Synthetic heterovalent inhibitors targeting recognition E3 components of the N-end rule pathway

Multivalent binding allows high selectivity and affinity in a ligand-protein interaction. The N-end rule pathway is a ubiquitin (Ub)-dependent proteolytic system in which specific E3s, called N-recognins, mediate ubiquitylation through the recognition of types 1 and 2, destabilizing N-terminal residues of substrates. We recently identified a set of E3 Ub ligases (named UBR1-UBR7) containing the 70-residue UBR box, and we demonstrated that UBR1, UBR2, UBR4, and UBR5 can bind to destabilizing N-terminal residues. To explore a model of heterovalent interaction to the N-recognin family, we synthesized the small-molecule compound RF-C11, which bears two heterovalent ligands designed to target N-recognins, together with control molecules with two homovalent ligands. We demonstrate that heterovalent ligands of RF-C11 selectively and cooperatively bind cognate-binding sites of multiple N-recognins and thereby inhibit both types 1 and 2 N-end rule activities. Furthermore, the efficacy of heterovalent RF-C11 was substantially higher than homovalent inhibitors, which can target either a type 1 or type 2 site, providing the molecular basis of designing multivalent inhibitors for the control of specific intracellular pathways. In addition, RF-C11 exhibited higher efficacy and stability, compared with dipeptides bearing destabilizing N-terminal residues, which are known competitive inhibitors of the pathway. We also used the heterovalent compound to study the function of N-recognins in cardiac signaling. Using mouse and rat cardiomyocytes, we demonstrate that the N-end rule pathway has a cell-autonomous function in cardiac proliferation and hypertrophy, explaining our earlier results implicating the pathway in cardiac development and proteolysis of multiple cardiovascular regulators.     

The N-end rule pathway counteracts cell death by destroying proapoptotic protein fragments

In the course of apoptosis, activated caspases cleave ～500 to ～1,000 different proteins in a mammalian cell. The dynamics of apoptosis involve a number of previously identified, caspase-generated proapoptotic protein fragments, defined as those that increase the probability of apoptosis. In contrast to activated caspases, which can be counteracted by inhibitor of apoptosis proteins, there is little understanding of antiapoptotic responses to proapoptotic protein fragments. One possibility is the regulation of proapoptotic fragments through their selective degradation. The previously identified proapoptotic fragments Cys-RIPK1, Cys-TRAF1, Asp-BRCA1, Leu-LIMK1, Tyr-NEDD9, Arg-BID, Asp-BCL(XL), Arg-BIM(EL), Asp-EPHA4, and Tyr-MET bear destabilizing N-terminal residues. Tellingly, the destabilizing nature (but not necessarily the actual identity) of N-terminal residues of proapoptotic fragments was invariably conserved in evolution. Here, we show that these proapoptotic fragments are short-lived substrates of the Arg/N-end rule pathway. Metabolic stabilization of at least one such fragment, Cys-RIPK1, greatly augmented the activation of the apoptosis-inducing effector caspase-3. In agreement with this understanding, even a partial ablation of the Arg/N-end rule pathway in two specific N-end rule mutants is shown to sensitize cells to apoptosis. We also found that caspases can inactivate components of the Arg/N-end rule pathway, suggesting a mutual suppression between this pathway and proapoptotic signaling. Together, these results identify a mechanistically specific and functionally broad antiapoptotic role of the Arg/N-end rule pathway. In conjunction with other apoptosis-suppressing circuits, the Arg/N-end rule pathway contributes to thresholds that prevent a transient or otherwise weak proapoptotic signal from reaching the point of commitment to apoptosis.     

