<p>Throughout their development in the thymus and during their maintenance and the immunological response in the periphery, T cells rely on the regulation of classical apoptotic pathways to promote cell survival or death. Several proteins of the Bcl-2 family have been shown to be critical in thymocyte and T cell survival and consequently, in T cell function. Among these proteins, the antiapoptotic proteins Bcl-2 and Mcl-1 are critical for promoting T cell survival at multiple stages of the T cell "life cycle." While these proteins have been reported to interact with several of the proapoptotic members of the Bcl-2 family, the specific interactions by which Mcl-1 in particular promotes T cell survival in vivo were not well understood. Further, how different stimuli (for example, cytokine signaling and T cell activation) modulate the specific functions of Mcl-1 had also not been thoroughly explored. </p><p>We utilized mouse models to dissect the roles of Mcl-1 at multiple stages of T cell development and function. We utilized conditional knockout and double knockout strategies to build genetic pathways for Mcl-1 activity during thymocyte development and in peripheral T cells under a variety of conditions. In the thymus, the major role of Mcl-1 is to inhibit the activity of proapoptotic Bak because the loss of Bak, but not the loss of Bax or Bim, rescued the survival of Mcl-1-deficient thymocytes at both the double negative and single positive stages. Further, we concluded that this role is not shared with Bcl-2 because overexpression of Bcl-2 did not rescue DN or SP survival. </p><p>In peripheral T cells, the loss of Bak rescued T cell survival in the presence of IL 7, but not during conditions of cytokine withdrawal. Interestingly, the overexpression of Bcl-2 or the loss of Bim partially rescued the survival of T cells during cytokine withdrawal, indicating that Mcl-1 has dual roles in T cells: cytokine-dependent and cytokine independent. Additionally, we found that cytokines of the common gamma chain family have different effects on the activity of Mcl-1 due to the differential regulation of other proteins of the Bcl-2 family, most notably Bim.</p><p>Finally, we utilized a Bcl-2 reporter mouse model to examine the role of Bcl-2 in the establishment of CD8+ T cell memory to infection. Although it is known that Bcl-2 is dynamically regulated in response to activation, the importance of this regulation in the establishment of T cell memory is not yet clear. We show that a subset of effector T cells within a previously defined memory precursor population retained high Bcl-2 expression at the peak of the immune response. Using adoptive transfer of sorted effector T cells, we provide preliminary evidence that the cells with memory potential lie within a strict range of Bcl-2 expression. These studies indicate that the regulation of Bcl 2 is likely critical in establishing T cell memory and provide a platform for the future study of the factors that influence T cell memory.</p> / Dissertation

XIAP and Bcl-xL are critical anti-apoptotic molecules that directly inhibit caspases and block mitochondrial membrane permeabilization, respectively. In addition to preventing apoptosis, both XIAP and Bcl-xL can be generated by cap-independent translation via the utilization of an IRES in the 5'-UTR of their mRNAs. In recent years it has been shown that activation of S6K2 induces the translational upregulation of these two apoptotic regulators. Here I have determined that activation of S6K2 enhances IRES-mediated translation of XIAP and Bcl-xL by inducing the degradation of PDCD4, which I have identified as a novel regulator of XIAP and Bcl-xL IRES elements. Furthermore, I have shown that PDCD4 is a positive modulator of the Apaf-1 IRES element. The concurrent regulation of XIAP, Bcl-xL and Apaf-1 by PDCD4 suggests a model in which the level of PDCD4 expression alters the apoptotic threshold by specifically impacting IRES-mediated translation of the XIAP, Bcl-xL and Apaf-1 mRNAs.

