Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death

Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.     

Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response

The unfolded protein response (UPR) detects the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and adjusts the protein-folding capacity to the needs of the cell. Under conditions of ER stress, the transmembrane protein Ire1 oligomerizes to activate its cytoplasmic kinase and ribonuclease domains. It is unclear what feature of ER stress Ire1 detects. We found that the core ER-lumenal domain (cLD) of yeast Ire1 binds to unfolded proteins in yeast cells and to peptides primarily composed of basic and hydrophobic residues in vitro. Mutation of amino acid side chains exposed in a putative peptide-binding groove of Ire1 cLD impaired peptide binding. Peptide binding caused Ire1 cLD oligomerization in vitro, suggesting that direct binding to unfolded proteins activates the UPR.     

Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1

Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1alpha-/- fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals.     

Regulation of MAPK function by direct interaction with the mating-specific Galpha in yeast

The mating response of the budding yeast Saccharomyces cerevisiae is mediated by a prototypical heterotrimeric GTP-binding protein (G protein) and mitogen-activated protein kinase (MAPK) cascade. Although signal transmission by such pathways has been modeled in detail, postreceptor down-regulation is less well understood. The pheromone-responsive G protein alpha subunit (Galpha) of yeast down-regulates the mating signal, but its targets are unknown. We have found that Galpha binds directly to the mating-specific MAPK in yeast cells responding to pheromone. This interaction contributes both to modulation of the mating signal and to the chemotropic response, and it demonstrates direct communication between the top and bottom of a Galpha-MAPK pathway.     

Acquisition by innervated cardiac myocytes of a pertussis toxin-specific regulatory protein linked to the alpha 1-receptor

During development, the chronotropic response of rat ventricular myocardium to alpha 1-adrenergic stimulation changes from positive to negative. The alpha 1-agonist phenylephrine increases the rate of contraction of neonatal rat myocytes cultured alone but decreases the rate of contraction when the myocytes are cultured with functional sympathetic neurons. The developmental induction of the inhibitory myocardial response to alpha 1-adrenergic stimulation in intact ventricle and in cultured myocytes was shown to coincide with the functional acquisition of a substrate for pertussis toxin. A 41-kilodalton protein from myocytes cultured with sympathetic neurons and from adult rat myocardium showed, respectively, 2.2- and 16-fold increases in pertussis toxin-associated ADP-ribosylation (ADP, adenosine diphosphate) as compared to controls. In nerve-muscle cultures, inhibition of the actions of this protein by pertussis toxin-specific ADP-ribosylation reversed the mature inhibitory alpha 1-adrenergic response to an immature stimulatory pattern. The results suggest that innervation is associated with the appearance of a functional pertussis toxin substrate by which the alpha 1-adrenergic response becomes linked to a decrease in automaticity.     

Regulation of myosin phosphatase by a specific interaction with cGMP- dependent protein kinase Ialpha

Contraction and relaxation of smooth muscle are regulated by myosin light-chain kinase and myosin phosphatase through phosphorylation and dephosphorylation of myosin light chains. Cyclic guanosine monophosphate (cGMP)-dependent protein kinase Ialpha (cGKIalpha) mediates physiologic relaxation of vascular smooth muscle in response to nitric oxide and cGMP. It is shown here that cGKIalpha is targeted to the smooth muscle cell contractile apparatus by a leucine zipper interaction with the myosin-binding subunit (MBS) of myosin phosphatase. Uncoupling of the cGKIalpha-MBS interaction prevents cGMP-dependent dephosphorylation of myosin light chain, demonstrating that this interaction is essential to the regulation of vascular smooth muscle cell tone.     

Herpesviral protein networks and their interaction with the human proteome

The comprehensive yeast two-hybrid analysis of intraviral protein interactions in two members of the herpesvirus family, Kaposi sarcoma-associated herpesvirus (KSHV) and varicella-zoster virus (VZV), revealed 123 and 173 interactions, respectively. Viral protein interaction networks resemble single, highly coupled modules, whereas cellular networks are organized in separate functional submodules. Predicted and experimentally verified interactions between KSHV and human proteins were used to connect the viral interactome into a prototypical human interactome and to simulate infection. The analysis of the combined system showed that the viral network adopts cellular network features and that protein networks of herpesviruses and possibly other intracellular pathogens have distinguishing topologies.     

Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response

The BRCT repeats of the breast and ovarian cancer predisposition protein BRCA1 are essential for tumor suppression. Phosphopeptide affinity proteomic analysis identified a protein, Abraxas, that directly binds the BRCA1 BRCT repeats through a phospho-Ser-X-X-Phe motif. Abraxas binds BRCA1 to the mutual exclusion of BACH1 (BRCA1-associated C-terminal helicase) and CtIP (CtBP-interacting protein), forming a third type of BRCA1 complex. Abraxas recruits the ubiquitin-interacting motif (UIM)-containing protein RAP80 to BRCA1. Both Abraxas and RAP80 were required for DNA damage resistance, G(2)-M checkpoint control, and DNA repair. RAP80 was required for optimal accumulation of BRCA1 on damaged DNA (foci) in response to ionizing radiation, and the UIM domains alone were capable of foci formation. The RAP80-Abraxas complex may help recruit BRCA1 to DNA damage sites in part through recognition of ubiquitinated proteins.     

Bmf: a proapoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis

Bcl-2 family members bearing only the BH3 domain are essential inducers of apoptosis. We identified a BH3-only protein, Bmf, and show that its BH3 domain is required both for binding to prosurvival Bcl-2 proteins and for triggering apoptosis. In healthy cells, Bmf is sequestered to myosin V motors by association with dynein light chain 2. Certain damage signals, such as loss of cell attachment (anoikis), unleash Bmf, allowing it to translocate and bind prosurvival Bcl-2 proteins. Thus, at least two mammalian BH3-only proteins, Bmf and Bim, function to sense intracellular damage by their localization to distinct cytoskeletal structures.     

Sequence and expression of mRNAs encoding the alpha 1 and alpha 2 subunits of a DHP-sensitive calcium channel

Complementary DNAs were isolated and used to deduce the primary structures of the alpha 1 and alpha 2 subunits of the dihydropyridine-sensitive, voltage-dependent calcium channel from rabbit skeletal muscle. The alpha 1 subunit, which contains putative binding sites for calcium antagonists, is a hydrophobic protein with a sequence that is consistent with multiple transmembrane domains and shows structural and sequence homology with other voltage-dependent ion channels. In contrast, the alpha 2 subunit is a hydrophilic protein without homology to other known protein sequences. Nucleic acid hybridization studies suggest that the alpha 1 and alpha 2 subunit mRNAs are expressed differentially in a tissue-specific manner and that there is a family of genes encoding additional calcium channel subtypes.     

P1 of strawberry vein banding virus,a multilocalized protein,functions as a movement protein and interacts with the coat protein

Although the complete nucleotide sequence of strawberry vein banding virus (SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of each protein is unclear. This study provided evidence that the P1 protein of SVBV (SVBV-P1) possesses the following features. Bioinformatic and subcellular localization analyses showed that SVBV-P1 is localized in the cytoplasm and cell walls of epidermal cells in Nicotiana benthamiana, and it forms inclusion bodies associated with microtubules and the endoplasmic reticulum. Dilution experiments demonstrated that SVBV-P1 could move from the original agro-infiltrated cells to adjacent cells in N. benthamiana leaves. Further trans-complementation experiments demonstrated that SVBV-P1 could facilitate the intercellular movement of a movement-deficient potato virus X mutant in N. benthamiana leaves. Finally, yeast two-hybrid and bimolecular fluorescence complementation assays revealed that SVBV-P1 could interact with the SVBV coat protein, which is a major component of Caulimovirus virions. Results of the electrophoretic mobility shift assay indicated that SVBV-P1 lacks DNA-binding capability. In summary, the results suggest that SVBV-P1 is probably a movement protein of SVBV, providing new insights into the function of movement proteins of the Caulimovirus genus.     

