in a comparative study , uchida et al . found that brain extract from alzheimer 's disease ( ad ) patient had an enhanced neurotrophic effect when compared to normal brain extract . this was subsequently attributed to the reduction of a protein originally called , growth inhibitory factor ( gif ) . the latter was first thought to be involved in the formation of the senile plaques , which leads to the enhanced sprouting and the exhaustion of the neurons and , therefore , increases the susceptibility of the neurons to exocitotoxic substances . however , in a followup study , a correlation was found between the reduction of the gif activity and the presence of neurofibrillary tangles and not with the senile plaques . further isolation , purification , and sequencing of the gif revealed that it belonged to the metallothionein ( mt ) protein family , the third isoform isolated and purified in this family , thus the name metallothionein-3 ( mt-3 ) . mts are a class of small ( 6 - 7  kda ) cysteine - rich ( 30% ) proteins binding both essential ( cu and zn ) and nonessential ( cd , ag and hg ) metal ions . while metal binding is very tight , there is a facile metal exchange between mts and other metalloproteins . in mammalian species , four isoforms are currently known , which are designated as mt-1 , 2 , 3 , and 4 . mt-1 and 2 are expressed in almost every organ , particularly high in the kidney and liver and both exhibit similar properties as far as metal - binding characteristics are concerned . mt-3 is expressed mainly in the brain , though some studies have reported finding mt-3 in the kidney and some cancer cells . mt-4 is expressed in certain stratified tissues . since the discovery of mt-1 and mt-2 , there have been numerous publications describing the biochemical characteristics and possible function of these isoforms . although the essential physiological functions remain elusive , mt 's have been shown to be involved in these vital processes : ( 1 ) the modulation of free oxygen radicals and nitric oxide , ( 2 ) apoptosis , and ( 3 ) homeostasis of heavy metals . the binding of metal ions occurs , specifically through cysteine thiolate bonds , in two separate domains . the n - terminal domain , called -domain , binds 3 divalent metals in a me3s9 tetrahedral coordination . the c - terminal domain , called -domain , binds 4 divalent metals in a me4s11 tetrahedral coordination [ 12 , 13 ] . while mt-1 and mt-2 have been shown to be inducible at the transcriptional level by the very heavy metals that they bind , such as cd , cu , and zn , mt-3 is not induced by metal ions . indeed , mt-3 and mt-1 respond differentially to zinc deprivation of cells in culture with only mt-1 being downregulated in these cells . thus , it is reasonable to conclude that mt-3 is not involved in metal ion homeostasis , as both the mt-1 and mt-2 isoforms appears to be [ 4 , 14 ] . studies have shown that the mt-3 -domain was sufficient to exhibit the gif activity [ 1618 ] . however , mt-3 by itself does not exhibit gif activity when added to neuronal cell cultures . , one can conclude that the inhibitory activity is invoked by specific interaction(s ) between mt-3 and other component(s ) in the brain extract . indeed , we and others have previously described some other components in the brain interacting with mt-3 [ 1923 ] . immunoaffinity chromatography is a highly sensitive and selective method for the isolation of target proteins and for the identification of protein - protein interactions . the results from these experiments presented in this current paper represent a significant extension with 7 new proteins positively identified interacting with mt3 , along with the 5 proteins identified in the preliminary mass spectra analysis of the bands in the tryptic digested gel . a recent paper appeared by chung et al . , which showed the release of mt-1 and 2 from astrocytes and uptake in neurons in response to brain injury . the mechanism of the secretion of these two mt isoforms , however , is quite distinct from the mechanism proposed here for mt-3 . in this paper , we present a detailed description of new proteins found interacting with mt-3 , which leads us to propose a new model for the secretion of mt-3 from the astrocytes and uptake in neurons , thus providing the first mechanistic description of its neuroinhibitory activity . whole mouse ( 810 weeks ) brains ( swiss webster , pel - freez ) were used for total protein extraction . the brains were homogenized at a ratio of 2  :  1 ( v / w ) in 10  mm mops , 10  mm nacl , ph 7.0 containing 1  mm pmsf and edta - free protease inhibitor cocktail ( roche ) . the homogenate was then centrifuged at 30000  g , 4c for 30  min . affi - gel hz ( biorad ) was used to immobilize the mt-3 antibody previously purified . the column was then rinsed with 10  mm mops , 150  mm nacl ph 7.0 containing 1  mm pmsf and the protease inhibitor cocktail until no protein was eluted from the column ( see figure 1 ) . the mt-3 protein complex was then chaotropically eluted using 10  mm mops , 3  m nascn , ph 7.0 . the mt-3 protein complex was then concentrated using a spin - filter with mwco of 3  kda and the buffer exchanged to the homogenization buffer . the mt-3 protein complex fraction was then sds - paged using nupage novex 412% bis - tris gel and run for 30  min at 200  v using mes - sds buffer ( invitrogen ) . the gel was then silver - stained using an enhanced silver stain kit ( biorad ) . briefly , bands were excised and destained using 30  mm potassium ferricyanide and 100  mm sodium thiosulphate , washed with milli - q and then with 100% acetonitrile , followed by speedvac for 30  min . trypsin ( sequence - grade modified porcine , promega ) was then added to a final concentration of 12.5  ng/l in ice - cold 50  mm ammonium bicarbonate containing 5  mm cacl2 . the supernatant was removed and the gel slices washed once with 20  mm ammonium bicarbonate for 20  min and then pooled together . the gel slices were washed three times with 30  l of 50% acetonitrile/5% formic acid for 20  min each , and then pooled with the previous supernatant . the collected supernatant was then speedvac to near dryness and subjected to cysteine reduction and alkylation . this was done by rehydration in 10  mm dtt/100  mm nh4hco3 at 56c for 45  min . after cooling the sample to room temperature , alkylation was conducted using freshly made 55  mm iodoacetamide , 100  mm nh4hco3 , for 30  min at room temperature in dark . maldi - tof data were collected on a biflex iii instrument ( bruker daltonics ) . full mass scans were collected , in the positive ion mode between m / z 500 and 3500 , for the peptide mixtures of each sample placed on an anchor chip target . each peak list of measured peptide masses was then used to search the national center of biotechnology information ( ncbi ) sequence database for protein identification using mascot ( http://www.matrixscience.com/ ) . searches were performed allowing modifications that included methionine oxidation and carbamidomethyl addition , as well as up to 1 missed cleavage site and up to 250  ppm mass error . only hits with p < .05 were considered positive hits , where p is the probability that the observed match is a random event . ms - ms experiments were preformed on a linear ion trap ( ltq , thermo electron corp . , san jose , ca ) using nanoscale microcapillary reversed - phase liquid chromatography electrospray ionization tandem mass spectrometry ( lc - ms / ms ) . peptide mixtures were auto sampled on a paradigm ms4 system ( michrom bioresources , inc . , auburn , ca ) . samples were desalted and concentrated on a paradigm platinum peptide nanotrap precolumn ( 0.15  50  mm , michrom bioresources , inc . ) and subsequently on a fused silica microcapillary column ( 75  m i. d. ) packed in - house with magic c18aq reversed - phase material on a flow splitter ( michrom bioresources , inc . ) at a flow rate of approximately 250  nl / min . the samples were subjected to a 60  min ( 1040% acn ) gradient and directly eluted into the microcapillary column set to 2.0  kv . the ltq was operated in the positive - ion mode using data - dependent acquisition methods initiated by a survey ms scan , which was followed by ms / ms ( collision energy of 29% ) on the 4 most abundant ions detected in the survey scan . m / z values selected in the survey scan for ms / ms were excluded for subsequence ms / ms for 30  sec . the signal intensity threshold for an ion to be selected for ms / ms was set to a lower limit of 1000 . mass spectra were analyzed using bioworks sequest analysis software package ( abi , inc . , proteins that contained peptides with sequest cross - correlation scores greater than 2 and contained 2 or more peptides were further examined manually . the analysis of the mt-3 protein complex(es ) with sds - page revealed the presence of about 16 bands ( figure 2 ) . twelve proteins have been confidently identified thus far using maldi - tof and ms / ms . all the proteins reported previously to interact with mt-3 [ 19 , 21 ] were reproducibly detected . new proteins found in the current study include : exocyst complex component 8 ( exo84p ) ,  and -enolase , 14 - 3 - 3 zeta , malate dehydrogenase , fructose - biphosphate aldolase 3 , and atp synthase ( table 1 ) . the low mw protein region ( < 24  kda ) did not yield sufficiently significant scores for protein identification with high confidence . rab3a [ 22 , 23 ] was not identified although a faint band was seen in the gel with a mw of about 20  kda . no significant protein ( p < .05 ) was found for this band when searching the database , however the hit returned was for a gtp - binding protein . to confirm the i d of the newly identified proteins , we conducted esi - ms / ms on the trypsin digest of the bands . the sequence of the peptide was considered a positive hit if the cross - correlation score ( xcorr ) was higher than 2 and the number of matching ions higher than 60% . ms / ms results allowed not only the confirmation of the proteins previously identified by maldi - tof , but also the specific isoform of the proteins interacting with mt-3 ( see supplemental material , tables 2 and 3 available online at doi:10.4061/2011/208634 ) . moreover , the ms / ms allowed for the detection of proteins that were not confidently detected with maldi - tof ( valosin containing protein , hsp70 glucose - regulated protein and protein 8 , pyruvate kinase 3 , tubulin alpha 3 ) each with at least two peptides and an xcorr value higher than 2 . table 1 summarizes the regrouped results of the maldi - tof and ms / ms for all proteins identified and their known function . this information allowed for the classification of the partner proteins into three groups : ( 1 ) proteins that are involved in transport , ( 2 ) chaperoning / scaffolding and regulation , and ( 3 ) glucose metabolism related proteins ( metabolic enzymes ) . a brief description of a representative protein from each group is provided below . exo84p is a component of the exocyst multiprotein complex that plays an important role in targeting and docking the secretory vesicle , containing proteins and lipids , at the plasma membrane . the exocyst multiprotein complex is composed of sec3p , sec5p , sec6p , sec8p , sec10p , sec15p , exo70p and exo84p . the exocyst complex plays an essential role in tethering secretory vesicles to specific domains of the plasma membrane for exocytosis . exo84p plays a key role in the organization and the polarized localization of the exocyst complex . exo84p does interact with sec5p and/or sec10p , proteins that we were not able to identify with confidence in our data . the ral - binding domain of exo84p adopts a plekstrin homology ( ph ) domain fold , which consists of a seven - stranded -sandwich containing two orthogonal antiparallel -sheets and an abutting c - terminal -helix . dihydropyrimidinase - related protein 2 ( drp-2 ) is a protein that is related to dihyropyrimidinase and is usually referred to as collapsing response mediator protein ( crmp2 ) . five isoforms of crmps exists in humans ( crmp1crmp5 ) , which are approximately 70% identical to each other . there is no known enzymatic activity for the drp-2 protein family , although it is 58% identical to the human dihydropyrimidinase that catalyzes the second step in pyrimidine degradation . drp-2 is known to be involved in neuronal differentiation , axonal guidance and neuronal polarity . overexpression of drp-2 induces the growth of numerous axons and it is also involved in the maturation of neurites and preexisting dendrites to axons . however , this binding is inactivated by the phosphorylation of thr514 by glycogen synthase kinase 3 ( gsk-3 ) . 14 - 3 - 3 proteins were first identified in 1967 during a study of the soluble acidic proteins from mammalian brains . they are small proteins ( ~30  kda ) that form both homo- and heterodimers . the importance of these proteins was not recognized until later when it was discovered that these proteins can activate tyrosine , tryptophan hydroxylases and raf and can inhibit protein kinase c . 14 - 3 - 3 proteins bind to specific phosphoserine or phosphothreonine motifs ( rsxpsxp and rxxxpsxp ) in protein targets . 14 - 3 - 3 proteins can alter their target proteins using three ways : ( 1 ) conformational change , ( 2 ) physical occlusion of sequence - specific or structural protein features , and ( 3 ) scaffolding . they can , thereby , prevent the interaction with other proteins , regulate the subcellular distribution of proteins or protect proteins from proteolysis . 14 - 3 - 3 zeta modulates the action of proteins that are involved in the cell cycle , transcriptional control , signal transduction , intracellular trafficking and regulation of ion channels . recently , 14 - 3 - 3 zeta was proposed to be a  sweeper  of misfolded proteins . enolase , also called 2-phospho - d - glycerate hydrolase , is a metal - activated enzyme that catalyzes the dehydration of 2-phospho - d - glycerate to phosphoenolpyruvate ( pyruvate kinase substrate ) . in vertebrates , three tissue - specific isoform are found : nonneuronal enolase (  ) , neuron - specific enolase (  ) and muscle - specific enolase (  ) . enolase exists in a physiological state as a dimer , with the two monomer subunits oriented in an antiparallel manner . the dimerization