Contribution of membrane localization to the apoptotic activity of PUMA

The BH3-only protein PUMA plays an important role in the activation of apoptosis in response to p53. In different studies, PUMA has been described to function by either directly activating the pro-apoptotic proteins Bax and Bak, or by neutralizing anti-apoptotic members of the Bcl2 family. We have examined the contribution of regions of PUMA other than the BH3 domain to its localization and function. Although the hydrophobic domain in the C-terminus of PUMA is necessary for efficient mitochondrial localization of PUMA itself, PUMA proteins lacking this region can still induce apoptosis and localize to the mitochondria through binding to Bcl2. Even a nuclear localization signal (NLS)-tagged PUMA protein retains apoptotic activity and can be efficiently relocalized from the nucleus after interaction with ectopically expressed Bcl2, underscoring the efficiency of this interaction. Interestingly, unlike the Bcl2 interaction, the binding of PUMA to Bax is severely compromised by the loss of the C-terminal domain of PUMA. However, since the loss of the C-terminus does not compromise the ability of PUMA to induce cell death, our results indicate that the key apoptotic function of PUMA is through interaction with anti-apoptotic proteins such as Bcl2.     

The product of the UTH1 gene,required for Bax-induced cell death in yeast,is involved in the response to rapamycin

A yeast mutant was isolated that was resistant to Bax-induced cell death. It supports a mutation leading to decreased amounts of the protein Uth1p. A strain in which the UTH1 gene is disrupted also exhibits resistance to Bax expression. The absence of Uth1p does not change the mitochondrial localization of Bax, its insertion in the mitochondrial outer membrane or its cytochrome c-releasing activity. On the other hand, the absence of Uth1p does prevent the appearance of other hallmarks related to Bax expression in yeast, such as oxidation of mitochondrial lipid, production of reactive oxygen species and maintenance of plasma membrane properties after ethanol stress. The absence of Uth1p was also found to induce resistance to rapamycin, a specific inducer of autophagy. This resistance only appears when cells are grown under respiratory conditions, but not under fermentative conditions, suggesting that Uth1p acts in an autophagic pathway involving mitochondria, in accordance with its main localization in the outer mitochondrial membrane. Taken together, these data show that Bax is able to activate a death pathway related to autophagy in yeast, which also exhibits typical hallmarks of apoptosis, revealing a possible dual function of Bax in both types of death. This hypothesis is discussed in the light of observations suggesting a co-regulation of apoptosis and autophagy in mammalian cells.     

The noncatalytic C-terminus of AtPOLK Y-family DNA polymerase affects synthesis fidelity, mismatch extension and translesion replication

Cell survival depends not only on the ability to repair damaged DNA but also on the capability to perform DNA replication on unrepaired or imperfect templates. Crucial to this process are specialized DNA polymerases belonging to the Y family. These enzymes share a similar catalytic fold in their N-terminal region, and most of them have a less-well-conserved C-terminus which is not required for catalytic activity. Although this region is essential for appropriate localization and recruitment in vivo, its precise role during DNA synthesis remains unclear. Here we have compared the catalytic properties of AtPOLK, an Arabidopsis orthologue of mammalian pol kappa, and a truncated version lacking 193 amino acids from its C-terminus. We found that C-terminally truncated AtPOLK is a high-efficiency mutant protein, the DNA-binding capacity of which is not affected but it has higher catalytic efficiency and fidelity than the full-length enzyme. The truncated protein shows increased propensity to extend mispaired primer termini through misalignment and enhanced error-free bypass activity on DNA templates containing 7,8-dihydro-8-oxoGuanine. These results suggest that, in addition to facilitating recruitment to the replication fork, the C-terminus of Y-family DNA polymerases may also play a role in the kinetic control of their enzymatic activity.     

Ku70 suppresses the apoptotic translocation of Bax to mitochondria

Bax induces mitochondrial-dependent cell death signals in mammalian cells. However, the mechanism of how Bax is kept inactive has remained unclear. Yeast-based functional screening of Bax inhibitors from mammalian cDNA libraries identified Ku70 as a new Bax suppressor. Bax-mediated apoptosis was suppressed by overexpression of Ku70 in mammalian cells, but enhanced by downregulation of Ku70. We found that Ku70 interacts with Bax, and that the carboxyl terminus of Ku70 and the amino terminus of Bax are required for this interaction. Bax is known to translocate from the cytosol to mitochondria when cells receive apoptotic stimuli. We found that Ku70 blocks the mitochondrial translocation of Bax. These results suggest that in addition to its previously recognized DNA repair activity in the nucleus, Ku70 has a cytoprotective function in the cytosol that controls the localization of Bax.     

