蛋白质是生命的物质基础。蛋白质是人体的主要“建筑材料”。婴幼儿靠它形成肌肉、血液、骨骼、神经、毛发等；成年人需要它更新组织，修补损伤、老化的机体。没有蛋白质的供给，人就不可能从3～4千克的新生儿长成50～60千克重的成年人，所以说蛋白质是人体生命得以延续的主要物质基础。它在人体内的功能共有6 个方面： ◎ 结构功能与催化调节功能 蛋白质是构成体内各组织的主要成分，蛋白质在人体内的主要功能是构成组织和修补组织。人的大脑、神经、肌肉、内脏、血液、皮肤乃至指甲、头发等都是以蛋白质为主要成分构成的。人体发育成长后，随着机体内新陈代谢的不断进行，部分蛋白质分解，组织衰老更新以及损伤后的组织修补等都需要不断补充蛋白质。所以，人每天都要补充一定量的蛋白质，以满足身体的正常需要。人体内的化学变化几乎都是在酶的催化下不断进行的。激素对代谢的调节作用也具有重要意义，而酶和激素都直接或间接来自于蛋白质。 ◎ 防御功能与运动功能 机体抵抗力的强弱，取决于抵抗疾病的抗体的多少，抗体的生成与蛋白质有密切关系。近年来被誉为抑制病毒的法宝和抗癌生力军的干扰素，也是一种复合蛋白质（糖和蛋白质结合而成）。肌肉收缩依赖于肌球蛋白和肌动蛋白，有肌肉收缩才有躯体运动、呼吸、消化及血液循环等生理活动。 ◎ 供给热能与运输和存储功能 人体每日需要的能量，主要来自于糖类及脂肪。当蛋白质的量超过人体的需要，或者饮食中的糖类、脂肪供给不足时，蛋白质亦可作为热量的来源。另外，在人体新陈代谢过程中，被更新的组织蛋白亦可氧化产生热能，供给人体的需要。不论是营养素的吸收、运输和储存以及其他物质的运输和储存，都有特殊蛋白质作为载体。如氧和二氧化碳在血液中的运输、脂类的运输、铁的运输和储存都与蛋白质有密切的关系。

蛋白质结构预测在线软件

蛋白质预测分析网址集锦? 物理性质预测：? Compute PI/MW?? ?? SAPS?? 基于组成的蛋白质识别预测? AACompIdent???PROPSEARCH?? 二级结构和折叠类预测? nnpredict?? Predictprotein??? SSPRED?? 特殊结构或结构预测? COILS?? MacStripe?? 与核酸序列一样，蛋白质序列的检索往往是进行相关分析的第一步，由于数据库和网络技校术的发展，蛋白序列的检索是十分方便，将蛋白质序列数据库下载到本地检索和通过国际互联网进行检索均是可行的。? 由NCBI检索蛋白质序列? 可联网到：“”进行检索。? 利用SRS系统从EMBL检索蛋白质序列? 联网到：”，可利用EMBL的SRS系统进行蛋白质序列的检索。? 通过EMAIL进行序列检索?

当网络不是很畅通时或并不急于得到较多数量的蛋白质序列时，可采用EMAIL方式进行序列检索。? 蛋白质基本性质分析? 蛋白质序列的基本性质分析是蛋白质序列分析的基本方面，一般包括蛋白质的氨基酸组成，分子质量，等电点，亲水性，和疏水性、信号肽，跨膜区及结构功能域的分析等到。蛋白质的很多功能特征可直接由分析其序列而获得。例如，疏水性图谱可通知来预测跨膜螺旋。同时，也有很多短片段被细胞用来将目的蛋白质向特定细胞器进行转移的靶标（其中最典型的例子是在羧基端含有KDEL序列特征的蛋白质将被引向内质网。WEB中有很多此类资源用于帮助预测蛋白质的功能。? 疏水性分析? 位于ExPASy的ProtScale程序（?）可被用来计算蛋白质的疏水性图谱。该网站充许用户计算蛋白质的50余种不同属性，并为每一种氨基酸输出相应的分值。输入的数据可为蛋白质序列或SWISSPROT数据库的序列接受号。需要调整的只是计算窗口的大小（n）该参数用于估计每种氨基酸残基的平均显示尺度。? 进行蛋白质的亲/疏水性分析时，也可用一些windows下的软件如，bioedit,dnamana等。? 跨膜区分析? 有多种预测跨膜螺旋的方法，最简单的是直接，观察以20个氨基酸为单位的疏水性氨基酸残基的分布区域，但同时还有多种更加复杂的、精确的算法能够预测跨膜螺旋的具体位置和它们的膜向性。这些技术主要是基于对已知