process depends on the two mg that are bound to the monomers and the divalent metal ion plays a critical role in catalysis . although mg is the primary activator of enolase , the enzyme also binds zinc strongly , which has a lower activity . studies in yeast , vertebrates and mammalian cells have shown that -enolase may have other functions such as thermal tolerance , growth control [ 42 , 43 ] , and hypoxia tolerance . enolase also functions as a cell surface receptor for plasminogen ( pgn ) , which has a neurotrophic - like effect on neurons and enhances the neurite outgrowth of neocortical explants [ 42 , 43 , 45 ] . the cellular localization of -enolase is known to be predominantly cytosolic   and can translocate to the plasma membrane in either the homodimeric (  ) or heterodimeric (  ) form . while the  isoform is mainly detected in cells of neuronal origin , the  isoform is widely distributed among different tissues . the  isoenzyme ( nonneuronal ) is expressed in many tissues , while the  isoenzyme occurs exclusively in muscle . the  and  isoforms are the predominant dimers in the brain , representing the neuron - specific enolases . specifically , the -type enolase subunit is mainly located in neurons while -type subunits are mostly found in glial cells . mt-3 by itself is not toxic nor does it inhibit the growth of neurons . indeed , it maintains the survival of neurons in culture . mt-3 knockout mice show a higher susceptibility to kainic acid induced neuronal injury , while mice overexpressing mt-3 show enhanced resistance . mt-3 is abundantly expressed in astrocytes of normal brain [ 1 , 52 ] . moreover , when added to neuronal cell cultures that are exposed to amyloid- peptides , mt-3 enhanced the resistance of the cells against the amyloid- toxicity . indeed , although mt-3 does not have any signal sequence , a 23-day - old astrocytes cell culture did secrete mt-3 into the media . a recently published results   showed a very important difference in the secretion of mt-1 and 2 being triggered only in the presence of zn and interleukin-1 , neither of these factors have been implicated in mt-3 secretion , which we propose is mediated by one or more of the specific partner protein interactions identified in this study . the subcellular localization of mt-3 in rat brain astrocytes showed an association with organelles involved in the secretion pathway - free ribosomes , rough endoplasmic reticulum , small vesicles , the outer membrane of the mitochondria , plasma membrane , and also around the blood vessels . the localization in neurons was mostly found in the axons , dendrites , the synaptic vesicles , and the postsynaptic densities . it is , therefore , reasonable to hypothesize that mt-3 may well be expressed in the astrocytes . we propose that through its interaction with some of the proteins that we have identified , mt-3 is secreted to the extracellular milieu ( blood vessels ) and then absorbed by the neurons . strengthening this argument is the fact that some of the proteins interacting with mt-3 are in fact involved in the transport process : rab3a , exo84p . indeed , exo84p is a subunit of the exocyst complex , which is important in intracellular trafficking . in metazoa ph domains share little sequence conservation , but all have a common fold , which is electrostatically polarized . they are often involved in targeting proteins to the plasma membrane , with a few displaying strong specificity in lipid binding . ph domains are found in cellular signaling proteins such as serine / threonine kinase , tyrosine kinases , regulators of g - proteins , endocytotic gtpases adaptors , as well as in cytoskeletal associated molecules and in lipid - associated enzymes . rabphilin3a , a downstream effector of rab3a that binds the gtp - bound form of rab3a , also binds 14 - 3 - 3 , another protein found in the mt-3 multiprotein complex . 14 - 3 - 3 has also been shown to interact with sec23 , a gtpase activating protein and a component of the sec23p - sec24p heterodimeric complex of the copii vesicle coat , which is involved in er to golgi transport in s. cerevisiae . the secretion is regulated through the  mitochondrial pathway  and involves mt-3 's interaction with rab3a , exo84p and the exocyst complex , 14 - 3 - 3 zeta , and rabphilin3a ( figure 3 , left panel ) . once mt-3 is secreted , pgn and/or -enolase could facilitate its uptake by the neurons , which have been shown to uptake mt-1/2 . indeed , -enolase is located on the cell surface of a certain population of neurons and has been shown to interact with the microglia - derived pgn , which has a neurotrophic - like effect on neurons . the neuronal mt-3 could then regulate the growth of the neurons through either or both of the following mechanisms : ( i ) considering