Differential regulation of cell death programs in males and females by Poly (ADP-Ribose) Polymerase-1 and 17 β estradiol

Cell death can be divided into the anti-inflammatory process of apoptosis and the pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP) 1/3 are major mediators. We previously showed that absence or inhibition of PARP-1 protects mice from nephritis, however only the male mice. We therefore hypothesized that there is an inherent difference in the cell death program between the sexes. We show here that in an immune-mediated nephritis model, female mice show increased apoptosis compared to male mice. Treatment of the male mice with estrogens induced apoptosis to levels similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male and female mice are prone to different types of cell death. Our data also suggest that estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore propose that targeting cell death based on sex will lead to tailored and better treatments for each gender.     

Structure and Expression of a Dhurrinase (β-Glucosidase) from Sorghum

Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic β-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) β-glucosidase isozymes. Southern-blot data suggested that β-glu-cosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root—only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-β-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression.     

Spermine Synthase Deficiency Leads to Deafness and a Profound Sensitivity to ��-Difluoromethylornithine

Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the SpmS and Phex genes, were found to be profoundly hearing impaired. This defect was due to alteration in polyamine content due to the absence of spermine synthase, the product of the SpmS gene. It was reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that ubiquitously expresses spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken β-actin promoter. There was an almost complete loss of the endocochlear potential in the Gy mice, which parallels the hearing deficiency, and this was also reversed by the production of spermine from the spermine synthase transgene. Gy mice showed a striking toxic response to treatment with the ornithine decarboxylase inhibitor α-difluoromethylornithine (DFMO). Within 2–3 days of exposure to DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor function resulting in death within 5 days. This effect was due to an inability to maintain normal balance and was also prevented by the transgenic expression of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no effect on balance. The loss of balance in Gy mice treated with DFMO was due to inhibition of polyamine synthesis because it was prevented by administration of putrescine. Our results are consistent with a critical role for polyamines in regulation of Kir channels that maintain the endocochlear potential and emphasize the importance of normal spermidine:spermine ratio in the hearing and balance functions of the inner ear.Polyamines are essential for viability in mammals. Knockouts of the genes for ornithine decarboxylase and S-adenosylmethionine decarboxylase, which are enzymes needed for the synthesis of putrescine, spermidine, and spermine, are lethal at early stages of embryonic development (1, 2). There is convincing evidence that the formation of hypusine in eIF5A, which requires spermidine as a precursor, is essential for eukaryotes (3). However, the function(s) of spermine is not so well established. Yeast mutants with inactivated spermine synthase grow at a normal rate (4). Mammalian cells in culture also grow normally in the presence of inhibitors of spermine synthase (5) or after inactivation of the spermine synthase gene (SpmS) (6–8). Inactivation of both of the genes that were originally described as encoding spermine synthases in plants leads to profound developmental defects (9–11), but recently it was discovered that one of these genes actually encodes a thermospermine synthase, and it appears that the lack of thermospermine may be responsible for these defects (12).In contrast, spermine is clearly required for normal development in mammals. The rare human Snyder-Robinson syndrome is caused by mutations in SpmS located in the X chromosome that drastically reduces the amount of spermine synthase (13, 14). This leads to mental retardation, hypotonia, cerebellar circuitry dysfunction, facial asymmetry, thin habitus, osteoporosis, and kyphoscoliosis. Male mice, which have an X chromosomal deletion that includes SpmS and have no detectable spermine synthase activity, do survive but are only viable on the B6C3H background (15–17). This mouse strain having an X-linked dominant mutation was isolated from a female offspring of an irradiated mouse and was termed gyro (Gy)² based on a circling behavior pattern in affected males (18). Subsequent studies have shown that the Gy mice have a deletion of part of the X chromosome that inactivates both Phex, a gene that regulates phosphate metabolism, and SpmS (16, 19). The lack of SpmS causes a total absence of spermine (6, 7, 15, 16). Such Gy mice suffer from hypophosphatemia, have a greatly reduced size, sterility, and neurological abnormalities, and have a short life span (6, 16, 18). All of these changes except the hypophosphatemia are reversed when spermine synthase activity is restored (20).The original characterization of Gy mice also reported preliminary indications that these mice had hearing defects lacking the Preyer reflex (21, 22). This is of particular interest in the context of polyamine metabolism because a drug, α-difluoromethylornithine (DFMO, Eflornithine), that targets ornithine decarboxylase has been shown to cause occasional hearing loss in some patients (23–26). Although DFMO was ineffective for cancer treatment, it is an extremely promising agent for cancer chemoprevention (27, 28). When combined with sulindac, DFMO treatment produced a substantial reduction in the recurrence of colorectal adenomas in a large clinical trial (27). DFMO is a major drug for the treatment of African sleeping sickness caused by Trypanosoma brucei (29, 30). It is also used as a topically applied cream for treatment of unwanted facial hair in women (31, 32). DFMO is generally well tolerated even at high doses, but reversible hearing loss has been reported in multiple clinical trials (25, 33), and a rarer irreversible defect has also been reported (34). These side effects are not observed at lower doses of DFMO (26, 27).Ototoxicity has been demonstrated to occur in experimental animals treated with DFMO including rats (35), guinea pigs (36), gerbils (37), and mice (38). Using immunohistochemistry, a high level of ornithine decarboxylase was observed in the inner ear of the rat, with the highest in the organ of Corti and lateral wall followed by the cochlear nerve (39). Measurements of polyamines in the relevant structures are very difficult due to the small amount of tissue available, but as expected, DFMO treatment reduced polyamine levels and ornithine decarboxylase activity in the inner ear of the guinea pig (36). A plausible explanation for the importance of polyamines in auditory physiology is based on their well documented role as regulators of potassium channels (38). The inward rectification of Kir channels is caused by blockage of the outward current by polyamines (40–42). Studies of the cloned mouse cochlear lateral wall-specific Kir4.1 channel showed that inward rectification was reduced and that there was a marked reduction in endocochlear potential (EP). It was proposed that DFMO treatment increases the outward Kir4.1 current, resulting in a drop in EP (38).In the experiments reported here, we have studied in more detail the role of polyamines in auditory physiology using Gy mice and crosses of these mice with transgenic CAG/SpmS mice (43). These mice express spermine synthase under the control of a composite cytomegalovirus-IE enhancer/chicken β-actin promoter, which was designed to provide ubiquitous expression (44–46). Assays of the spermine synthase activity in CAG/SpmS line 8 confirmed that there was a high level of expression of the transgene in many different organs and that this level was maintained for at least 1 year (43). Our studies confirm that Gy mice are totally deaf and that this condition is reversed by the expression of the SpmS gene. These changes are due to a virtually complete loss of the EP in the Gy mice. We have also examined the effect of DFMO on the Gy mice. Unexpectedly, it was found that these mice show a rapid and profound toxicity to this drug, leading to death within a few days. Within 5 days of exposure to DFMO in the drinking water, the DFMO-treated mice suffered a catastrophic loss of balance due to inner ear effects. This toxicity was also prevented by the transgenic expression of spermine synthase in the Gy background.     