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?体内蛋白质的代谢状况可用氮平衡描述 氮平衡(nitrogen balance)指每日氮的摄入量与排出量之间的关系。粪便尿液 氮正平衡氮负平衡正常成人 饥饿、严重烧伤、出血、消耗性疾病患者 氮总平衡 儿童、孕妇、恢复期病人

食物蛋白质的营养需求 ?量：蛋白质的生理需要量 ●成人每日蛋白质最低生理需 要量为30g-50g ●我国营养学会推荐成人每日 蛋白质需要量为80g ?质：蛋白质的营养价值 营养必需氨基酸（nutritional essential amino acid）指体内需要而又不能自身合成，必须由食物供给的氨基酸。 通常认为有8种必需氨基酸，分别是亮氨酸、异亮氨酸、苏氨酸、缬氨酸、赖氨酸、甲硫氨酸、苯丙氨酸和色氨酸。现在认为，组氨酸也是一种必需氨基酸。

蛋白质的营养价值（nutrition value ） 蛋白质的营养价值是指食物蛋白质在体内的利用率，取决于必需氨基酸的数量、种类、量质比。 动物性蛋白质所含必需氨基酸的种类和比例与人体需要接近，所以营养 价值较高。 蛋白质的互补作用指营养价值较低的蛋白质混合食用，其必需氨基酸可以互相补充而提高营养价值。 Complementary 鱼和豆腐同吃，提高营养价值

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13第二节 蛋白质的生理功能

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蛋白质功能-结构-相互作用预测网站工具合集

蛋白质组学 蛋白质是生物体的重要组成部分，参与几乎所有生理和细胞代谢过程。此外，与基因组学和转录组学比较，对一个细胞或组织中表达的所有蛋白质，及其修饰和相互作用的大规模研究称为蛋白质组学。 蛋白质组学通常被认为是在基因组学和转录组学之后，生物系统研究的下一步。然而，蛋白质组的研究远比基因组学复杂，这是由于蛋白质内在的复杂特点，如蛋白质各种各样的翻译后修饰所决定的。并且，研究基因组学的技术要比研究蛋白质组学的技术强得多，虽然在蛋白质组学研究中，质谱技术的研究已取得了一些进展。 尽管存在方法上的挑战，蛋白质组学正在迅速发展，并且对癌症的临床诊断和疾病治疗做出了重要贡献。几项研究鉴定出了一些蛋白质在乳腺癌、卵巢癌、前列腺癌和食道癌中表达变化。例如，通过蛋白质组学技术，人们可以在患者血液中明确鉴定出肿瘤标志物。表1列出了更多的蛋白质组学技术用于研究癌症的例子。 另外，高尔基体功能复杂。最新研究表明，它除了参与蛋白加工外，还能参与细胞分化及细胞间信号传导的过程，并在凋亡中扮演重要角色，其功能障碍也许和肿瘤的发生、发展有某种联系。根据人类基因组研究，约1000多种人类高尔基体蛋白质中仅有500~600种得到了鉴定，建立一条关于高尔基体蛋白质组成的技术路线将有助于其功能的深入研究。 蛋白质组学是一种有效的研究方法，特别是随着亚细胞器蛋白质组学技术的迅猛发展，使高尔基体的全面研究变为可能。因此研究人员希望能以胃癌细胞中的高尔基体为研究对象，通过亚细胞器蛋白质组学方法，建立胃癌细胞中高尔基体的蛋白质组方法学。 研究人员采用蔗糖密度梯度的超速离心方法分离纯化高尔基体，双向凝胶电泳（2-DE）分离高尔基体蛋白质，用ImageMaster 2D软件分析所得图谱，基质辅助激光解吸离子化飞行时间质谱（MALDI-TOF MS）鉴定蛋白质点等一系列亚细胞器蛋白质组学方法建立了胃癌细胞内高尔基体的蛋白图谱。 最后，人们根据分离出的纯度较高的高尔基体建立了分辨率和重复性均较好的双向电泳图谱，运用质谱技术鉴定出12个蛋白质，包括蛋白合成相关蛋白、膜融合蛋白、调节蛋白、凋亡相关蛋白、运输蛋白和细胞增殖分化相关蛋白。通过亚细胞器分离纯化、双向电泳的蛋白分离及MALDI-TOF MS蛋白鉴定分析，研究人员首次成功建立了胃癌细胞SGC7901中高尔基体的蛋白质组学技术路线。 3.1 蛋白质功能预测工具 也许生物信息学方法在癌症研究中最常用的就是基因功能预测方法，但是这些数据库只存储了基因组的大约一半基因的功能。为了在微阵列资料基础上完成功能性的富集分析，基因簇的功能注解是非常重要的。近几年生物学家研发了一些基因功能预测的方法，这些方法旨在超越传统的BLAST搜索来预测基因的功能。基因功能预测可以以氨基酸序列、三级结构、与之相互作用的配体、相互作用过程或基因的表达方式为基础。其中最重要的是基于氨基酸序列的分析，因为这种方法适合于微阵列分析的全部基因。 在表3中，前三项列举了三种同源搜索方法。FASTA方法虽然应用还不太广泛，但它要优于BLAST，或者至少相当。FASTA程序是第一个使用的数据库相似性搜索程序。为了达到较高的敏感程度，程序引用取代矩阵实行局部比对以获得最佳搜索。美国弗吉尼亚大学可以提供这项程序的地方版本，当然数据库搜索结果依赖于要搜索的数据库序列。如果最近的序列数据库版本在弗吉尼亚大学不能获得，那么就最好试一下京都大学（Kyoto University）的KEGG站点。PSI-BLAST（位点特异性反复BLAST）是BLAST的转化版本，PSI-BLAST的特色是每次用profile 搜索数据库后再利用搜索的结果重新构建profile，然后用新的profile再次搜索数据库，如此反复直至没有新的结果产生为止。PSI-BLAST先用带空位的BLAST搜索数据库，将获得的序列通过多序列比对来构建第一个profile。PSI-BLAST自然地拓展了BLAST方法，能寻找蛋白质序列中的隐含模式，有研究表明这种方法可以有效地找到很多序列差异较大而结构功能相似的相关蛋白，所以它比BLAST和FASTA有更好的敏感性。PSI-BLAST服务可以