Repair of the secretion defect in the Z form of alpha 1-antitrypsin by addition of a second mutation

Homozygous inheritance of the Z-type mutant form of the alpha 1-antitrypsin (alpha 1AT) gene results in the most common form of alpha 1AT deficiency, a human hereditary disease associated with a high risk for the development of emphysema and an increased incidence of neonatal hepatitis. The alpha 1AT-synthesizing cells of individuals with the Z gene have normal alpha 1AT messenger RNA levels, but alpha 1AT secretion is markedly reduced secondary to accumulation of newly synthesized alpha 1AT in the rough endoplasmic reticulum. Crystallographic analysis of alpha 1AT predicts that in normal alpha 1AT, a negatively charged Glu342 is adjacent to positively charged Lys290. Thus the Glu342----Lys342 Z mutation caused the loss of a normal salt bridge, resulting in the intracellular aggregation of the Z molecule. The prediction was made that a second mutation in the alpha 1AT genet that changed the positively charged Lys290 to a negatively charged Glu290 would correct the secretion defect. When the second mutation was added to the Z-type complementary DNA, the resulting gene directed the synthesis and secretion of amounts of alpha 1AT similar to that directed by the normal alpha 1AT complementary DNA in an in vitro eukaryotic expression system. This suggests the possibility that a human hereditary disease can be corrected by inserting an additional mutation in the same gene.     

小麦黄花叶病毒编码VPg蛋白N端结构是VPg与核仁蛋白Fibrillarin互作区域

小麦黄花叶病毒（Wheat yellow mosaic virus,WYMV）属于马铃薯Y病毒科大麦黄花叶病毒属成员,其基因组是由两条正义单链RNA组成,共编码10个蛋白。其中,VPg（Viral protein genome\|linked）作为病毒末端结合蛋白,与病毒基因组RNA 5’端共价连接。利用软件分析WYMV VPg蛋白氨基酸序列发现其N端具有核定位信号（NLS）序列,C端具有核输出信号（NES）序列。通过激光共聚焦显微镜观察VPg与GFP融合蛋白在烟草细胞中的表达情况发现,GFP\|VPg主要定位于细胞核中。利用eYFP（n）标记核仁结构蛋白Fibrillarin与VPg进行双分子荧光互补实验发现,VPg与Fibrillarin互作且定位于细胞核中。根据VPg结构特点构建一系列缺失突变体与Fibrillarin的互作实验验证了它们之间的互作区域发生在N端NLS区域。     

我国苹果产业70年发展历程与展望

目的研究干旱胁迫对苹果不同中间砧及其上嫁接品种的叶片光合特性及抗氧化酶活性的影响。方法以河北农业大学自主选育的苹果矮化砧木冀砧3号及优系15-1、1-8、14-7、10-1和22-46为试验材料,研究干旱胁迫下苹果砧木及其上嫁接天红2号的叶片光合参数和抗氧化酶活性的变化,并利用隶属函数法分析不同中间砧对干旱胁迫的响应差异。结果干旱胁迫下,不同苹果砧木及砧穗组合的光合作用受到不同程度的影响;叶片初始荧光与可变荧光和最大荧光比值与对照组相比差异显著;随着干旱胁迫时间的延长,砧木叶片抗氧化物酶(SOD、POD和CAT)活性呈现先升高后降低的趋势,嫁接品种叶片的SOD和CAT活性整体呈升高的趋势;砧木14-7及其上嫁接天红2号叶片的光合作用和3种抗氧化酶活性对干旱胁迫响应相对较弱,而砧木22-46和其上嫁接品种响应较强。隶属函数分析表明：6种砧木抗旱性为14-7>15-1>冀砧3号>10-1>1-8>22-46。结论干旱胁迫下,苹果6种不同中间砧及其上嫁接品种的光合特性和抗氧化酶活性存在差异。