The Bcl-2 family of proteins contains both anti-apoptotic (e.g.Bcl-2, Bcl-xL) and pro-apoptotic (e.g.Bad, Bcl-xS) proteins. The Bcl-xL and Bcl-xS are splice variants, but have different functions during apoptosis. The pro-survival kinase Akt can phosphorylate certain Bcl-2-related proteins, specifically on serine residues, to regulate their function and localization. This is an extension of the work from our laboratorys finding that haloperidol induces PC12 cell death by inducing Bcl-xS which then translocates from cytosol to mitochondria where it facilitates the release of cytochrome c. The toxicity induced by Bcl-xS is reversed by expression of constitutively active Akt. I hypothesized that Akt-mediated post-translational modification may be important for regulating the function of Bcl-xS and Bcl-xL.<p> Three specific serine residues were ultimately chosen for the characterization of Bcl-xS/xL function: Ser62 (inactivation mutant), Ser106 (putative Akt phosphorylation motif), and Ser165 in Bcl-xS (and the corresponding Ser228 in Bcl-xL) (immediately upstream of hydrophobic tail). The individual substitution of all three Serines with Alanines (which precludes phosphorylation at that site) in Bcl-xS did not affect the expression of the protein, but they did induce varying degrees of cytotoxicity in both PC12 and HEK cultures. I focused on the Ser106 substitution mutant given my hypothesis that Akt targeted this site. Overexpression of Bcl-xS(S106A) was toxic in both PC12 and HEK cultures, as expected, and this coincided with the appearance of the Bcl-xS(S106A) protein in the mitochondrial fraction. The release of cytochrome c from PC12 cell mitochondria coincided with the co-immunoprecipitation of the Bcl-xS protein with VDAC (voltage-dependent anion channel), a channel-forming protein that is known to mediate cytochrome c release, and with the initiation of caspase-dependent events. This was not the case in HEK cells, where the mitochondrial VDAC seemed to be diminished and the toxicity was cytochrome c-independent as well as caspase-independent. In addition, I was able to demonstrate that the S106A substituted protein was not able to co-immunoprecipitate with Akt, supporting Ser106 as a potential target for the Akt protein. I then studied the effects of the homologous substitutions in Bcl-xL on cell function. I chose to use treatment with the potent inducer of apoptosis, staurosporine, as a model of cytotoxicity. Again, substituted proteins exerted toxicity, but they did not potentiate the effects of staurosporine, at least not on MTT conversion. I did notice, however, that there was a clear morphological change with certain concentrations of staurosporine, and subsequently demonstrated that the Bcl-xL(S106A) protein potentiated PC12 cell differentiation induced by staurosporine. This protein also co-immunoprecipitated better with Akt, which was unexpected given my results with the Bcl-xS(S106A) protein described above. Perhaps the extra amino acids in Bcl-xL account for this.<p> It is clear that the phosphorylation of Bcl-xS and Bcl-xL proteins is an important means of regulating their function and localization within the cell. These data support the S106 residues in both Bcl-xS and Bcl-xL as novel targets for the pro-survival Akt kinase, and indicate a role for this/these residue(s) in cellular functions as diverse as apoptosis and differentiation.

XIAP and Bcl-xL are critical anti-apoptotic molecules that directly inhibit caspases and block mitochondrial membrane permeabilization, respectively. In addition to preventing apoptosis, both XIAP and Bcl-xL can be generated by cap-independent translation via the utilization of an IRES in the 5'-UTR of their mRNAs. In recent years it has been shown that activation of S6K2 induces the translational upregulation of these two apoptotic regulators. Here I have determined that activation of S6K2 enhances IRES-mediated translation of XIAP and Bcl-xL by inducing the degradation of PDCD4, which I have identified as a novel regulator of XIAP and Bcl-xL IRES elements. Furthermore, I have shown that PDCD4 is a positive modulator of the Apaf-1 IRES element. The concurrent regulation of XIAP, Bcl-xL and Apaf-1 by PDCD4 suggests a model in which the level of PDCD4 expression alters the apoptotic threshold by specifically impacting IRES-mediated translation of the XIAP, Bcl-xL and Apaf-1 mRNAs.