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实验名称：蛋白质结构与功能的生物信息学研究 实验目的：1.掌握运用BLAST工具对指定蛋白质的氨基酸序列同源性搜索的方法。 2.掌握用不同的工具分析蛋白质的氨基酸序列的基本性质 3掌握蛋白质的氨基酸序列进行三维结构的分析 4.熟悉对蛋白质的氨基酸序列所代表蛋白的修饰情况、所参与的 代谢途径、相互作用的蛋白，以及与疾病的相关性的分析。实验方法和流程： 一、同源性搜索 同源性从分子水平讲则是指两个核酸分子的核苷酸序列或两个蛋白质分子的氨基酸序列间的相似程度。BLAST工具能对生物不同蛋白质的氨基酸序列或不同的基因的DNA序列极性比对，并从相应数据库中找到相同或相似序列。对指定的蛋白质的氨基酸序列进行同源性搜索步骤如下： ↓ 登录网址https://www.sodocs.net/doc/5d11989217.html,/blast/ ↓ 输入序列后,运行blast工具 ↓ 序列比对的图形结果显示

序列比对的图形结果：用相似性区段（Hit）覆盖输入序列的范围判断两个序列 的相似性。如果图形中包含低得分的颜色（主要是红色） 区段，表明两序列的并非完全匹配。 ↓ 匹配序列列表及得分

各序列得分 可选择不同的比对工具 备注: Clustal是一款用来对()的软件。可以用来发现特征序列，进行蛋白分类，证明序列间的同源性，帮助预测新序列二级结构与三级结构，确定PCR引物，以及 在分子进化分析方面均有很大帮助。Clustal包括Clustalx和Clustalw(前者是 图形化界面版本后者是命令界面)，是生物信息学常用的多序列比对工具。 该序列的比对结果有100条，按得分降序排列，其中最大得分2373，最小得分 分为1195. ↓ 详细的比对序列的排列情况 第一个匹配 序列 第一个序列的匹配率为100% Score表示打分矩阵计算出来的值，由搜索算法决定的，值越大说明匹配程度