Mechanism of Activation and Functional Role of Protein Kinase C�� in Human Platelets

The novel class of protein kinase C (nPKC) isoform η is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCη using pharmacological and gene knock-out approaches. nPKCη was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y₁ receptor antagonist, or YM-254890, a G_q blocker, abolished 2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to activate nPKCη in platelets isolated from P2Y₁ and G_q knock-out mice. However, pretreatment of platelets with P2Y₁₂ receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCη phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCη was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin α_IIbβ₃ mediated outside-in signaling. Moreover, in the presence of SC-57101, a α_IIbβ₃ receptor antagonist, nPKCη dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cγ, a catalytic subunit of serine/threonine phosphatase, α_IIbβ₃ failed to dephosphorylate nPKCη. Thus, we conclude that ADP activates nPKCη via P2Y₁ receptor and is subsequently dephosphorylated by PP1γ phosphatase activated by α_IIbβ₃ integrin. In addition, pretreatment of platelets with η-RACK antagonistic peptides, a specific inhibitor of nPKCη, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCη positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.Platelets are the key cellular components in maintaining hemostasis (1). Vascular injury exposes subendothelial collagen that activates platelets to change shape, secrete contents of granules, generate thromboxane, and finally aggregate via activated α_IIbβ₃ integrin, to prevent further bleeding (2, 3). ADP is a physiological agonist of platelets secreted from dense granules and is involved in feedback activation of platelets and hemostatic plug stabilization (4). It activates two distinct G-protein-coupled receptors (GPCRs) on platelets, P2Y₁ and P2Y₁₂, which couple to G_q and G_i, respectively (5–8). G_q activates phospholipase Cβ (PLCβ), which leads to diacyl glycerol (DAG)² generation and calcium mobilization (9, 10). On the other hand, G_i is involved in inhibition of cAMP levels and PI 3-kinase activation (4, 6). Synergistic activation of G_q and G_i proteins leads to the activation of the fibrinogen receptor integrin α_IIbβ₃. Fibrinogen bound to activated integrin α_IIbβ₃ further initiates feed back signaling (outside-in signaling) in platelets that contributes to the formation of a stable platelet plug (11).Protein kinase Cs (PKCs) are serine/threonine kinases known to regulate various platelet functional responses such as dense granule secretion and integrin α_IIbβ₃ activation (12, 13). Based on their structure and cofactor requirements, PKCs are divided in to three classes: classical (cofactors: DAG, Ca²⁺), novel (cofactors: DAG) and atypical (cofactors: PIP₃) PKC isoforms (14). All the members of the novel class of PKC isoforms (nPKC), viz. nPKC isoforms δ, θ, η, and ε, are expressed in platelets (15), and they require DAG for activation. Among all the nPKCs, PKCδ (15, 16) and PKCθ (17–19) are fairly studied in platelets. Whereas nPKCδ is reported to regulate protease-activated receptor (PAR)-mediated dense granule secretion (15, 20), nPKCθ is activated by outside-in signaling and contributes to platelet spreading on fibrinogen (18). On the other hand, the mechanism of activation and functional role of nPKCη is not addressed as yet.PKCs are cytoplasmic enzymes. The enzyme activity of PKCs is modulated via three mechanisms (14, 21): 1) cofactor binding: upon cell stimulus, cytoplasmic PKCs mobilize to membrane, bind cofactors such as DAG, Ca²⁺, or PIP3, release autoinhibition, and attain an active conformation exposing catalytic domain of the enzyme. 2) phosphorylations: 3-phosphoinositide-dependent kinase 1 (PDK1) on the membrane phosphorylates conserved threonine residues on activation loop of catalytic domain; this is followed by autophosphorylations of serine/threonine residues on turn motif and hydrophobic region. These series of phosphorylations maintain an active conformation of the enzyme. 3) RACK binding: PKCs in active conformation bind receptors for activated C kinases (RACKs) and are lead to various subcellular locations to access the substrates (22, 23). Although various leading laboratories have elucidated the activation of PKCs, the mechanism of down-regulation of PKCs is not completely understood.The premise of dynamic cell signaling, which involves protein phosphorylations by kinases and dephosphorylations by phosphatases has gained immense attention over recent years. PP1, PP2A, PP2B, PHLPP are a few of the serine/threonine phosphatases reported to date. Among them PP1 and PP2 phosphatases are known to regulate various platelet functional responses (24, 25). Furthermore, PP1c, is the catalytic unit of PP1 known to constitutively associate with α_IIb and is activated upon integrin engagement with fibrinogen and subsequent outside-in signaling (26). Among various PP1 isoforms, recently PP1γ is shown to positively regulate platelet functional responses (27). Thus, in this study we investigated if the above-mentioned phosphatases are involved in down-regulation of nPKCη. Furthermore, reports from other cell systems suggest that nPKCη regulates ERK/JNK pathways (28). In platelets ERK is known to regulate agonist induced thromboxane generation (29, 30). Thus, we also investigated if nPKCη regulates ERK phosphorylation and thereby agonist-induced platelet functional responses.In this study, we evaluated the activation of nPKCη downstream of ADP receptors and its inactivation by an integrin-associated phosphatase PP1γ. We also studied if nPKCη regulates functional responses in platelets and found that this isoform regulates ADP-induced thromboxane generation, but not fibrinogen receptor activation in platelets.     