The main auxin biosynthesis pathway in Arabidopsis

The phytohormone auxin plays critical roles in the regulation of plant growth and development. Indole-3-acetic acid (IAA) has been recognized as the major auxin for more than 70 y. Although several pathways have been proposed, how auxin is synthesized in plants is still unclear. Previous genetic and enzymatic studies demonstrated that both TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) flavin monooxygenase-like proteins are required for biosynthesis of IAA during plant development, but these enzymes were placed in two independent pathways. In this article, we demonstrate that the TAA family produces indole-3-pyruvic acid (IPA) and the YUC family functions in the conversion of IPA to IAA in Arabidopsis (Arabidopsis thaliana) by a quantification method of IPA using liquid chromatography-electrospray ionization-tandem MS. We further show that YUC protein expressed in Escherichia coli directly converts IPA to IAA. Indole-3-acetaldehyde is probably not a precursor of IAA in the IPA pathway. Our results indicate that YUC proteins catalyze a rate-limiting step of the IPA pathway, which is the main IAA biosynthesis pathway in Arabidopsis.     

Impaired neurogenesis and cardiovascular development in mice lacking the E3 ubiquitin ligases UBR1 and UBR2 of the N-end rule pathway

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. A subset of degradation signals recognized by the N-end rule pathway comprises the signals, called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified a family of at least four mammalian E3 ubiquitin ligases, including UBR1 and UBR2, that share the UBR box and recognize N-degrons. These E3 enzymes mediate the multifunctional N-end rule pathway, but their individual roles are just beginning to emerge. Mutations of UBR1 in humans are the cause of Johanson-Blizzard syndrome. UBR1 and UBR2 are 46% identical and appear to be indistinguishable in their recognition of N-degrons. UBR1-/- mice are viable but have defects that include pancreatic insufficiency, similarly to UBR1-/- human patients with Johanson-Blizzard syndrome. UBR2-/- mice are inviable in some strain backgrounds and are defective in male meiosis. To examine functional relationships between UBR1 and UBR2, we constructed mouse strains lacking both of these E3s. We report here that UBR1-/-UBR2-/- embryos die at midgestation, with defects in neurogenesis and cardiovascular development. These defects included reduced proliferation as well as precocious migration and differentiation of neural progenitor cells. The expression of regulators such as D-type cyclins and Notch1 was also altered in UBR1-/-UBR2-/- embryos. We conclude that the functions of UBR1 and UBR2 are significantly divergent, in part because of differences in their expression patterns and possibly also because of differences in their recognition of protein substrates that contain degradation signals other than N-degrons.     

UBR box N-recognin-4 (UBR4), an N-recognin of the N-end rule pathway,and its role in yolk sac vascular development and autophagy

The N-end rule pathway is a proteolytic system in which destabilizing N-terminal residues of short-lived proteins act as degradation determinants (N-degrons). Substrates carrying N-degrons are recognized by N-recognins that mediate ubiquitylation-dependent selective proteolysis through the proteasome. Our previous studies identified the mammalian N-recognin family consisting of UBR1/E3α, UBR2, UBR4/p600, and UBR5, which recognize destabilizing N-terminal residues through the UBR box. In the current study, we addressed the physiological function of a poorly characterized N-recognin, 570-kDa UBR4, in mammalian development. UBR4-deficient mice die during embryogenesis and exhibit pleiotropic abnormalities, including impaired vascular development in the yolk sac (YS). Vascular development in UBR4-deficient YS normally advances through vasculogenesis but is arrested during angiogenic remodeling of primary capillary plexus associated with accumulation of autophagic vacuoles. In the YS, UBR4 marks endoderm-derived, autophagy-enriched cells that coordinate differentiation of mesoderm-derived vascular cells and supply autophagy-generated amino acids during early embryogenesis. UBR4 of the YS endoderm is associated with a tissue-specific autophagic pathway that mediates bulk lysosomal proteolysis of endocytosed maternal proteins into amino acids. In cultured cells, UBR4 subpopulation is degraded by autophagy through its starvation-induced association with cellular cargoes destined to autophagic double membrane structures. UBR4 loss results in multiple misregulations in autophagic induction and flux, including synthesis and lipidation/activation of the ubiquitin-like protein LC3 and formation of autophagic double membrane structures. Our results suggest that UBR4 plays an important role in mammalian development, such as angiogenesis in the YS, in part through regulation of bulk degradation by lysosomal hydrolases.     