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蛋白质结构与功能的关系 专业：植物学 摘要：蛋白质特定的功能都是由其特定的构象所决定的，各种蛋白质特定的构象又与其一级结构密切相关。天然蛋白质的构象一旦发生变化，必然会影响到它的生物活性。由于蛋白质的构象的变化引起蛋白质功能变化，可能导致蛋白质构象紊乱症，当然也能引起生物体对环境的适应性增强。而分子模拟技术为蛋白质的研究提供了一种崭新的手段。在理论上解决了结构预测和功能分析以及蛋白质工程实施方面所面临的难题。它在蛋白质的结构预测和模建工作中占有举足轻重的地位，实现了生物技术与计算机技术的完美结合。 关键词：蛋白质的结构、功能；折叠/功能关系；蛋白质构象紊乱症；分子模拟技术；同源建模 RNase是由124个氨基酸残基组成的单肽链，分子中 8 个Cys的-SH构成4对二硫键，形成具有一定空间构象的蛋白质分子。在蛋白质变性剂和一些还原剂存在下，酶分子中的二硫键全部被还原，酶的空间结构破坏，肽链完全伸展，酶的催化活性完全丧失。当用透析的方法除去变性剂和巯基乙醇后，发现酶大部分活性恢复，所有的二硫键准确无误地恢复原来状态。若用其他的方法改变分子中二硫键的配对方式，酶完全丧失活性。这个实验表明，蛋白质的一级结构决定它的空间结构，而特定的空间结构是蛋白质具有生物活性的保证。前体与活性蛋白质一级结构的关系，由108个氨基酸残基构成的前胰岛素原，在合成的时候完全没有活性，当切去N-端的24个氨基酸信号肽，形成84个氨基酸的胰岛素原，胰岛素原也没活性，在包装分泌时，A、B链之间的33个氨基酸残基被切除，才形成具有活性的胰岛素。 功能不同的蛋白质总是有着不同的序列；种属来源不同而功能相同的蛋白质的一级结构，可能有某些差异，但与功能相关的结构也总是相同。若一级结构变化，蛋白质的功能可能发生很大的变化。蛋白质特定的功能都是由其特定的构象所决定的，各种蛋白质特定的构象又与其一级结构密切相关。天然蛋白质的构象一旦发生变化，必然会影响到它的生物活性。由于蛋白质的构象的变化引起蛋白质功能变化，可能导致蛋白质构象紊乱症，当然也能引起生物体对环境的适应性增强。 虽然蛋白质结构与生物功能的关系比序列与功能的关系更加紧密，但结构与功能的这种关联亦若隐若现，并不能排除折叠差别悬殊的蛋白质执行相似的功能，折叠相似的蛋白质执行差别悬殊功能的现象的存在。无奈，该领域仍不得不将100多年前Fisher提出的“锁一钥

WB02 蛋白质的生理功能.

公共营养师考试讲义：蛋白质的生理功能 1、构成人体结构：是肌体细胞的重要组成部分，是人体组织更新和修补的主要原料。人体的每个组织：毛发、皮肤、肌肉、骨骼、内脏、大脑、血液、神经、内分泌等都是由蛋白质组成，所以说饮食造就人本身。蛋白质对人的生长发育非常重要。 2、修补人体组织：人的细胞处于永不停息的衰老、死亡、新生的新陈代谢过程中。例如年轻人的表皮28天更新一次，而胃黏膜两三天就要全部更新。所以一个人如果蛋白质的摄入、吸收、利用都很好，那么皮肤就是光泽而又有弹性的。反之，人则经常处于亚健康状态。组织受损后，包括外伤，不能得到及时和高质量的修补，便会加速机体衰退。 胶原蛋白：占身体蛋白质的1/3，生成结缔组织，构成身体骨架。如骨骼、血管、韧带等，决定了皮肤的弹性，保护大脑（在大脑脑细胞中，很大一部分是胶原细胞，并且形成血脑屏障保护大脑） 3、维持肌体正常的新陈代谢和各类物质在体内的输送。 载体蛋白对维持人体的正常生命活动是至关重要的。 可以在体内运载各种物质。比如血红蛋白—输送氧（红血球更新速率250万/秒）、脂蛋白—输送脂肪、细胞膜上的受体还有转运蛋白等。 白蛋白：维持机体内的渗透压的平衡及体液平衡。 免疫细胞和免疫蛋白：有白细胞、淋巴细胞、巨噬细胞、抗体（免疫球蛋白）、补体、干扰素等。七天更新一次。当蛋白质充足时，这个部队就很强，在需要时，数小时内可以增加100倍。 4、构成人体必需的催化和调节功能的各种酶。