Molecular Basis for Class V ��-Tubulin Effects on Microtubule Assembly and Paclitaxel Resistance

Vertebrates produce at least seven distinct β-tubulin isotypes that coassemble into all cellular microtubules. The functional differences among these tubulin isoforms are largely unknown, but recent studies indicate that tubulin composition can affect microtubule properties and cellular microtubule-dependent behavior. One of the isotypes whose incorporation causes the largest change in microtubule assembly is β5-tubulin. Overexpression of this isotype can almost completely destroy the microtubule network, yet it appears to be required in smaller amounts for normal mitotic progression. Moderate levels of overexpression can also confer paclitaxel resistance. Experiments using chimeric constructs and site-directed mutagenesis now indicate that the hypervariable C-terminal region of β5 plays no role in these phenotypes. Instead, we demonstrate that two residues found in β5 (Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and confer paclitaxel resistance when introduced into β1-tubulin; yet the single mutation of residue Ser-239 in β5 eliminates its ability to confer these phenotypes. Despite the high degree of conservation among β-tubulin isotypes, mutations affecting residue 365 demonstrate that amino acid substitutions can be context sensitive; i.e. an amino acid change in one isotype will not necessarily produce the same phenotype when introduced into a different isotype. Modeling studies indicate that residue Cys-239 of β1-tubulin is close to a highly conserved Cys-354 residue suggesting the possibility that disulfide formation could play a significant role in the stability of microtubules formed with β1- but not with β5-tubulin.Microtubules are needed to organize the Golgi apparatus and endoplasmic reticulum, maintain cell shape, construct ciliary and flagellar axonemes, and ensure the accurate segregation of genetic material prior to cell division. These cytoskeletal structures assemble from α- and β-tubulin heterodimers to form long cylindrical filaments that exist in a state of dynamic equilibrium characterized by stochastic episodes of slow growth and rapid shrinkage (1). Impairment of normal dynamic behavior has serious consequences for cell proliferation and thus makes microtubules an attractive target for drug development (2).Vertebrates express multiple β-tubulin genes that produce highly homologous proteins differing most notably in their C-terminal 15–20 amino acids (3, 4). These variable C-terminal sequences are conserved across vertebrate species and have been used to classify β-tubulin genes into distinct isotypes (5). In mammals, for example, there are seven known isotypes designated by the numbers I, II, III, IVa, IVb, V, and VI. The functional significance of the C-terminal sequences is uncertain, but some studies suggest that they may be involved in binding or modulating the action of microtubule-interacting proteins (6–14). Additional amino acid differences are scattered throughout the primary sequence, but the functional role of these differences, if any, has not been elucidated. Although some β-tubulin isotypes are expressed in a tissue-specific manner (3), evidence indicates that microtubules incorporate all available isotypes, including transfected isotypes that are not normally produced in those cells (5, 15–17). Genetic experiments designed to test potential functional differences among the various β-tubulin isotypes have only demonstrated isotype-specific effects on the assembly of specialized microtubule-containing structures such as flagellar axonemes in Drosophila or 15-protofilament microtubules in Caenorhabditis elegans (18, 19). Thus, the consequences, if any, of producing multiple β-tubulin isoforms in vertebrate organisms remain elusive.Our recent work showed that conditional overexpression of isotypes β1, β2, and β4b has no effect on microtubule assembly or drug sensitivity in transfected Chinese hamster ovary (CHO)² cells (20). Similarly, expression of neuronal-specific β4a produced very minor effects on microtubule assembly but was able to increase sensitivity to paclitaxel, most likely through increased binding of the drug (21). On the other hand, high expression of neuronal-specific β3 reduced microtubule assembly, conferred low level resistance to paclitaxel, and inhibited cell growth (22). The most dramatic effects, however, were seen in cells transfected with β5, a minor but widely expressed isotype (23). Even modest overexpression of this isotype reduced microtubule assembly and conferred paclitaxel resistance, whereas high levels of expression (∼50% of total tubulin) caused fragmentation and a near complete loss of the microtubule cytoskeleton (24). Despite the toxicity associated with β5 overexpression, this isotype was recently shown to be required for normal mitotic progression and cell proliferation (25).Because of its importance for cell division, and the extreme phenotype associated with its overexpression, we sought to identify the structural differences between β5-tubulin and its more “normal” homolog, β1. Although there are 40 amino acid differences between the 2 isotypes, we report that most of the unique properties of β5 can be attributed to the presence of serine in place of cysteine at residue 239. This residue faces the colchicine binding pocket and is very close to a highly conserved Cys-354 residue. We propose that Ser-239 found in β5-tubulin may prevent formation of a disulfide bond that normally stabilizes microtubules.     

