A Reaction-Diffusion Model of ROS-Induced ROS Release in a Mitochondrial Network

Loss of mitochondrial function is a fundamental determinant of cell injury and death. In heart cells under metabolic stress, we have previously described how the abrupt collapse or oscillation of the mitochondrial energy state is synchronized across the mitochondrial network by local interactions dependent upon reactive oxygen species (ROS). Here, we develop a mathematical model of ROS-induced ROS release (RIRR) based on reaction-diffusion (RD-RIRR) in one- and two-dimensional mitochondrial networks. The nodes of the RD-RIRR network are comprised of models of individual mitochondria that include a mechanism of ROS-dependent oscillation based on the interplay between ROS production, transport, and scavenging; and incorporating the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and Ca²⁺ handling. Local mitochondrial interaction is mediated by superoxide (O₂ ^.−) diffusion and the O₂ ^.−-dependent activation of an inner membrane anion channel (IMAC). In a 2D network composed of 500 mitochondria, model simulations reveal ΔΨ_m depolarization waves similar to those observed when isolated guinea pig cardiomyocytes are subjected to a localized laser-flash or antioxidant depletion. The sensitivity of the propagation rate of the depolarization wave to O₂^.− diffusion, production, and scavenging in the reaction-diffusion model is similar to that observed experimentally. In addition, we present novel experimental evidence, obtained in permeabilized cardiomyocytes, confirming that ΔΨ_m depolarization is mediated specifically by O₂ ^.−. The present work demonstrates that the observed emergent macroscopic properties of the mitochondrial network can be reproduced in a reaction-diffusion model of RIRR. Moreover, the findings have uncovered a novel aspect of the synchronization mechanism, which is that clusters of mitochondria that are oscillating can entrain mitochondria that would otherwise display stable dynamics. The work identifies the fundamental mechanisms leading from the failure of individual organelles to the whole cell, thus it has important implications for understanding cell death during the progression of heart disease.     

Pin1 modulates p63α protein stability in regulation of cell survival,proliferation and tumor formation

The homolog of p53 gene, p63, encodes multiple p63 protein isoforms. TAp63 proteins contain an N-terminal transactivation domain similar to that of p53 and function as tumor suppressors; whereas ΔNp63 isoforms, which lack the intact N-terminal transactivation domain, are associated with human tumorigenesis. Accumulating evidence demonstrating the important roles of p63 in development and cancer development, the regulation of p63 proteins, however, is not fully understood. In this study, we show that peptidyl-prolyl isomerase Pin1 directly binds to and stabilizes TAp63α and ΔNp63α via inhibiting the proteasomal degradation mediated by E3 ligase WWP1. We further show that Pin1 specifically interacts with T₅₃₈P which is adjacent to the P₅₅₀PxY₅₄₃ motif, and disrupts p63α–WWP1 interaction. In addition, while Pin1 enhances TAp63α-mediated apoptosis, it promotes ΔNp63α-induced cell proliferation. Furthermore, knockdown of Pin1 in FaDu cells inhibits tumor formation in nude mice, which is rescued by simultaneous knockdown of WWP1 or ectopic expression of ΔNp63α. Moreover, overexpression of Pin1 correlates with increased expression of ΔNp63α in human oral squamous cell carcinoma samples. Together, these results suggest that Pin1-mediated modulation of ΔNp63α may have a causative role in tumorigenesis.     

Efflux and Influx of Erythrocyte Water

Rabbit erythrocytes were washed in buffered NaCl solutions isotonic with rabbit serum (Δ^t -0.558°C.) and suspended in buffered NaCl solutions of tonicity equidistant from intracellular tonicity (Δ^t = -0.558°C. ± 0.112°C.) of varying pH and incubated at varying temperatures. After incubation, the freezing point depression (Δ^t) was measured on the supernatant. Change in the Δ^t measured change in the water content of the extracellular solutions—water being withdrawn by erythrocytes (W_I) from the hypotonic solutions and added (W_E) to the hypertonic solutions. W_E was always less than W_I and was inversely proportional to the pH in the range 6.5–8.0. W_E was significantly increased by lowering the temperature of the cell suspension to 4°C. W_I was increased by raising or lowering the pH or raising the temperature of the cell suspension. W_E x W_I ≠ k. W_E and W_I were affected differently by changes in pH and temperature. It was concluded that W_E and W_E were probably under different physicochemical control.     

