Mutations in isocitrate dehydrogenases (IDHs) have a gain‐of‐function effect leading to R (−)‐2‐hydroxyglutarate ( R‐ 2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R ‐ and S ‐2HG inhibit 2‐oxoglutarate (2OG)‐dependent oxygenases with varying potencies. Half‐maximal inhibitory concentration (IC 50 ) values for the R ‐form of 2HG varied from approximately 25 μM for the histone N ε ‐lysine demethylase JMJD2A to more than 5 mM for the hypoxia‐inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH‐associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation. The oncometabolite 2‐hydroxyglutarate (2‐HG) inhibits chromatin‐modifying oxygenases (as histone lysine demethylases) with greater potency than HIF hydroxylases. This suggests that 2‐HG‐associated oncogenic pathways involve the regulation of histone methylation, rather than an elevated HIF response.
Allergic asthma rates have increased steadily in developed countries, arguing for an environmental aetiology. To assess the influence of gut microbiota on experimental murine allergic asthma, we treated neonatal mice with clinical doses of two widely used antibiotics—streptomycin and vancomycin—and evaluated resulting shifts in resident flora and subsequent susceptibility to allergic asthma. Streptomycin treatment had little effect on the microbiota and on disease, whereas vancomycin reduced microbial diversity, shifted the composition of the bacterial population and enhanced disease severity. Neither antibiotic had a significant effect when administered to adult mice. Consistent with the ‘hygiene hypothesis’, our data support a neonatal, microbiota‐driven, specific increase in susceptibility to experimental murine allergic asthma. Allergic asthma rates are increasing in developed countries, arguing for an environmental aetiology. Profound changes in gut microbiota are reported in response to treatment in early life with the widely used antibiotic vancomycin, influencing asthma susceptibility.
Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin‐like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP‐L1 to damaged mitochondria through its amino‐terminal LC3‐interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation. How specific cellular components are targeted for autophagy is poorly understood. The Dikic lab reports that the mitochondrial protein Nix acts as a selective autophagy receptor by binding to LC3/GABARAP proteins and recruiting them to damaged mitochondria. Moreover, ablation of the Nix:LC3/GABARAP interaction retards mitophagy in maturing murine reticulocytes.
The embryonic programme ‘epithelial–mesenchymal transition’ (EMT) is thought to promote malignant tumour progression. The transcriptional repressor zinc‐finger E‐box binding homeobox 1 (ZEB1) is a crucial inducer of EMT in various human tumours, and was recently shown to promote invasion and metastasis of tumour cells. Here, we report that ZEB1 directly suppresses transcription of microRNA‐200 family members miR‐141 and miR‐200c, which strongly activate epithelial differentiation in pancreatic, colorectal and breast cancer cells. Notably, the EMT activators transforming growth factor β2 and ZEB1 are the predominant targets downregulated by these microRNAs. These results indicate that ZEB1 triggers an microRNA‐mediated feedforward loop that stabilizes EMT and promotes invasion of cancer cells. Alternatively, depending on the environmental trigger, this loop might switch and induce epithelial differentiation, and thus explain the strong intratumorous heterogeneity observed in many human cancers.
R‐spondins are secreted Wnt signalling agonists, which regulate embryonic patterning and stem cell proliferation, but whose mechanism of action is poorly understood. Here we show that R‐spondins bind to the orphan G‐protein‐coupled receptors LGR4 and LGR5 by their Furin domains. Gain‐ and loss‐of‐function experiments in mammalian cells and Xenopus embryos indicate that LGR4 and LGR5 promote R‐spondin‐mediated Wnt/β‐catenin and Wnt/PCP signalling. R‐spondin‐triggered β‐catenin signalling requires Clathrin, while Wnt3a‐mediated β‐catenin signalling requires Caveolin‐mediated endocytosis, suggesting that internalization has a mechanistic role in R‐spondin signalling. R‐spondins are secreted proteins known to synergize with Wnt signalling. The authors now show that R‐spondins are ligands for LGR4 and LGR5 orphan G‐protein‐coupled receptors. Binding of R‐spondins to LGRs positively regulates both Wnt/ ‐catenin and Wnt/PCP signalling pathways in vivo.