Pen2 and Presenilin-1 Modulate the Dynamic Equilibrium of Presenilin-1 and Presenilin-2 ��-Secretase Complexes

γ-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic Aβ42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of γ-secretase, invariably increase the Aβ42:Aβ40 ratio. However, the mechanism by which these mutations affect γ-secretase complex formation and cleavage specificity is poorly understood. We show that our in vitro assay system recapitulates the effect of PS1 mutations on the Aβ42:Aβ40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active γ-secretase complexes. Furthermore we reveal that the equilibrium of PS1- and PS2-containing active complexes is dynamic and altered by overexpression of Pen2 or PS1 mutants and that formation of PS2 complexes is positively correlated with increased Aβ42:Aβ40 ratios. These data suggest that perturbations to γ-secretase complex equilibrium can have a profound effect on enzyme activity and that increased PS2 complexes along with mutated PS1 complexes contribute to an increased Aβ42:Aβ40 ratio.β-Amyloid (Aβ)⁵ peptides are believed to play a causative role in Alzheimer disease (AD). Aβ peptides are generated from the processing of the amyloid precursor protein (APP) by two proteases, β-secretase and γ-secretase. Although γ-secretase generates heterogenous Aβ peptides ranging from 37 to 46 amino acids in length, significant work has focused mainly on the Aβ40 and Aβ42 peptides that are the major constituents of amyloid plaques. γ-Secretase is a multisubunit membrane aspartyl protease comprised of at least four known subunits: presenilin (PS), nicastrin (Nct), anterior pharynx-defective (Aph), and presenilin enhancer 2 (Pen2). Presenilin is thought to contain the catalytic core of the complex (1–4), whereas Aph and Nct play critical roles in the assembly, trafficking, and stability of γ-secretase as well as substrate recognition (5, 6). Lastly Pen2 facilitates the endoproteolysis of PS into its N-terminal (NTF) and C-terminal (CTF) fragments thereby yielding a catalytically competent enzyme (5, 7–10). All four proteins (PS, Nct, Aph1, and Pen2) are obligatory for γ-secretase activity in cell and animal models (11, 12). There are two homologs of PS, PS1 and PS2, and three isoforms of Aph1, Aph1aS, Aph1aL, and Aph1b. At least six active γ-secretase complexes have been reported (two presenilins × three Aph1s) (13, 14). The sum of apparent molecular masses of the four proteins (PS1-NTF/CTF ≈ 53 kDa, Nct ≈ 120 kDa, Aph1 ≈ 30 kDa, and Pen2 ≈ 10kDa) is ∼200 kDa. However, active γ-secretase complexes of varying sizes, ranging from 250 to 2000 kDa, have been reported (15–19). Recently a study suggested that the γ-secretase complex contains only one of each subunit (20). Collectively these studies suggest that a four-protein complex around 200–250 kDa may be the minimal functional γ-secretase unit with additional cofactors and/or varying stoichiometry of subunits existing in the high molecular weight γ-secretase complexes. CD147 and TMP21 have been found to be associated with the γ-secretase complex (21, 22); however, their role in the regulation of γ-secretase has been controversial (23, 24).Mutations of PS1 or PS2 are associated with familial early onset AD (FAD), although it is debatable whether these familial PS mutations act as “gain or loss of function” alterations in regard to γ-secretase activity (25–27). Regardless the overall outcome of these mutations is an increased ratio of Aβ42:Aβ40. Clearly these mutations differentially affect γ-secretase activity for the production of Aβ40 and Aβ42. Despite intensive studies of Aβ peptides and γ-secretase, the molecular mechanism controlling the specificity of γ-secretase activity for Aβ40 and Aβ42 production has not been resolved. It has been found that PS1 mutations affect the formation of γ-secretase complexes (28). However, the precise mechanism by which individual subunits alter the dynamics of γ-secretase complex formation and activity is largely unresolved. A better mechanistic understanding of γ-secretase activity associated with FAD mutations has been hindered by the lack of suitable assays and probes that are necessary to recapitulate the effect of these mutations seen in cell models and to characterize the active γ-secretase complex.In our present studies, we have determined the overall effect of Pen2 and PS1 expression on the dynamics of PS1- and PS2-containing complexes and their association with γ-secretase activity. Using newly developed biotinylated small molecular probes and activity assays, we revealed that expression of Pen2 or PS1 FAD mutants markedly shifts the equilibrium of PS1-containing active complexes to that of PS2-containing complexes and results in an overall increase in the Aβ42:Aβ40 ratio in both stable cell lines and animal models. Our studies indicate that perturbations to the equilibrium of active γ-secretase complexes by an individual subunit can greatly affect the activity of the enzyme. Moreover they serve as further evidence that there are multiple and distinct γ-secretase complexes that can exist within the same cells and that their equilibrium is dynamic. Additionally the affinity probes developed here will facilitate further study of the expression and composition of endogenous active γ-secretase from a variety of model systems.     