that mt-3 has a gif activity , one could speculate that mt-3 might inhibit pgn . ( ii ) drp-2 is known to regulate neuronal differentiation , axonal guidance and neuronal polarity . moreover , drp-2 is involved in the maturation of neurites and preexisting dendrites to axons , and its overexpression induces the outgrowth of numerous axons . on the other hand , mt-3 suppresses the neurite extension of neurons in the early period of differentiation and suppresses cell death of more differentiated neurons . based on this , we hypothesize that through its interaction with drp-2 , mt-3 could regulate the growth of the neurons ( figure 3 , right panel ) . the above model conforms to the neuroactivity of mt3 and its reported reduction in ad brain tissue , where the above regulatory interactions would be disrupted . the trigger might well be the oxidative stress or other hallmark pathology associated with the disease : the neurofibrillary tangles or the plaques . most proteins found in the multiprotein complex(es ) with mt3 have in fact been shown to be altered in ad patients . 14 - 3 - 3 zeta is overexpressed in the temporal cortex of patient with cognitive impairment , leading to its implication in the ad neuropathology . glyceraldehyde-3-phosphate dehydrogenase , pyruvate kinase , malate dehydrogenase and 14 - 3 - 3 zeta were found to be significantly oxidized both in rat brains and in cell cultures that were exposed to amyloid-42 [ 59 , 60 ] . -enolase and malate dehydrogenase were found to be downregulated in the tg2576 ad mouse model , whereas pyruvate kinase , -enolase , and drp-2 were upregulated . this latter function seems to depend upon the phosphorylation of drp-2 by gsk-3 [ 31 , 32 , 62 , 63 ] . 14 - 3 - 3 zeta has been proposed to be an effector of tau phosphorylation by gsk-3 and hyperphosphorylated drp-2 has been detected within neurofibrillary tangles from the brains of alzheimer 's disease patients [ 62 , 64 ] . the hyperphosphorylation of drp-2 could inhibit the interaction with mt-3 leading to the alteration of the gif activity of mt-3 and , therefore , to the outgrowth of the neurons . it is noteworthy , to reemphasize the positive correlation found between the gif activity and the neurofibrillary tangles .       to summarize , novel new partner proteins for mt3 the identities of a couple of these associated proteins , 14 - 3 - 3 and exo84p , provide a basis for a biologically relevant model for the processing of mt3 . in view of the size of mt3 the interaction model we envision is a thiol / disulfide interchange model , which could involve one or more of the many cysteines in mt3 . indeed , rab3a , 14 - 3 - 3z , exo84p , -enolase , all have cysteines that could participate in this thiol / disulfide interchange model . in this regard , it is important to note that published work   has established that the beta domain of mt3 binds 4 copper leaving two cysteines free to participate in such a mechanism . this is in contrast to mt1 and 2 , both of which bind either 3 zinc or 6 coppers to the beta domain leaving no free cysteines to participate in such a thiol exchange mechanism . the proposed model , along with rab3a [ 22 , 23 ] , promotes the secretion of mt3 from astrocytes . now in the extracellular milieu , mt-3 is positioned to function consistent with its reported activities : the regulation of neuronal growth and the antagonism of -amyloid . regulation of the growth of neurons could be mediated through two possible mechanisms : ( i ) direct inhibition of the pgn at the surface of neurons , or ( ii ) the uptake of mt3 into neurons facilitated by the interaction with enolase located in the cell surface of the neurons . once taken up by the neurons , mt-3 further studies are in progress to validate this newly proposed functional model for mt3 's role in regulating neuronal growth .
metallothionein 3 ( mt-3 ) , also known as growth inhibitory factor ( gif ) , exhibits a neuroinhibitory activity . our lab and others 
 have previously shown that this biological activity involves interacting protein partners in the brain . 
 however , nothing specific is yet known about which of these interactions is responsible for the gif activity . in this paper 
 , we are reporting upon new proteins found interacting with mt-3 as determined through immunoaffinity chromatography and mass spectrometry . these new partner proteins 
 exo84p , 14 - 3 - 3 zeta ,  and  enolase , aldolase c , malate dehydrogenase , atp synthase , and pyruvate kinase  along with those previously identified have now been classified into three functional groups : transport and signaling , chaperoning and scaffolding , and glycolytic metabolism . when viewed together 
 , these interactions support a proposed model for the regulation of the gif activity of mt-3 .