Mutations in phosphatase and tensin homologue‐induced kinase 1 (PINK1) cause recessively inherited Parkinson's disease (PD), a neurodegenerative disorder linked to mitochondrial dysfunction. In healthy mitochondria, PINK1 is rapidly degraded in a process involving both mitochondrial proteases and the proteasome. However, when mitochondrial import is compromised by depolarization, PINK1 accumulates on the mitochondrial surface where it recruits the PD‐linked E3 ubiquitin ligase Parkin from the cytosol, which in turn mediates the autophagic destruction of the dysfunctional organelles. Using an unbiased RNA‐mediated interference (RNAi)‐based screen, we identified four mitochondrial proteases, mitochondrial processing peptidase (MPP), presenilin‐associated rhomboid‐like protease (PARL), m‐AAA and ClpXP, involved in PINK1 degradation. We find that PINK1 turnover is particularly sensitive to even modest reductions in MPP levels. Moreover, PINK1 cleavage by MPP is coupled to import such that reducing MPP activity induces PINK1 accumulation at the mitochondrial surface, leading to Parkin recruitment and mitophagy. These results highlight a new role for MPP in PINK1 import and mitochondrial quality control via the PINK1–Parkin pathway. Dysfunctional mitochondria express high surface levels of the Parkinson's disease‐linked protein PINK1, which in turn recruits Parkin for mitophagy. Fon and colleagues now show that levels of PINK1 are kept low in normal mitochondria through degradation by the mitochondrial processing peptidase (MPP).
The breast cancer 2, early onset protein (BRCA2) is central to the repair of DNA damage by homologous recombination. BRCA2 recruits the recombinase RAD51 to sites of damage, regulates its assembly into nucleoprotein filaments and thereby promotes homologous recombination. Localization of BRCA2 to nuclear foci requires its association with the partner and localizer of BRCA2 (PALB2), mutations in which are associated with cancer predisposition, as well as subtype N of Fanconi anaemia. We have determined the structure of the PALB2 carboxy‐terminal β‐propeller domain in complex with a BRCA2 peptide. The structure shows the molecular determinants of this important protein–protein interaction and explains the effects of both cancer‐associated truncating mutants in PALB2 and missense mutations in the amino‐terminal region of BRCA2.
Propagation of tau pathology is linked with progressive neurodegeneration, but the mechanism underlying trans‐synaptic spread of tau is unknown. We show that stimulation of neuronal activity, or AMPA receptor activation, induces tau release from healthy, mature cortical neurons. Notably, phosphorylation of extracellular tau appears reduced in comparison with intracellular tau. We also find that AMPA‐induced release of tau is calcium‐dependent. Blocking pre‐synaptic vesicle release by tetanus toxin and inhibiting neuronal activity with tetrodotoxin both significantly impair AMPA‐mediated tau release. Tau secretion is therefore a regulatable process, dysregulation of which could lead to the spread of tau pathology in disease. This report provides evidence that stimulation of neuronal activity, or AMPA receptor activation, induces tau release from cortical neurons via a calcium‐dependent mechanism. Dysregulation of this process could lead to the spread of tau pathology in disease.
Mitochondria form intricate networks through fission and fusion events. Here, we identify mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively) anchored in the mitochondrial outer membrane. MiD49/51 form foci and rings around mitochondria similar to the fission mediator dynamin‐related protein 1 (Drp1). MiD49/51 directly recruit Drp1 to the mitochondrial surface, whereas their knockdown reduces Drp1 association, leading to unopposed fusion. Overexpression of MiD49/51 seems to sequester Drp1 from functioning at mitochondria and cause fused tubules to associate with actin. Thus, MiD49/51 are new mediators of mitochondrial division affecting Drp1 action at mitochondria. Mitochondria undergo fission and fusion events that are essential for proper cellular function. Here, MiD49 and MiD51 are identified as novel regulators of mitochondrial division that act on the fission mediator Drp1.