Engineering Functional Antithrombin Exosites in ��1-Proteinase Inhibitor That Specifically Promote the Inhibition of Factor Xa and Factor IXa

We have previously shown that residues Tyr-253 and Glu-255 in the serpin antithrombin function as exosites to promote the inhibition of factor Xa and factor IXa when the serpin is conformationally activated by heparin. Here we show that functional exosites can be engineered at homologous positions in a P1 Arg variant of the serpin α₁-proteinase inhibitor (α₁PI) that does not require heparin for activation. The combined effect of the two exosites increased the association rate constant for the reactions of α₁PI with factors Xa and IXa 11–14-fold, comparable with their rate-enhancing effects on the reactions of heparin-activated antithrombin with these proteases. The effects of the engineered exosites were specific, α₁PI inhibitor reactions with trypsin and thrombin being unaffected. Mutation of Arg-150 in factor Xa, which interacts with the exosite residues in heparin-activated antithrombin, abrogated the ability of the engineered exosites in α₁PI to promote factor Xa inhibition. Binding studies showed that the exosites enhance the Michaelis complex interaction of α₁PI with S195A factor Xa as they do with the heparin-activated antithrombin interaction. Replacement of the P4-P2 AIP reactive loop residues in the α₁PI exosite variant with a preferred IEG substrate sequence for factor Xa modestly enhanced the reactivity of the exosite mutant inhibitor with factor Xa by ∼2-fold but greatly increased the selectivity of α₁PI for inhibiting factor Xa over thrombin by ∼1000-fold. Together, these results show that a specific and selective inhibitor of factor Xa can be engineered by incorporating factor Xa exosite and reactive site recognition determinants in a serpin.The ubiquitous proteins of the serpin superfamily share a common structure and mostly function as inhibitors of intracellular and extracellular serine and cysteine-type proteases in a vast array of physiologic processes (1, 2). Serpins inhibit their target proteases by a suicide substrate inhibition mechanism in which an exposed reactive loop of the serpin is initially recognized as a substrate by the protease. Subsequent cleavage of the reactive loop by the protease up to the acyl-intermediate stage of proteolysis triggers a massive conformational change in the serpin that kinetically traps the acyl-intermediate (3, 4). Although it is well established that serpins recognize their cognate proteases through a specific reactive loop “bait” sequence, it has more recently become clear that serpin exosites outside the reactive loop provide crucial determinants of protease specificity (5–7). In the case of the blood clotting regulator antithrombin and its target proteases, physiological rates of protease inhibition are only possible with the aid of exosites generated upon activation of the serpin by heparin binding (5). Mutagenesis studies have shown that the antithrombin exosites responsible for promoting the interaction of heparin-activated antithrombin with factor Xa and factor IXa map to two key residues, Tyr-253 and Glu-255, in strand 3 of β-sheet C (8, 9). Parallel mutagenesis studies of factor Xa and factor IXa have shown that the protease residues that interact with the antithrombin exosites reside in the autolysis loop, arginine 150 in this loop being most important (10, 11). The crystal structures of the Michaelis complexes of heparin-activated antithrombin with catalytically inactive S195A variants of thrombin and factor Xa have confirmed that these complexes are stabilized by exosites in antithrombin and in heparin (12–14). In particular, the Michaelis complex with S195A factor Xa revealed that Tyr-253 of antithrombin and Arg-150 of factor Xa comprise a critical protein-protein interaction of the antithrombin exosite, in agreement with mutagenesis studies. Binding studies of antithrombin interactions with S195A proteases have shown that the exosites in heparin-activated antithrombin increase the binding affinity for proteases minimally by ∼1000-fold in the Michaelis complex (15, 16).In this study, we have grafted the two exosites in strand 3 of β-sheet C of antithrombin onto their homologous positions in a P1 Arg variant of α₁-proteinase inhibitor (α₁PI)² and shown that the exosites are functional in promoting α₁PI inhibition of factor Xa and factor IXa. The exosites specifically promote factor Xa and factor IXa inhibition and do not affect the inhibition of trypsin or thrombin. Moreover, mutation of the complementary exosite residue in factor Xa, Arg-150, largely abrogates the rate-enhancing effect of the engineered exosites in α₁PI on factor Xa inhibition. Binding studies show that the exosites function by promoting the binding of α₁PI and factor Xa in the Michaelis complex. Replacing the P4-P2 residues of the P1 Arg α₁PI with an IEG factor Xa recognition sequence modestly enhances the reactivity of the exosite mutant of α₁PI with factor Xa and greatly increases the selectivity of the mutant α₁PI for inhibiting factor Xa over thrombin. These findings demonstrate that a potent and selective inhibitor of factor Xa can be engineered by grafting exosite and reactive site determinants for the protease on a serpin scaffold.     