Delayed activation of Bax by DNA damage in embryonic stem cells with knock-in mutations of the Abl nuclear localization signals

The non-receptor tyrosine kinase Abl contains nuclear localization (NLS) and nuclear export signals that drive its nucleo-cytoplasmic shuttling. The nuclear Abl tyrosine kinase is activated by DNA damage through ataxia telangiectasia mutated (ATM). Previous studies have suggested nuclear Abl to have proapoptotic activity. To determine the requirement for Abl nuclear import in DNA damage-induced apoptosis, we took a genetic approach by mutating the three NLS (muNLS) of abl1 in mouse embryonic stem (ES) cells through homologous recombination. Exposure of ES cells to genotoxins caused an ATM-dependent nuclear accumulation of Abl but not Abl muNLS. ES cells expressing Abl muNLS exhibited delayed Bax activation, reduced cytochrome c release and decreased caspase-9 activity in response to DNA damage. These results provide a genetic proof that Abl nuclear entry contributes to DNA damage-induced activation of the intrinsic apoptotic pathway.     

The phosphatidylinositol 3-kinase (PI3K)-Akt pathway suppresses Bax translocation to mitochondria

Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but redistributes to mitochondria in response to apoptotic stimuli, where it induces cytochrome c release. In this study, we show that the phosphatidylinositol 3-OH kinase (PI3K)-Akt pathway plays an important role in the regulation of Bax subcellular localization. We found that LY294002, a PI3K inhibitor, blocked the effects of serum to prevent Bax translocation to mitochondria and that expression of an active form of PI3K suppressed staurosporine-induced Bax translocation, suggesting that PI3K activity is essential for retaining Bax in the cytoplasm. In contrast, both U0126, a MEK inhibitor, and active MEK had little effect on Bax localization. In respect to downstream effectors of PI3K, we found that expression of active Akt, but not serum and glucocorticoid-induced protein kinase (SGK), suppressed staurosporine-induced translocation of Bax, whereas dominant negative Akt moderately promoted Bax translocation. Expression of Akt did not alter the levels of Bax, Bcl-2, Bcl-X(L), or phosphorylated JNK under the conditions used, suggesting that there were alternative mechanisms for Akt in the suppression of Bax translocation. Collectively, these results suggest that the PI3K-Akt pathway inhibits Bax translocation from cytoplasm to mitochondria and have revealed a novel mechanism by which the PI3K-Akt pathway promotes survival.     

An anti-apoptotic viral protein that recruits Bax to mitochondria

The viral mitochondria-localized inhibitor of apoptosis (vMIA), encoded by the UL37 gene of human cytomegalovirus, inhibits apoptosis-associated mitochondrial membrane permeabilization by a mechanism different from that of Bcl-2. Here we show that vMIA induces several changes in Bax that resemble those found in apoptotic cells yet take place in unstimulated, non-apoptotic vMIA-expressing cells. These changes include the constitutive localization of Bax at mitochondria, where it associates tightly with the mitochondrial membrane, forming high molecular weight aggregates that contain vMIA. vMIA recruits Bax to mitochondria but delays relocation of caspase-8-activated truncated Bid-green fluorescent protein (GFP) (t-Bid-GFP) to mitochondria. The ability of vMIA and its deletion mutants to associate with Bax and to induce relocation of Bax to mitochondria correlates with their anti-apoptotic activity and with their ability to suppress mitochondrial membrane permeabilization. Taken together, our data indicate that vMIA blocks apoptosis via its interaction with Bax. vMIA neutralizes Bax by recruiting it to mitochondria and "freezing" its pro-apoptotic activity. These data unravel a novel strategy of subverting an intrinsic pathway of apoptotic signaling.     