Conserved endoplasmic reticulum-associated degradation system to eliminate mutated receptor-like kinases in Arabidopsis

Endoplasmic reticulum (ER)-associated degradation (ERAD) is an integral part of the ER quality-control system that removes toxic misfolded proteins via ubiquitin/proteasome-mediated degradation. Most of our knowledge on ERAD comes from biochemical and genetic studies in yeast and mammalian cells. Although ERAD is known to operate in plant cells, little is known about its molecular components and its biochemical mechanism. A genetic screen for suppressors of the Arabidopsis bri1-9, a weak dwarf mutant caused by ER retention of a structurally defective yet biochemically competent brassinosteroid (BR) receptor BRI1, resulted in identification of the EMS-mutagenized bri1 suppressor 5 (EBS5) gene that encodes an Arabidopsis homolog of the yeast Hrd3/mammalian Sel1L protein known to be involved in ERAD. Loss-of-function ebs5 mutations block the ERAD of bri1-9 and bri1-5, another ER-retained BR receptor. We showed that EBS5 complemented the ERAD defect of the yeast Δhrd3 mutant and interacted with the two mutated BR receptors in plant cells. Using a reverse genetic approach, we discovered that two Arabidopsis homologs of the yeast/mammalian Hrd1, an ER membrane-localized ubiquitin ligase, function redundantly in the ERAD of bri1-9. Together, our results revealed functional roles of two conserved ERAD components in degrading mutated/misfolded receptor-like kinases in Arabidopsis.     

Brassinosteroid signaling and auxin transport are required to establish the periodic pattern of Arabidopsis shoot vascular bundles

The plant vascular system provides transport and support capabilities that are essential for plant growth and development, yet the mechanisms directing the arrangement of vascular bundles within the shoot inflorescence stem remain unknown. We used computational and experimental biology to evaluate the role of auxin and brassinosteroid hormones in vascular patterning in Arabidopsis. We show that periodic auxin maxima controlled by polar transport and not overall auxin levels underlie vascular bundle spacing, whereas brassinosteroids modulate bundle number by promoting early procambial divisions. Overall, this study demonstrates that auxin polar transport coupled to brassinosteroid signaling is required to determine the radial pattern of vascular bundles in shoots.     

T-bet concomitantly controls migration, survival, and effector functions during the development of Valpha14i NKT cells

Valpha14i natural killer T (NKT)-cell function has been implicated in a number of disease conditions. The molecular events that drive Valpha14i NKT-cell development remain elusive. We recently showed that T-bet is required for the terminal maturation of these cells. Here we identify some of the genetic targets of T-bet during Valpha14i NKT-cell lineage development. Microarray gene-expression analyses on developing Valpha14i NKT cells were performed and provide a molecular framework to study these maturation events. In vitro ectopic expression of T-bet in immature Valpha14i NKT cells, which do not yet express T-bet, was sufficient to promote Valpha14i NKT-cell maturation, driving the expression of multiple genes, including those that participate in migration, survival, and effector functions. By regulating the expression of T-helper 1 (Th1)-associated cytokines, chemokines, chemokine receptors, and molecules involved in cytolysis, T-bet defines the unique lineage attributes of mature Valpha14i NKT cells and acts to link these attributes to a developmental process.     