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides: I. EQUILIBRIUM AND MODELING STUDIES*

We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c₁ in detergent-solubilized bc₁ complex from Rhodobacter sphaeroides. Imidazole binding to cyt c₁ substantially lowers the midpoint potential of the heme and fully inhibits bc₁ complex activity. Temperature dependences showed that binding of Im (K_d ≈ 330 μm, 25 °C, pH 8) is enthalpically driven (ΔH⁰ = −56 kJ/mol, ΔS⁰ = −121 J/mol/K), whereas binding of MeIm is 30 times weaker (K_d ≈ 9.3 mm) and is entropically driven (ΔH⁰ = 47 kJ/mol, ΔS⁰° = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c₁ triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c₂ suggested that Im binding to cyt c₁ is assisted by formation of hydrogen bonds within the heme cleft. This was strongly supported by molecular dynamics simulations of Im adducts of cyts c, c₂, and c₁, which showed hydrogen bonds formed between the N_δH of Im and the cyt c₁ protein, or with a water molecule sequestered with the ligand in the heme cleft.     

Mitogen-activated protein kinase Hog1 mediates adaptation to G1 checkpoint arrest during arsenite and hyperosmotic stress

Cells slow down cell cycle progression in order to adapt to unfavorable stress conditions. Yeast (Saccharomyces cerevisiae) responds to osmotic stress by triggering G₁ and G₂ checkpoint delays that are dependent on the mitogen-activated protein kinase (MAPK) Hog1. The high-osmolarity glycerol (HOG) pathway is also activated by arsenite, and the hog1Δ mutant is highly sensitive to arsenite, partly due to increased arsenite influx into hog1Δ cells. Yeast cell cycle regulation in response to arsenite and the role of Hog1 in this process have not yet been analyzed. Here, we found that long-term exposure to arsenite led to transient G₁ and G₂ delays in wild-type cells, whereas cells that lack the HOG1 gene or are defective in Hog1 kinase activity displayed persistent G₁ cell cycle arrest. Elevated levels of intracellular arsenite and “cross talk” between the HOG and pheromone response pathways, observed in arsenite-treated hog1Δ cells, prolonged the G₁ delay but did not cause a persistent G₁ arrest. In contrast, deletion of the SIC1 gene encoding a cyclin-dependent kinase inhibitor fully suppressed the observed block of G₁ exit in hog1Δ cells. Moreover, the Sic1 protein was stabilized in arsenite-treated hog1Δ cells. Interestingly, Sic1-dependent persistent G₁ arrest was also observed in hog1Δ cells during hyperosmotic stress. Taken together, our data point to an important role of the Hog1 kinase in adaptation to stress-induced G₁ cell cycle arrest.     

Recombinant, Replication-Defective Adenovirus Gene Transfer Vectors Induce Cell Cycle Dysregulation and Inappropriate Expression of Cyclin Proteins

First-generation adenovirus (Ad) vectors that had been rendered replication defective by removal of the E1 region of the viral genome (ΔE1) or lacking the Ad E3 region in addition to E1 sequences (ΔE1ΔE3) induced G₂ cell cycle arrest and inhibited traverse across G₁/S in primary and immortalized human bronchial epithelial cells. Cell cycle arrest was independent of the cDNA contained in the expression cassette and was associated with the inappropriate expression and increase in cyclin A, cyclin B1, cyclin D, and cyclin-dependent kinase p34^cdc2 protein levels. In some instances, infection with ΔE1 or ΔE1ΔE3 Ad vectors produced aneuploid DNA histogram patterns and induced polyploidization as a result of successive rounds of cell division without mitosis. Cell cycle arrest was absent in cells infected with a second-generation ΔE1Ad vector in which all of the early region E4 except the sixth open reading frame was also deleted. Consequently, E4 viral gene products present in ΔE1 or ΔE1ΔE3 Ad vectors induce G₂ growth arrest, which may pose new and unintended consequences for human gene transfer and gene therapy.     

Δ122p53, a mouse model of Δ133p53α, enhances the tumor-suppressor activities of an attenuated p53 mutant