The cytostatic function of c-Abl is controlled by multiple nuclear localization signals and requires the p53 and Rb tumor suppressor gene products.

c-Abl is a non-receptor protein-tyrosine kinase lacking a clear physiological role. A clue to its normal function is suggested by overexpression of Abl in fibroblasts, which leads to inhibition of cell growth. This effect requires tyrosine kinase activity and the Abl C-terminus. c-Abl is localized to the cell nucleus, where it can bind DNA, and interacts with the retinoblastoma protein, a potential mediator of the growth-inhibitory effect. Nuclear localization of Abl can be directed by a pentalysine nuclear localization signal in the Abl C-terminus. Here, we have identified two additional basic motifs in the Abl C-terminus, either of which can function independently of the pentalysine signal to localize Abl to the nucleus. Using a quantitative transfection assay, we show that both c-Abl and transforming Abl proteins inhibit entry into S phase and this effect is absolutely dependent on nuclear localization. Further, we demonstrate that the Abl cytostatic effect requires both the Rb and p53 tumor suppressor gene products. These results indicate that Abl inhibits cell proliferation by interacting with central elements of the cell cycle control apparatus in the nucleus, and suggest a direct connection between p53 and Rb in this growth-inhibitory pathway.     

Mono- or double-site phosphorylation distinctly regulates the proapoptotic function of Bax

Bax is the major multidomain proapoptotic molecule that is required for apoptosis. It has been reported that phosphorylation of Bax at serine(S) 163 or S184 activates or inactivates its proapoptotic function, respectively. To uncover the mechanism(s) by which phosphorylation regulates the proapoptotic function of Bax, a series of serine (S)→ alanine/glutamate (A/E) Bax mutants, including S163A, S184A, S163E, S184E, S163E/S184A (EA), S163A/S184E (AE), S163A/S184A (AA) and S163E/S184E (EE), were created to abrogate or mimic, respectively, either single or double-site phosphorylation. The compound Bax mutants (i.e. EA and AE) can flesh out the functional contribution of individual phosphorylation site(s). WT and each of these Bax mutants were overexpressed in Bax(-/-) MEF or lung cancer H157 cells and the proapoptotic activities were compared. Intriguingly, expression of any of Bax mutants containing the mutation S→A at S184 (i.e. S184A, EA or AA) represents more potent proapoptotic activity as compared to WT Bax in association with increased 6A7 epitope conformational change, mitochondrial localization/insertion and prolonged half-life. In contrast, all Bax mutants containing the mutation S→E at S184 (i.e. S184E, AE or EE) have a mobility-shift and fail to insert into mitochondrial membranes with decreased protein stability and less apoptotic activity. Unexpectedly, mutation either S→A or S→E at S163 site does not significantly affect the proapoptotic activity of Bax. These findings indicate that S184 but not S163 is the major phosphorylation site for functional regulation of Bax's activity. Therefore, manipulation of the phosphorylation status of Bax at S184 may represent a novel strategy for cancer treatment.     

Bacillus subtilis SepF Binds to the C-Terminus of FtsZ

Bacterial cell division is mediated by a multi-protein machine known as the "divisome", which assembles at the site of cell division. Formation of the divisome starts with the polymerization of the tubulin-like protein FtsZ into a ring, the Z-ring. Z-ring formation is under tight control to ensure bacteria divide at the right time and place. Several proteins bind to the Z-ring to mediate its membrane association and persistence throughout the division process. A conserved stretch of amino acids at the C-terminus of FtsZ appears to be involved in many interactions with other proteins. Here, we describe a novel pull-down assay to look for binding partners of the FtsZ C-terminus, using a HaloTag affinity tag fused to the C-terminal 69 amino acids of B. subtilis FtsZ. Using lysates of Escherichia coli overexpressing several B. subtilis cell division proteins as prey we show that the FtsZ C-terminus specifically pulls down SepF, but not EzrA or MinC, and that the interaction depends on a conserved 16 amino acid stretch at the extreme C-terminus. In a reverse pull-down SepF binds to full-length FtsZ but not to a FtsZΔC16 truncate or FtsZ with a mutation of a conserved proline in the C-terminus. We show that the FtsZ C-terminus is required for the formation of tubules from FtsZ polymers by SepF rings. An alanine-scan of the conserved 16 amino acid stretch shows that many mutations affect SepF binding. Combined with the observation that SepF also interacts with the C-terminus of E. coli FtsZ, which is not an in vivo binding partner, we propose that the secondary and tertiary structure of the FtsZ C-terminus, rather than specific amino acids, are recognized by SepF.     