The shoot meristem identity gene TFL1 is involved in flower development and trafficking to the protein storage vacuole

Plants are unique in their ability to store proteins in specialized protein storage vacuoles (PSVs) within seeds and vegetative tissues. Although plants use PSV proteins during germination, before photosynthesis is fully functional, the roles of PSVs in adult vegetative tissues are not understood. Trafficking pathways to PSVs and lytic vacuoles appear to be distinct. Lytic vacuoles are analogous evolutionarily to yeast and mammalian lysosomes. However, it is unclear whether trafficking to PSVs has any analogy to pathways in yeast or mammals, nor is PSV ultrastructure known in Arabidopsis vegetative tissue. Therefore, alternative approaches are required to identify components of this pathway. Here, we show that an Arabidopsis thaliana mutant that disrupts PSV trafficking identified TERMINAL FLOWER 1 (TFL1), a shoot meristem identity gene. The tfl1-19/mtv5 (for "modified traffic to the vacuole") mutant is specifically defective in trafficking of proteins to the PSV. TFL1 localizes to endomembrane compartments and colocalizes with the putative delta-subunit of the AP-3 adapter complex. Our results suggest a developmental role for the PSV in vegetative tissues.     

The ferritin Fe2 site at the diiron catalytic center controls the reaction with O2 in the rapid mineralization pathway

Oxidoreduction in ferritin protein nanocages occurs at sites that bind two Fe(II) substrate ions and O₂, releasing Fe(III)₂–O products, the biomineral precursors. Diferric peroxo intermediates form in ferritins and in the related diiron cofactor oxygenases. Cofactor iron is retained at diiron sites throughout catalysis, contrasting with ferritin. Four of the 6 active site residues are the same in ferritins and diiron oxygenases; ferritin-specific Gln¹³⁷ and variable Asp/Ser/Ala¹⁴⁰ substitute for Glu and His, respectively, in diiron cofactor active sites. To understand the selective functions of diiron substrate and diiron cofactor active site residues, we compared oxidoreductase activity in ferritin with diiron cofactor residues, Gln¹³⁷ → Glu and Asp¹⁴⁰ → His, to ferritin with natural diiron substrate site variations, Asp¹⁴⁰, Ser¹⁴⁰, or Ala¹⁴⁰. In Gln¹³⁷ → Glu ferritin, diferric peroxo intermediates were undetectable; an altered Fe(III)–O product formed, ΔA₃₅₀ = 50% of wild type. In Asp¹⁴⁰ → His ferritin, diferric peroxo intermediates were also undetectable, and Fe(II) oxidation rates decreased 40-fold. Ferritin with Asp¹⁴⁰, Ser¹⁴⁰, or Ala¹⁴⁰ formed diferric peroxo intermediates with variable kinetic stabilities and rates: t_1/2 varied 1- to 10-fold; k_cat varied approximately 2- to 3-fold. Thus, relatively small differences in diiron protein catalytic sites determine whether, and for how long, diferric peroxo intermediates form, and whether the Fe–active site bonds persist throughout the reaction cycle (diiron cofactors) or break to release Fe(III)₂–O products (diiron substrates). The results and the coding similarities for cofactor and substrate site residues—e.g., Glu/Gln and His/Asp pairs share 2 of 3 nucleotides—illustrate the potential simplicity of evolving active sites for diiron cofactors or diiron substrates.     

Overlapping functions of the four class XI myosins in Arabidopsis growth,root hair elongation,and organelle motility

Flowering plants have evolved multigene families of the class XI myosin motors, the functions of which remain poorly understood. Here, we investigated functional profiles of the Arabidopsis myosins that belong to two paralogous pairs, XI-K/XI-1 and XI-2/XI-B, using single and double gene-knockout mutants. It was found that the myosins XI-K, XI-2, and XI-B, but not XI-1 have overlapping and additive roles in the root hair elongation. A nonidentical set of the three myosins, XI-K, XI-1, and XI-2, exhibited partially redundant and additive roles in the transport of Golgi stacks, peroxisomes, and mitochondria. Conspicuously, the double xi-k/1 knockout plants that showed the largest cumulative reduction of the organelle velocities also exhibited a stunted plant growth and reduced fecundity phenotype. Collectively, these results suggest that the rapid, myosin-powered organelle trafficking is required for the optimal plant growth, whereas a distinct myosin function, presumably the vesicular transport, is involved in elongation of the root hairs. In addition, our data imply that the myosin gene duplication in plants has been followed by a gradual functional specialization of the resulting pairs of myosin paralogs.     