Growing evidence suggests the Δ133p53α isoform may function as an oncogene. It is overexpressed in many tumors, stimulates pathways involved in tumor progression, and inhibits some activities of wild-type p53, including transactivation and apoptosis. We hypothesized that Δ133p53α would have an even more profound effect on p53 variants with weaker tumor-suppressor capability. We tested this using a mouse model heterozygous for a Δ133p53α-like isoform (Δ122p53) and a p53 mutant with weak tumor-suppressor function (mΔpro). The Δ122p53/mΔpro mice showed a unique survival curve with a wide range of survival times (92–495 days) which was much greater than mΔpro/- mice (range 120–250 days) and mice heterozygous for the Δ122p53 and p53 null alleles (Δ122p53/-, range 78–150 days), suggesting Δ122p53 increased the tumor-suppressor activity of mΔpro. Moreover, some of the mice that survived longest only developed benign tumors. In vitro analyses to investigate why some Δ122p53/mΔpro mice were protected from aggressive tumors revealed that Δ122p53 stabilized mΔpro and prolonged the response to DNA damage. Similar effects of Δ122p53 and Δ133p53α were observed on wild-type of full-length p53, but these did not result in improved biological responses. The data suggest that Δ122p53 (and Δ133p53α) could offer some protection against tumors by enhancing the p53 response to stress.The p53 tumor suppressor is most important for preventing cancers. p53 controls cell fate in response to stress by inducing apoptosis, cell cycle arrest/senescence, DNA repair (reviewed in Braithwaite et al.,^{1, 2} Oren,³ and Speidel⁴) or possibly restricting supply of basic substrates for metabolism.^{5, 6, 7} The regulation of p53 function has recently become more complex with the discovery of 13 isoforms, which may interfere with the normal functioning of full-length (FL) p53.^{8, 9, 10, 11, 12, 13, 14} An alternative promoter in intron 4 generates the Δ133p53 isoforms (Δ133p53α, and with additional alternative splicing in intron 9, Δ133p53β, and Δ133p53γ¹¹).The Δ133p53α isoform is expressed in many tissues, but elevated levels have been found in several cancers.^{11, 15, 16} Although the function(s) of Δ133p53α are not fully understood, growing evidence suggests it may have tumor-promoting capacities. Reducing Δ133p53α levels in the U87MG glioblastoma cell line reduced its ability to migrate and stimulate angiogenesis.¹⁷ Δ133p53α may also interfere with the tumor-suppressor functions of FLp53. The zebrafish ortholog of Δ133p53α, Δ113p53, inhibited p53-mediated apoptosis,¹⁸ and overexpression of Δ133p53α inhibited p53-directed G₁ cell cycle arrest.¹⁶Previously, we reported the construction and characterization of a mouse expressing an N-terminal truncation mutant of p53 (designated Δ122p53) that is very similar to Δ133p53α, providing the first mouse model of the Δ133p53α isoform.^{19, 20} Δ122p53 was found to increase cell proliferation and in p53 null cells transduced with a Δ122p53 expressing retrovirus, inhibited the transactivation of CDKN1a (encoding) p21^CIP1 and MDM2 by FLp53.^{19, 20} As well as elevating cell proliferation, homozygote Δ122p53 mice exhibited a profound pro-inflammatory phenotype, including increased serum interleukin-6 (IL-6) and γ-interferon (γ-IFN), and features of autoimmune disease.^{19, 20} The mice were tumor-prone displaying a complex tumor spectrum, but predominantly B-cell lymphomas and osteosarcomas. Thus, most evidence supports a role for the Δ133p53α isoform as a dominant oncogene that may interfere with normal FLp53 tumor-suppressor functions, but also has additional ''gain-of-function'' properties to promote tumor progression, probably through inflammatory mechanisms.²¹Given the above data, we reasoned that in an environment where p53 tumor-suppression capacity is compromised, such as in the context of the R72P allele^{22, 23, 24} or where p53 levels are reduced,^{25, 26, 27} the influence of Δ133p53α isoform on FLp53 function would be greater, leading to rapid tumor formation with a phenotype that would resemble that of the isoform alone. To test this, we generated mice heterozygous for Δ122p53 and a p53 mutant (mΔpro) that we previously described, that has attenuated tumor-suppressor activity.^{28, 29} The mΔpro mouse model is missing part of the p53 proline rich domain (PRD, amino acids 58–88). These mice are defective for DNA damage-induced apoptosis, and show a delayed and impaired cell cycle arrest response. Homozygous mΔpro mice develop late onset follicular B-cell tumors, while mΔpro heterozygotes developed few tumors in the presence of a wild-type p53 allele, or an early onset T-cell lymphoma in a p53-null background. In the latter case, the onset and tumor spectrum are indistinguishable from p53-null mice.²⁸In the current study, we found that, in contrast to our hypothesis, many Δ122p53/mΔpro mice showed extended survival compared with Δ122p53 homozygotes. In vitro analyses to explain this phenomenon suggested that Δ122p53 allele can enhance mΔpro tumor-suppressor functions, in particular cell cycle arrest.     

Bacterial Porin Disrupts Mitochondrial Membrane Potential and Sensitizes Host Cells to Apoptosis

The bacterial PorB porin, an ATP-binding β-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (ΔΨ_m). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of β-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of ΔΨ_m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce ΔΨ_m loss and apoptosis, demonstrating that dissipation of ΔΨ_m is a requirement for cell death caused by neisserial infection.     