Per a que el desenvolupament i la maduració testicular tinguin lloc d'una manera correcta, es fa necessari que existeixi un equilibri entre els fenomens de proliferació, diferenciació i mort cel.lular. Tot i que desde fa uns anys es coneix la importància de l'apoptosi en l'eliminació de les cèl.lules germinals anòmales durant l'espermatogènesi dels mamifers, incloent l'home, els mecanismes i gens que la regulen han estat poc estudiats. El desenvolupament d'animals transgènics ha permès conèixer la participació d'alguns gens en la meiosi com són Bax, PMS2, CREM, Mhl1, ATM i HSP70-2. El fenotip comú que presenten aquests animals consisteix en aturada espermatogènica durant la primera divisió meiòtica, inducció d'apoptosi en cèl.lules pre-meiòtiques o meiòtiques i aparició de cèl.lules germinals multinucleades, donant lloc a una disminució de la fertilitat. Amb la finalitat d'identificar gens reguladors de l'apoptosi durant l'espermatogènesi en la rata així com l'estudi de la relevància del mecanisme d'apoptosi i de l'expressió dels gens que la regulen en els problemes de fertilitat en els humans, es van posar a punt dos models d'inducció d'apoptosi en les cèl.lules germinals que la actuen mitjançant mecanismes diferents. Un model de deprivació hormonal, mitjançant l'administració de sulfonat d'etil dimetà (EDS), un tòxic de les cèl.lules de Leydig, que provoca la mort d'aquestes cèl.lules, la qual cosa condueix a la desaparició dels andrògens en el testicle, donant lloc a la mort per apoptosi de les cèl.lules germinals. Un model de toxicitat, mitjançant l'administració d'àcid metoxiacètic (MAA), un tòxic dels espermatocits primaris en fase de paquitè, que provoca la seva mort per apoptosi. Dels resultats obtinguts en el present estudi es desprén que, els membres de la familia de Bcl-2 (Bax i Bcl-2) i la Caspasa-3 participen activament en l'apoptosi induïda en els dos models, co-existint en el mateix estadi tubular i presentant un curs temporal similar. El tractament amb MAA indueix canvis en l'expressió del mRNA i les proteïnes AR, ABP i ERb, que es manifesten en forma de diferències en la distribució en funció de l'estadi del cicle cel.lular en el cas de les dues primeres, i en un notable augment d'expressió a nivell de les cèl.lules germinals en el cas de la tercera. Finalment, s'han aïllat dos nous gens que participen en l'apoptosi induïda a les cèl.lules germinals, Rdes i HARP. Rdes és un homòleg d'un grup de desaturases involucrades en processos meiòtics durant l'espermatogènesi, i HARP és un nou membre de la familia de factors de creixement HDGF, alguns dels quals han estat igualment involucrats en processos meiòtics durant l'espermatogènesi. / To reach a correct testicular development and maduration, it is necessary a balance between proliferation, differentiation and cell death. Since severals years ago it is known, that apoptosis is important to eliminate damaged cells during mammal spermatogenesis, including humans. However, the mechanisms and genes that regulate apoptosis during spermatogenesis are poor understood. Transgenic animals have proven to be useful tool for studying meiotic regulation in mammals. Among genes whose targeted disruption causes meiotic arrest in transgenic animals are Bax, PMS2, CREM, Mhl1, ATM and HSP70-2. Most of these animals show a common phenotype that includes, meiotic arrest, apoptotic induction in pre-meiotic or meiotic cells and multinucleated cells. In order to identify genes regulating apoptosis during spermatogenesis in the rat, as well as study of the apoptotic mechanisms involved, we used two different models of induction of apoptosis in rat germ cells, each model acting by different ways. One model of hormonal deprivation, using ethane dimethane sulphonate (EDS) as a toxicant, which induces death of Leydig cells and androgen withdrawal, that leads to apoptotic germ cell. One model of toxicity, using methoxyacetic acid (MAA) as a toxicant, which induces death of pachytene spermatocytes by apoptosis. The results obtained in this study confirm the active participation of the Bcl-2 family members (Bax and Bcl-2) and Caspasse-3 in the apoptosis induced in both models, co-existing in the same stage of the seminiferous tubule and presenting a similar time course. MAA treatment induces changes in the expression of AR and ABP mRNAs and proteins, showed by the differences in the seminiferous tubule stage distribution. MAA treatment also induces an elevation in the mRNA and protein levels of ERb in germ cells. Finally, we have isolated two new genes which participate in the germ cell induced apoptosis, Rdes and HARP. Rdes is a homologous of a desaturases group involved in meiotic processes during spermatogenesis, and HARP is a new member of the HDGF family, some of those have been involved in meiotic processes during spermatogenesis.