PUMA Dissociates Bax and Bcl-X(L) to induce apoptosis in colon cancer cells

PUMA is a BH3-only Bcl-2 family protein that plays an essential role in DNA damage-induced apoptosis. PUMA interacts with anti-apoptotic Bcl-2 and Bcl-X(L) and is dependent on Bax to induce apoptosis. In this study, we investigated how the interactions of PUMA with the antiapoptotic proteins coordinate with Bax to initiate apoptosis in HCT116 colon cancer cells. We found that Bcl-X(L) was most effective among several antiapoptotic proteins in suppressing PUMA-induced apoptosis and PUMA-dependent apoptosis induced by the DNA-damaging agent adriamycin. Mutant Bcl-X(L) that cannot interact with Bax was unable to protect cells from PUMA-mediated apoptosis. Knockdown of Bcl-X(L) by RNA interference significantly enhanced PUMA-mediated apoptosis in HCT116 cells but not in PUMA-knockout cells. Furthermore, Bax was found to be dissociated preferentially from Bcl-X(L) in HCT116 cells but not in the PUMA-knockout cells, in response to PUMA induction and adriamycin treatment. PUMA inhibited the association of Bax and Bcl-X(L) in vitro by directly binding to Bcl-X(L) through its BH3 domain. Finally, we found that wild-type Bax, but not mutant Bax deficient in either multimerization or mitochondrial localization, was able to restore PUMA-induced apoptosis in the BAX-knockout cells. Together, these results indicate that PUMA initiates apoptosis in part by dissociating Bax and Bcl-X(L), thereby promoting Bax multimerization and mitochondrial translocation.     

Purification and characterization of a thermostable carboxypeptidase (carboxypeptidase Taq) from Thermus aquaticus YT-1.

A thermostable carboxypeptidase, which we named carboxypeptidase Taq, was purified from Thermus aquaticus YT-1 and characterized. The molecular weight of the enzyme was estimated to be about 56,000 and 58,000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme has a monomeric structure. The optimum pH of the enzyme was 8.0, and the optimum temperature for the reaction was 80 degrees C. The enzyme activity was dependent on cobalt ion and was inhibited by metal-chelating reagents, indicating that the enzyme is a metalloenzyme. The enzyme had high thermostability independent of cobalt ion; about 90% of its activity remained even after treatment at 80 degrees C for 5 h. The enzyme showed broad substrate specificity, although proline at the C-terminus of peptides was not cleaved. The enzyme released amino acids sequentially from the C-terminus.     

The N-terminus and alpha-5, alpha-6 helices of the pro-apoptotic protein Bax,modulate functional interactions with the anti-apoptotic protein Bcl-x<Subscript>L</Subscript>

Background  

Bcl-2 family proteins are key regulators of mitochondrial integrity and comprise both pro- and anti-apoptotic proteins. Bax a pro-apoptotic member localizes as monomers in the cytosol of healthy cells and accumulates as oligomers in mitochondria of apoptotic cells. The Bcl-2 homology-3 (BH3) domain regulates interactions within the family, but regions other than BH3 are also critical for Bax function. Thus, the N-terminus has been variously implicated in targeting to mitochondria, interactions with BH3-only proteins as well as conformational changes linked to Bax activation. The transmembrane (TM) domains (α5-α6 helices in the core and α9 helix in the C-terminus) in Bax are implicated in localization to mitochondria and triggering cytotoxicity. Here we have investigated N-terminus modulation of TM function in the context of regulation by the anti-apoptotic protein Bcl-x_L.     

Characterization of the interaction between the plasma membrane H-ATPase of Arabidopsis thaliana and a novel interactor (PPI1)

Proton pump interactor, isoform 1 (PPI1) is a novel interactor of the C-terminus of Arabidopsis thaliana plasma membrane H(+)-ATPase (EC 3.6.3.6). We produced two fusion proteins consisting of, respectively, the first 88 amino acids or the entire protein deleted of the last 24 hydrophobic amino acids, and we show that the latter protein has a threefold higher affinity for the H(+)-ATPase. PPI1-induced stimulation of H(+)-ATPase activity dramatically decreased with the increase of pH above pH 6.8, but became largely pH-independent when the enzyme C-terminus was displaced by fusicoccin-induced binding of 14-3-3 proteins. The latter treatment did not affect PPI1 affinity for the H(+)-ATPase. These results indicate that PPI1 can bind the H(+)-ATPase independently of the C-terminus conformation, but is not able to suppress the C-terminus auto-inhibitory action.     