Src-Mediated Phosphorylation of Dynamin and Cortactin Regulates the “Constitutive” Endocytosis of Transferrin

The mechanisms by which epithelial cells regulate clathrin-mediated endocytosis (CME) of transferrin are poorly defined and generally viewed as a constitutive process that occurs continuously without regulatory constraints. In this study, we demonstrate for the first time that endocytosis of the transferrin receptor is a regulated process that requires activated Src kinase and, subsequently, phosphorylation of two important components of the endocytic machinery, namely, the large GTPase dynamin 2 (Dyn2) and its associated actin-binding protein, cortactin (Cort). To our knowledge these findings are among the first to implicate an Src-mediated endocytic cascade in what was previously presumed to be a nonregulated internalization process.Iron is an essential element for all mammalian organisms that plays essential roles in hemoglobin and myoglobin production (23). Altered iron transport can lead to disease states such as hemochromatosis (23), anemia (5, 23), and neuronal disorders (23). The transferrin receptor (TfR) is an important component of iron regulation in cells. There are two distinct TfRs in humans sharing 45% identity that are homodimeric and bind iron-associated transferrin (Tf) at markedly different affinities (26). While significant attention has been paid toward understanding the basic endocytic machinery that supports the efficient internalization and recycling of the TfR1 and its associated iron-bound ligand, it has been assumed that this transport process is constitutive in nature. This is in direct contrast to the highly regulated internalization pathway used by members of the receptor tyrosine kinase family (RTKs) and the family of G-coupled protein receptors (GPCRs) that utilize phosphorylation and/or ubiquination as signaling modules to regulate internalization.To test if TfR1 internalization might be regulated in a similar fashion, we focused on two essential components of the endocytic machinery: the large GTPase Dyn2 that mediates endocytic vesicle scission (35) and Cort that binds to Dyn2 via an SH3-PRD interaction and has been postulated to regulate actin dynamics to facilitate vesicle invagination and release (36, 40). Both Dyn2 and Cort have shown to be phosphorylated in vivo and in vitro by a variety of kinases (51, 58). Dyn1 interacts with (17) and is phosphorylated by Src in neuronal cells and in other excitable cells in response to activation of GPCRs and epidermal growth factor (EGF) (1, 2). While the Src phosphorylation motifs of dynamin are conserved in the epithelial expressed form of Dyn2, it is unclear if Dyn2 is phosphorylated in response to ligands that induce clathrin-based endocytosis.Cort possesses a series of C-terminal tyrosines that are heavily Src-phosphorylated and implicated in regulating actin remodeling during cell motility (20). In this study, we demonstrate that addition of Tf to cultured epithelial cells results in an internalization of the TfR1 mediated by a Src kinase-dependent phosphoactivation of the Dyn2-Cort-based endocytic machinery. In support of these findings, dominant negative forms of c-Src kinase, when expressed in a hepatocyte-derived cell line (Clone 9), attenuate Tf internalization. Remarkably, cells exposed to Tf showed a 3- to 4-fold increase in Dyn2 and Cort phosphorylation compared to that shown by untreated cells, an increase exceeding that observed in cells treated with EGF. These findings provide new insights into the regulation of what was thought to be a constitutive endocytic process.     

γCOP Is Required for Apical Protein Secretion and Epithelial Morphogenesis in Drosophila melanogaster

Background

There is increasing evidence that tissue-specific modifications of basic cellular functions play an important role in development and disease. To identify the functions of COPI coatomer-mediated membrane trafficking in Drosophila development, we were aiming to create loss-of-function mutations in the γCOP gene, which encodes a subunit of the COPI coatomer complex.

Principal Findings

We found that γCOP is essential for the viability of the Drosophila embryo. In the absence of zygotic γCOP activity, embryos die late in embryogenesis and display pronounced defects in morphogenesis of the embryonic epidermis and of tracheal tubes. The coordinated cell rearrangements and cell shape changes during tracheal tube morphogenesis critically depend on apical secretion of certain proteins. Investigation of tracheal morphogenesis in γCOP loss-of-function mutants revealed that several key proteins required for tracheal morphogenesis are not properly secreted into the apical lumen. As a consequence, γCOP mutants show defects in cell rearrangements during branch elongation, in tube dilation, as well as in tube fusion. We present genetic evidence that a specific subset of the tracheal defects in γCOP mutants is due to the reduced secretion of the Zona Pellucida protein Piopio. Thus, we identified a critical target protein of COPI-dependent secretion in epithelial tube morphogenesis.