5、激素的主要原料，调节体内各器官的生理活性。 6、构成神经递质乙酰胆碱、五羟色氨等。维持神经系统的正常功能--味觉、视觉和记忆。 7、提供热能 每克蛋白质产热16.81kJ（4kcal），占每日总能量的10%—15%。

蛋白质结构与功能的关系

蛋白质结构与功能的关系 （The relationship between protein structure and function） 摘要蛋白质特定的功能都是由其特定的构象所决定的，各种蛋白质特定的构象又与其一级结构密切相关。天然蛋白质的构象一旦发生变化，必然会影响到它的生物活性。由于蛋白质的构象的变化引起蛋白质功能变化，可能导致蛋白质构象紊乱症，当然也能引起生物体对环境的适应性增强！现而今关于蛋白质功能研究还有待发展，一门新兴学科正在发展，血清蛋白组学，生物信息学等！本文仅就蛋白质结构与其功能关系进行粗略阐述。 关键词：蛋白质结构；折叠/功能关系；蛋白质构象紊乱症；分子伴侣 Keywords：protein structure；fold／function relationship；protein conformational disorder；molecular chaperons 虽然蛋白质结构与生物功能的关系比序列与功能的关系更加紧密，但结构与功能的这种关联亦若隐若现，并不能排除折叠差别悬殊的蛋白质执行相似的功能，折叠相似的蛋白质执行差别悬殊功能的现象的存在。无奈，该领域仍不得不将100多年前Fisher提出的“锁一钥匙”模型(“lock—key”model)和50多年前Koshand提出的诱导契合模型(induce fitmodel)作为蛋白质实现功能的理论基础。这2个略显粗糙的模型只是认为蛋白质执行功能的部位局限在结构中的一个或几个小区域内，此类区域通常是蛋白质表面上的凹洞或裂隙。这种凹洞或裂隙被称为“活性部位(active site)”或“别构部位（fallosteric site）”，凹陷部位与配体分子在空间形状和静电上互补。此外，在酶的活性部位中还存在着几个作为催化基团(catalyticgroup)的氨基酸残基。对蛋白质未来的研究应从实验基本数据的归纳和统计入手，从原始的水平上发现蛋白质的潜藏机制【1】。 蛋白质结构与功能关系的研究主要是以力求刻画蛋白质的3D结构的几何学为基础的。蛋白质结构既非规则的几何形，又非完全的无规线团(randomcoil)，而是有序(α一螺旋和β一折叠)与无序(线团或环域loop)的混合体。理解蛋白质3D结构的技巧是将结构简化，只保留某种几何特征或拓扑模式，并将其数字化。探求数字中所蕴含的规律，且根据这一规律将蛋白质进行分类，再将分类的结构与蛋白质的功能进行比较，以检验蛋白质抽象结构的合理性。如果一种对蛋白质结构的简化、比较和分类能与蛋自质的功能有较好地对应关系，那么这就是一种对蛋白质结构的有价值的理解。蛋白质结构中，多种弱力(氢键、范德华力、静电相互作用、疏水相互作用、堆积力等)和可逆的二硫键使多肽链折叠成特定的构象。从某种意义上说，共价键维系了蛋白质的一级结构；主链上的氢键维系了蛋白质的二级结构；而氨基酸侧链的相互作用和二硫桥维系着蛋白质的三级结构。亚基(subunit)内部的侧链相互作用是构象稳定的基础，蛋白质链之间的侧链的相互作用是亚基组装(四级结构)的基础，而蛋白质中侧链与配体基团问的相互作用是蛋白质行使功能的基础。 牛胰核糖核酸酶(RNase)变性和复性的实验是蛋白质结构与功能关系的很好例证。蛋白质空间结构遭到破坏；，可导致蛋白质的理比性质和生物学性质的变化，这就是蛋白质变性。变性的蛋白质，只要其一级结构仍然完好，可在一定条件下恢复其空间结构，随之理化性质和生物学性质也可重现，这被称为复性。RNase是由124个氨基酸残基组成的一条肽链，分子中8个半胱氨酸的巯基构成4对二硫键，进而形成具有一定空间构象的活性蛋白质。天然RNase遇尿素和β巯基乙醇时发生变性，其分子中的氢键和4个二硫键解开，严密的空间结构遭破坏，丧失了生物学活性，但一级结构完整无损。若去除尿素和β巯基乙醇，RNase又可恢复其原有构象和生物学活性。RNase分子中的8个巯基若随机排列成二硫键可有105种方式。有活性的RNase只是其中的一种，复性时之所以选择了自