Separate functions for nuclear and cytoplasmic cryptochrome 1 during photomorphogenesis of Arabidopsis seedlings

Cryptochrome blue-light receptors mediate many aspects of plant photomorphogenesis, such as suppression of hypocotyl elongation and promotion of cotyledon expansion and root growth. The cryptochrome 1 (cry1) protein of Arabidopsis is present in the nucleus and cytoplasm of cells, but how the functions of one pool differ from the other is not known. Nuclear localization and nuclear export signals were genetically engineered into GFP-tagged cry1 molecules to manipulate cry1 subcellular localization in a cry1-null mutant background. The effectiveness of the engineering was confirmed by confocal microscopy. The ability of nuclear or cytoplasmic cry1 to rescue a variety of cry1 phenotypes was determined. Hypocotyl growth suppression by blue light was assessed by standard end-point analyses and over time with high resolution by a custom computer-vision technique. Both assays indicated that nuclear, rather than cytoplasmic, cry1 was the effective molecule in these growth inhibitions, as was the case for the mechanistically linked membrane depolarization, which occurs within several seconds of cry1 activation. Petiole elongation also was inhibited by nuclear, but not cytoplasmic, cry1. Conversely, primary root growth and cotyledon expansion in blue light were promoted by cytoplasmic cry1 and inhibited by nuclear cry1. Anthocyanin production in response to blue light was strongly stimulated by nuclear cry1 and, to a lesser extent, by cytoplasmic cry1. An important step toward elucidation of cry1 signaling pathways is the recognition that different subcellular pools of the photoreceptor have different functions.     

The crystal structure of the complex of PII and acetylglutamate kinase reveals how PII controls the storage of nitrogen as arginine

Photosynthetic organisms can store nitrogen by synthesizing arginine, and, therefore, feedback inhibition of arginine synthesis must be relieved in these organisms when nitrogen is abundant. This relief is accomplished by the binding of the PII signal transduction protein to acetylglutamate kinase (NAGK), the controlling enzyme of arginine synthesis. Here, we describe the crystal structure of the complex between NAGK and PII of Synechococcus elongatus, at 2.75-A resolution. We prove the physiological relevance of the observed interactions by site-directed mutagenesis and functional studies. The complex consists of two polar PII trimers sandwiching one ring-like hexameric NAGK (a trimer of dimers) with the threefold axes of these molecules aligned. The binding of PII favors a narrow ring conformation of the NAGK hexamer that is associated with arginine sites having low affinity for this inhibitor. Each PII subunit contacts one NAGK subunit only. The contacts map in the inner circumference of the NAGK ring and involve two surfaces of the PII subunit. One surface is on the PII body and interacts with the C-domain of the NAGK subunit, helping widen the arginine site found on the other side of this domain. The other surface is at the distal region of a protruding large loop (T-loop) that presents a novel compact shape. This loop is inserted in the interdomain crevice of the NAGK subunit, contacting mainly the N-domain, and playing key roles in anchoring PII on NAGK, in activating NAGK, and in complex formation regulation by MgATP, ADP, 2-oxoglutarate, and by phosphorylation of serine-49.     

MAPK信号通路在肝癌发生发展及治疗中的作用

丝裂原活化蛋白激酶(MAPK)信号通路的异常活化与肝癌的发生、发展、转移密切相关。介绍了MAPK通路蛋白在肝癌中的表达及其在肝癌增殖、分化、转移中的作用,阐述了MAPK信号通路在肝癌治疗及预后评价中的价值。认为MAPK信号通路在肝癌的发生发展及治疗中发挥非常重要的作用,是肝癌治疗及预后评价的潜在分子靶点。