Conclusions/Significance

These studies highlight the role of COPI coatomer-mediated vesicle trafficking in both general and tissue-specific secretion in a multicellular organism. Although COPI coatomer is generally required for protein secretion, we show that the phenotypic effect of γCOP mutations is surprisingly specific. Importantly, we attribute a distinct aspect of the γCOP phenotype to the effect on a specific key target protein.     

Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase C�� to Associate with Microtubules and to Recruit Dynein

Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Cι (aPKCι) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCι in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCι were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCι. Because GAPDH binds to the carboxyl terminus of α-tubulin, we characterized the distribution of tyrosinated/detyrosinated α-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated α-tubulin; however, aPKCι was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCι-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters.The small GTPase Rab2 is essential for membrane trafficking in the early secretory pathway and associates with vesicular tubular clusters (VTCs)² located between the endoplasmic reticulum (ER) and the cis-Golgi compartment (1, 2). VTCs are pleomorphic structures that sort anterograde-directed cargo from recycling proteins and trafficking machinery retrieved to the ER (3-6). Rab2 bound to a VTC microdomain stimulates recruitment of soluble factors that results in the release of vesicles containing the recycling protein p53/p58 (7). In that regard, we have previously reported that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical PKC ι (aPKCι) are Rab2 effectors that interact directly with the Rab2 amino terminus and with each other (8, 9). Their interaction requires Src-dependent tyrosine phosphorylation of GAPDH and aPKCι (10). Moreover, GAPDH is a substrate for aPKCι (11). GAPDH catalytic activity is not required for ER to Golgi transport indicating that GAPDH provides a specific function essential for membrane trafficking from VTCs independent of glycolytic function (9). Indeed, phospho-GAPDH influences MT dynamics in the early secretory pathway (11).GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs) (12) and subsequently was shown to interact with the carboxyl terminus of α-tubulin (13). The binding of GAPDH to MTs promotes formation of cross-linked parallel MT arrays or bundles (14, 15). GAPDH has also been reported to possess membrane fusogenic activity, which is inhibited by tubulin (16). Similarly, aPKC associates directly with tubulin and promotes MT stability and MT remodeling at specific intracellular sites (17-21). It may not be coincidental that these two Rab2 effectors influence MT dynamics because recent studies indicate that the cytoskeleton plays a central role in the organization and operation of the secretory pathway (22).MTs are dynamic structures that grow or shrink by the addition or loss of α- and β-tubulin heterodimers from the ends of protofilaments (23). Their assembly and stability is regulated by a variety of proteins traditionally referred to as microtubule-associated proteins (MAPs). In addition to the multiple α/β isoforms that are present in eukaryotes, MTs undergo an assortment of post-translational modifications, including acetylation, glycylation, glutamylation, phosphorylation, palmitoylation, and detyrosination, which further contribute to their biochemical heterogeneity (24, 25). It has been proposed that these tubulin modifications regulate intracellular events by facilitating interaction with MAPs and with other specific effector proteins (24). For example, the reversible addition of tyrosine to the carboxyl terminus of α-tubulin regulates MT interaction with plus-end tracking proteins (+TIPs) containing the cytoskeleton-associated protein glycine-rich (CAP-Gly) motif and with dynein-dynactin (27-29). Additionally, MT motility and cargo transport rely on the cooperation of the motor proteins kinesin and dynein (30). Kinesin is a plus-end directed MT motor, whereas cytoplasmic dynein is a minus-end MT-based motor, and therefore the motors transport vesicular cargo toward the opposite end of a MT track (31).Although MT assembly does not appear to be directly regulated by small GTPases, Rab proteins provide a molecular link for vesicle movement along MTs to the appropriate target (22, 32-34). In this study, the potential interaction of Rab2 with MTs and motor proteins was characterized. We found that Rab2 does not bind directly to preassembled MTs but does associate when both GAPDH and aPKCι are present and bound to MTs. Moreover, the MTs predominantly contained tyrosinated α-tubulin (Tyr-tubulin) suggesting that a dynamic pool of MTs that differentially binds MAPs/effector proteins/motors associates with VTCs in response to Rab2. To that end, we determined that Rab2-promoted dynein/dynactin binding to membranes and that the recruitment required aPKCι.     