Phosphatase Complex Pph3/Psy2 Is Involved in Regulation of Efficient Non-Homologous End-Joining Pathway in the Yeast Saccharomyces cerevisiae

One of the main mechanisms for double stranded DNA break (DSB) repair is through the non-homologous end-joining (NHEJ) pathway. Using plasmid and chromosomal repair assays, we showed that deletion mutant strains for interacting proteins Pph3p and Psy2p had reduced efficiencies in NHEJ. We further observed that this activity of Pph3p and Psy2p appeared linked to cell cycle Rad53p and Chk1p checkpoint proteins. Pph3/Psy2 is a phosphatase complex, which regulates recovery from the Rad53p DNA damage checkpoint. Overexpression of Chk1p checkpoint protein in a parallel pathway to Rad53p compensated for the deletion of PPH3 or PSY2 in a chromosomal repair assay. Double mutant strains Δpph3/Δchk1 and Δpsy2/Δchk1 showed additional reductions in the efficiency of plasmid repair, compared to both single deletions which is in agreement with the activity of Pph3p and Psy2p in a parallel pathway to Chk1p. Genetic interaction analyses also supported a role for Pph3p and Psy2p in DNA damage repair, the NHEJ pathway, as well as cell cycle progression. Collectively, we report that the activity of Pph3p and Psy2p further connects NHEJ repair to cell cycle progression.     

Reversible Cyclosporin A-sensitive Mitochondrial Depolarization Occurs within Minutes of Stroke Onset in Mouse Somatosensory Cortex in Vivo: A TWO-PHOTON IMAGING STUDY*

Neuronal structure and function are rapidly damaged during global ischemia but can in part recover during reperfusion. Despite apparent recovery in the hours/days following an ischemic episode, delayed cell death can be initiated, making it important to understand how initial ischemic events affect potential mediators of apoptosis. Mitochondrial dysfunction and the opening of the mitochondrial permeability transition pore (mPTP) are proposed to link ischemic ionic imbalance to mitochondrially mediated cell death pathways. Using two-photon microscopy, we monitored mitochondrial transmembrane potential (Δψ_m) in vivo within the somatosensory cortex during ischemia and reperfusion in a mouse global ischemia model. Our results indicated a synchronous loss of Δψ_m within 1–3 min of ischemic onset that was linked to within seconds of plasma membrane potential (Δψ_p) depolarization. Δψ_m recovered rapidly upon reperfusion, and no delayed depolarization was observed over 2 h. Cyclosporin A treatment largely blocked Δψ_m collapse during ischemia, suggesting a role for the mPTP. Blocking Δψ_m depolarization did not affect structural damage to dendrites, indicating that the opening of the mPTP and damage to dendrites are separable pathways that are activated during Δψ_p depolarization. Our findings using in vivo imaging suggest that mitochondrial dysfunction and specifically the activation of the mPTP are early reversible events during brain ischemia that could trigger delayed cell death.     

The Hect Domain E3 Ligase Tom1 and the F-box Protein Dia2 Control Cdc6 Degradation in G1 Phase

The accurate replication of genetic information is critical to maintaining chromosomal integrity. Cdc6 functions in the assembly of pre-replicative complexes and is specifically required to load the Mcm2-7 replicative helicase complex at replication origins. Cdc6 is targeted for protein degradation by multiple mechanisms in Saccharomyces cerevisiae, although only a single pathway and E3 ubiquitin ligase for Cdc6 has been identified, the SCF^Cdc4 (Skp1/Cdc53/F-box protein) complex. Notably, Cdc6 is unstable during the G₁ phase of the cell cycle, but the ubiquitination pathway has not been previously identified. Using a genetic approach, we identified two additional E3 ubiquitin ligase components required for Cdc6 degradation, the F-box protein Dia2 and the Hect domain E3 Tom1. Both Dia2 and Tom1 control Cdc6 turnover during G₁ phase of the cell cycle and act separately from SCF^Cdc4. Ubiquitination of Cdc6 is significantly reduced in dia2Δ and tom1Δ cells. Tom1 and Dia2 each independently immunoprecipitate Cdc6, binding to a C-terminal region of the protein. Tom1 and Dia2 cannot compensate for each other in Cdc6 degradation. Cdc6 and Mcm4 chromatin association is aberrant in tom1Δ and dia2Δ cells in G₁ phase. Together, these results present evidence for a novel degradation pathway that controls Cdc6 turnover in G₁ that may regulate pre-replicative complex assembly.