The transcription factor NF-κB interacts with many other co-regulator proteins that modulate its binding and transcriptional activity. One of these co-regulators, Pirin, is an iron-dependent metalloprotein that has been shown to enhance the DNA binding of NF-κB homodimers. Here, we characterize the interactions between Pirin and its known NF-κB binding partners and examined the role of Bcl-3, a protein that is required for Pirin’s interaction with p50. In addition, we use site-directed mutagenesis to alter conserved residues within Pirin’s metal binding environment and observed how it affected the DNA binding and conformation of the Pirin-NF-κB complex. These studies show that, while a similar enhancing effect on DNA binding is observed, the interactions of Pirin with different NF-κB members are distinct from each other and could possibly have different physiological purposes.

The Bcl-2 family of proteins contains both anti-apoptotic (e.g.Bcl-2, Bcl-xL) and pro-apoptotic (e.g.Bad, Bcl-xS) proteins. The Bcl-xL and Bcl-xS are splice variants, but have different functions during apoptosis. The pro-survival kinase Akt can phosphorylate certain Bcl-2-related proteins, specifically on serine residues, to regulate their function and localization. This is an extension of the work from our laboratorys finding that haloperidol induces PC12 cell death by inducing Bcl-xS which then translocates from cytosol to mitochondria where it facilitates the release of cytochrome c. The toxicity induced by Bcl-xS is reversed by expression of constitutively active Akt. I hypothesized that Akt-mediated post-translational modification may be important for regulating the function of Bcl-xS and Bcl-xL.<p> Three specific serine residues were ultimately chosen for the characterization of Bcl-xS/xL function: Ser62 (inactivation mutant), Ser106 (putative Akt phosphorylation motif), and Ser165 in Bcl-xS (and the corresponding Ser228 in Bcl-xL) (immediately upstream of hydrophobic tail). The individual substitution of all three Serines with Alanines (which precludes phosphorylation at that site) in Bcl-xS did not affect the expression of the protein, but they did induce varying degrees of cytotoxicity in both PC12 and HEK cultures. I focused on the Ser106 substitution mutant given my hypothesis that Akt targeted this site. Overexpression of Bcl-xS(S106A) was toxic in both PC12 and HEK cultures, as expected, and this coincided with the appearance of the Bcl-xS(S106A) protein in the mitochondrial fraction. The release of cytochrome c from PC12 cell mitochondria coincided with the co-immunoprecipitation of the Bcl-xS protein with VDAC (voltage-dependent anion channel), a channel-forming protein that is known to mediate cytochrome c release, and with the initiation of caspase-dependent events. This was not the case in HEK cells, where the mitochondrial VDAC seemed to be diminished and the toxicity was cytochrome c-independent as well as caspase-independent. In addition, I was able to demonstrate that the S106A substituted protein was not able to co-immunoprecipitate with Akt, supporting Ser106 as a potential target for the Akt protein. I then studied the effects of the homologous substitutions in Bcl-xL on cell function. I chose to use treatment with the potent inducer of apoptosis, staurosporine, as a model of cytotoxicity. Again, substituted proteins exerted toxicity, but they did not potentiate the effects of staurosporine, at least not on MTT conversion. I did notice, however, that there was a clear morphological change with certain concentrations of staurosporine, and subsequently demonstrated that the Bcl-xL(S106A) protein potentiated PC12 cell differentiation induced by staurosporine. This protein also co-immunoprecipitated better with Akt, which was unexpected given my results with the Bcl-xS(S106A) protein described above. Perhaps the extra amino acids in Bcl-xL account for this.<p> It is clear that the phosphorylation of Bcl-xS and Bcl-xL proteins is an important means of regulating their function and localization within the cell. These data support the S106 residues in both Bcl-xS and Bcl-xL as novel targets for the pro-survival Akt kinase, and indicate a role for this/these residue(s) in cellular functions as diverse as apoptosis and differentiation.