Kindlin-1 and -2 Directly Bind the C-terminal Region of �� Integrin Cytoplasmic Tails and Exert Integrin-specific Activation Effects

Integrin activation, the rapid conversion of integrin adhesion receptors from low to high affinity, occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin β subunits. Talin binding to integrin β tails provides one key activation signal, but additional factors are likely to cooperate with talin to regulate integrin activation. The integrin β tail-binding proteins kindlin-2 and kindlin-3 were recently identified as integrin co-activators. Here we report an analysis of kindlin-1 and kindlin-2 interactions with β1 and β3 integrin tails and describe the effect of kindlin expression on integrin activation. We demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin β tails in pulldown binding assays. Our mutational analysis shows that the second conserved NXXY motif (Tyr⁷⁹⁵), a preceding threonine-containing region (Thr⁷⁸⁸ and Thr⁷⁸⁹) of the integrin β1A tail, and a conserved tryptophan in the F3 subdomain of the kindlin FERM domain (kindlin-1 Trp⁶¹² and kindlin-2 Trp⁶¹⁵) are required for direct kindlin-integrin interactions. Similar interactions were observed for integrin β3 tails. Using fluorescence-activated cell sorting we further show that transient expression of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of endogenous α5β1 or stably expressed αIIbβ3 integrins. This inhibition is not dependent on direct kindlin-integrin interactions because mutant kindlins exhibiting impaired integrin binding activity effectively inhibit integrin activation. Consistent with previous reports, we find that when co-expressed with the talin head, kindlin-1 or -2 can activate αIIbβ3. This effect is dependent on an intact integrin-binding site in kindlin. Notably however, even when co-expressed with activating levels of talin head, neither kindlin-1 or -2 can cooperate with talin to activate β1 integrins; instead they strongly inhibit talin-mediated activation. We suggest that kindlins are adaptor proteins that regulate integrin activation, that kindlin expression levels determine their effects, and that kindlins may exert integrin-specific effects.Integrins are a family of αβ heterodimeric transmembrane receptors that mediate cell adhesion to extracellular matrix, cell surface, or soluble protein ligands and modulate a variety of intracellular signaling cascades. A key feature of integrins is their ability to dynamically regulate their affinity for extracellular ligands. In a tightly regulated process generally termed integrin activation, intracellular signals that impinge upon the β subunit cytoplasmic tail induce conformational rearrangements in the integrin extracellular domains, increasing the binding affinity for extracellular ligands (1-3). Ligand-bound integrins then recruit additional signaling, adaptor, and cytoskeletal proteins to the integrin cytoplasmic domains, providing mechanical connections to the actin cytoskeleton and a link to a variety of signal transduction pathways (2-8).Recent years have seen significant advances in our understanding of integrin activation. Notable among these is the identification of the actin- and integrin-binding protein talin as a key integrin activator (1, 9). The 50-kDa talin head contains the principal integrin-binding site, and expression of the talin head is sufficient to activate β1 and β3 integrins (10, 11). The talin head contains a FERM (four point one ezrin radixin moesin) domain. FERM domains consist of trefoil arrangement of three subdomains (F1, F2, and F3). The phosphotyrosine-binding domain-like F3 subdomain of the talin FERM directly binds a conserved NP(I/L)Y motif in integrin β tails, and this interaction is necessary for integrin activation in vitro and in vivo (10, 12-19). However, although abundant evidence supports the importance of talin binding to integrin β tails during integrin activation, differences in sensitivity of integrins to talin activation and submaximal activation by overexpressed talin suggested that other activating factors may cooperate with talin (10, 20). In an attempt to identify and characterize potential co-activators, we investigated the kindlin family of FERM domain-containing proteins.Kindlin family proteins (21) were first characterized in nematodes where the sole Caenorhabditis elegans kindlin, UNC-112, was identified in an embryonic screen for defective motility and shown to be essential for the assembly of proper cell-matrix adhesion structures, where it normally co-localized with β integrin (22-24). UNC-112 is conserved across many species, because the nematode, fly, and human homologs are ∼60% similar (∼41% identical) over their entire length (24). Humans express three known homologs of UNC-112: kindlin-1 (Kindlerin, URP1, and FERMT1), kindlin-2 (Mig2 and mig-2), and kindlin-3 (Mig2B and URP2) (25-27). Kindlin-1 and -2 are most closely related, sharing 60% identity and 74% similarity, whereas kindlin-3 shares 53% identity and 69% similarity to kindlin-1 and 49% identity and 67% similarity to kindlin-2 (28). The kindlin proteins all contain a predicted Pleckstrin homology domain and a FERM domain that is most closely related to the talin FERM domain, particularly within the integrin-binding F3 subdomain (29). Based on this sequence similarity we proposed that kindlin FERM domains may directly bind integrin β tails, and we previously showed that kindlin-1 could be pulled down from cell lysates using recombinant integrin β1 and β3 tails and that kindlin-1 co-localized with integrins in focal adhesions (29). A similar localization was reported for kindlin-2 (26, 30), and recent reports provided clear evidence implicating kindlin-2 and kindlin-3 in regulation of integrin activation (31-33). Here, we have used integrin pulldown assays to demonstrate direct binding of full-length kindlin-1 to the cytoplasmic tails of β1A and β3 integrins and to identify key binding residues within the integrin tails and the kindlin F3 subdomain. We confirm that these interactions are important for recruiting kindlin-1 to focal adhesions and show that, contrary to expectations, overexpressed kindlin-1 or -2 inhibit β1 and β3 integrin activation. Overexpressed kindlin-1 or -2 can, however, cooperate with expressed talin head to activate β3 but not β1 integrins. We therefore provide the first data suggesting that kindlin-1 and -2 effects on integrin activation may show β subunit specificity.