Characterization of RanBPM Molecular Determinants that Control Its Subcellular Localization

RanBPM/RanBP9 is a ubiquitous, nucleocytoplasmic protein that is part of an evolutionary conserved E3 ubiquitin ligase complex whose function and targets in mammals are still unknown. RanBPM itself has been implicated in various cellular processes that involve both nuclear and cytoplasmic functions. However, to date, little is known about how RanBPM subcellular localization is regulated. We have conducted a systematic analysis of RanBPM regions that control its subcellular localization using RanBPM shRNA cells to examine ectopic RanBPM mutant subcellular localization without interference from the endogenously expressed protein. We show that several domains and motifs regulate RanBPM nuclear and cytoplasmic localization. In particular, RanBPM comprises two motifs that can confer nuclear localization, one proline/glutamine-rich motif in the extreme N-terminus which has a dominant effect on RanBPM localization, and a second motif in the C-terminus which minimally contributes to RanBPM nuclear targeting. We also identified a nuclear export signal (NES) which mutation prevented RanBPM accumulation in the cytoplasm. Likewise, deletion of the central RanBPM conserved domains (SPRY and LisH/CTLH) resulted in the relocalization of RanBPM to the nucleus, suggesting that RanBPM cytoplasmic localization is also conferred by protein-protein interactions that promote its cytoplasmic retention. Indeed we found that in the cytoplasm, RanBPM partially colocalizes with microtubules and associates with α-tubulin. Finally, in the nucleus, a significant fraction of RanBPM is associated with chromatin. Altogether, these analyses reveal that RanBPM subcellular localization results from the combined effects of several elements that either confer direct transport through the nucleocytoplasmic transport machinery or regulate it indirectly, likely through interactions with other proteins and by intramolecular folding.     

The N-terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function

Due to unique features, proline residues may control protein structure and function. Here, we investigated the role of ⁵²PPQ⁵⁴ residues, indicated by the recently established experimental 3D structure of bovine herpesvirus 1-encoded UL49.5 protein as forming a characteristic proline hinge motif in its N-terminal domain. UL49.5 acts as a potent inhibitor of the transporter associated with antigen processing (TAP), which alters the antiviral immune response. Mechanisms employed by UL49.5 to affect TAP remain undetermined on a molecular level. We found that mutations in the ⁵²PPQ⁵⁴ region had a vast impact on its immunomodulatory function, increasing cell surface MHC class I expression, TAP levels, and peptide transport efficiency. This inhibitory effect was specific for UL49.5 activity towards TAP but not towards the viral glycoprotein M. To get an insight into the impact of proline hinge modifications on structure and dynamics, we performed all-atom and coarse-grained molecular dynamics studies on the native protein and PPQ mutants. The results demonstrated that the proline hinge sequence with its highly rigid conformation served as an anchor into the membrane. This anchor was responsible for the structural and dynamical behavior of the whole protein, constraining the mobility of the C-terminus, increasing the mobility of the transmembrane region, and controlling the accessibility of the C-terminal residues to the cytoplasmic environment. Those features appear crucial for TAP binding and inhibition. Our findings significantly advance the structural understanding of the UL49.5 protein and its functional regions and support the importance of proline motifs for the protein structure.     

Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells

G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3′-5′-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity.     

Nonspecific inhibition of proline dehydrogenase synthesis in Escherichia coli during osmotic stress

L-Proline, which is accumulated by Escherichia coli during growth in media of high osmolality, also induces the synthesis of the enzyme degrading it to glutamate. To determine if proline catabolism is inhibited during osmotic stress, proline utilization and the formation of proline dehydrogenase were examined in varying concentrations of NaCl and sucrose. Although the specific growth rate of E. coli with proline as the sole nitrogen source diminished as the solute osmolality increased, a comparable reduction in growth rate occurred with ammonium as the primary nitrogen source. Proline catabolism, as measured in whole cells by the conversion of [14C]proline to [14C]glutamate, was only slightly inhibited by solute osmolalities up to 1.0 osmol/kg; more than 50% of the initial activity was still found at 2.0 osmol/kg. By contrast, the specific activity of proline dehydrogenase in bacteria grown in the presence of added solutes decreased to less than 20% of the control level. This reduction was related to a lower rate of synthesis, but was independent of genes currently known to be involved in osmoregulation or proline metabolism. The specific activities of tryptophanase, beta-galactosidase, and histidinol dehydrogenase were also reduced under similar growth conditions. These results indicate that while proline catabolism is not directly inhibited by high solute concentrations, prolonged exposure to osmotic stress leads to its reduction as part of a more general metabolic response.