Leukemia is a hematological neoplasm with abnormal genetic mutation or chromosomal translocation in the myeloblast or lymphoblast, and characterized by accumulation of immature cells and malfunction of lymphocytes and myeloid-derived cells. The prognosis of treatment depends on genetic mutation, chromosomal aberration, disease progression and age of patients. Currently, bone marrow transplantation is a useful therapeutic strategy, but the success in therapy is limited by the bone marrow of donors and life-threatening events such as immune repulsion. Although chemotherapy improves leukemia treatment, long-term chemotherapy usually leads to the production of drug-resistant cancer cells. Thus, the development of new modality in overcoming drug-resistant should be beneficial for in leukemia therapy. In this thesis, Naja nigricollis toxin £^, piceatannol, caffeine, and Bungarus multicinctus protease inhibitor-like protein 1 (PILP-1) are employed to investigate the molecular mechanisms in regulating apoptosis and invasion of leukemic cell lines K562 and U937. Hopefully, the signaling pathways elicited by these treatments may be aid in identifying new targets in treating leukemia. Toxin £^ inducing cell death is found to evoke p38 MAPK-mediated Bcl-2 down-regulation, which facilitates mitochondria dysfunction, ROS generation and cytiochrome c release. Finally, activation of caspases leads to apoptotic death of toxin £^-treated cells. Piceatannol elicits Ca2+/p38£\ MAPK- mediated c-Jun and ATF-2 phosphorylation, leading to up-regulation of Fas/FasL protein expression and autocrine Fas-mediated death pathway activation. Caffeine treatment down-regulates MMP-2/-9 down-regulation via Ca2+/ROS-mediated inactivation of ERK/c-Fos and activation of p38 MAPK/c-Jun pathway. Consequently, caffeine treatment suppresses invasion of leukemia cells. PILP-1-induced ADAM17 down-regulation suppresses Lyn-mediated Akt phosphorylation, resulting in death of PILP-1-treated leukemia cells. Taken together, the results of the present study elucidate the signaling pathways responsible for apoptosis and invasion of leukemia cells. Moreover, these findings might suggest new targets in developing therapeutic strategy in treating leukemia.

Dissecting the Mechanisms of Direct Activation for Proapoptotic BAK and BAX / Chemistry and Chemical Biology

Programmed cell death by apoptosis is required for normal development and tissue homeostasis. Perturbations of the critical signaling pathways that regulate apoptosis drive a number of pathologic diseases; therefore, a deep understanding of the apoptotic regulatory networks and methods for therapeutically modulating them is highly warranted. As constituents of the intrinsic pathway of apoptosis, pro-apoptotic BCL-2 family proteins respond to internal signals of cell stress to activate cell death through permeabilization of the outer mitochondrial membrane to release cytochrome c and other apoptogenic factors. Anti-apoptotic BCL-2 proteins block apoptosis by forming stable heterodimers with pro-apoptotic BAX and BAK. Specifically, the C-terminal binding groove of BCL-2 sequesters the BCL-2 homology 3 (BH3) death domain of BAX to prevent BAX oligomerization. The N-terminal BH4 domain of BCL-2 is also believed to confer anti-apoptotic activity but the mechanism by which this occurs remains unknown. A direct, inhibitory interaction between the BCL-2 BH4 domain and BAX was established through application of a peptide stapling technology to preserve the &#945;-helical character of the BH4 domain outside the context of full-length BCL-2. Photoaffinity labeling identified a new mode of BH4 domain interaction at the C-terminal face of BAX, revealing an additional layer of apoptotic regulation. Examination of the conformational activation of full-length BAX in a lipid membrane by hydrogen-deuterium exchange mass spectrometry revealed that the BCL-2 BH4 helix blocks the BAX conformational changes triggered by an activating BH3 peptide to preserve the inactive BAX fold. Thus, the mechanistic paradigm for BCL-2 inhibition of BAX has been expanded to allow future opportunities for modulation of apoptosis by mimicking or inhibiting the BH4 motif.