Mitochondria manganese superoxide dismutase (SOD2) is an important antioxidant enzyme, deficiency of which is associated with various human diseases. The known primary regulation of SOD2 is through transcriptional activation. Here, we report that SOD2 is acetylated at Lys 68 and that this acetylation decreases SOD2 activity. Mitochondrial deacetylase SIRT3 binds to, deacetylates and activates SOD2. Increase of reactive oxygen species (ROS) levels stimulates SIRT3 transcription, leading to SOD2 deacetylation and activation. SOD2-mediated ROS reduction is synergistically increased by SIRT3 co-expression, but is cancelled by SIRT3 depletion. These results reveal a new post-translational regulation of SOD2 by means of acetylation and SIRT3-dependent deacetylation in response to oxidative stress.
In this study, we develop a simple assay to identify mitophagy inducers on the basis of the use of fluorescently tagged mitochondria that undergo a colour change on lysosomal delivery. Using this assay, we identify iron chelators as a family of compounds that generate a strong mitophagy response. Iron chelation-induced mitophagy requires that cells undergo glycolysis, but does not require PINK1 stabilization or Parkin activation, and occurs in primary human fibroblasts as well as those isolated from a Parkinson's patient with Parkin mutations. Thus, we have identified and characterized a mitophagy pathway, the induction of which could prove beneficial as a potential therapy for several neurodegenerative diseases in which mitochondrial clearance is advantageous.
Autophagy, an evolutionarily conserved process, has functions both in cytoprotective and programmed cell death mechanisms. Beclin 1, an essential autophagic protein, was recently identified as a BH3-domain-only protein that binds to Bcl-2anti-apoptotic family members. The dissociation of beclin 1 from its Bcl-2 inhibitors is essential for its autophagic activity, and therefore should be tightly controlled. Here, we show that death-associated protein kinase ( DAPK) regulates this process. The activated form of DAPK triggers autophagy in a beclin-1-dependent manner. DAPK phosphorylates beclin 1 on Thr 119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl-X-L and the induction of autophagy. These results reveal a substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and they provide a new phosphorylation-based mechanism that reduces the interaction of beclin 1 with its inhibitors to activate the autophagic machinery.
At least eight types of ubiquitin chain exist, and individual linkages affect distinct cellular processes. The only distinguishing feature of differently linked ubiquitin chains is their structure, as polymers of the same unit are chemically identical. Here, we have crystallized Lys 63‐linked and linear ubiquitin dimers, revealing that both adopt equivalent open conformations, forming no contacts between ubiquitin molecules and thereby differing significantly from Lys 48‐linked ubiquitin chains. We also examined the specificity of various deubiquitinases (DUBs) and ubiquitin‐binding domains (UBDs). All analysed DUBs, except CYLD, cleave linear chains less efficiently compared with other chain types, or not at all. Likewise, UBDs can show chain specificity, and are able to select distinct linkages from a ubiquitin chain mixture. We found that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO (NF‐κB essential modifier) binds to linear chains exclusively, whereas the NZF (Npl4 zinc finger) domain of TAB2 (TAK1 binding protein 2) is Lys 63 specific. Our results highlight remarkable specificity determinants within the ubiquitin system.
Priming of defence genes for amplified response to secondary stress can be induced by application of the plant hormone salicylic acid or its synthetic analogue acibenzolar S ‐methyl. In this study, we show that treatment with acibenzolar S ‐methyl or pathogen infection of distal leaves induce chromatin modifications on defence gene promoters that are normally found on active genes, although the genes remain inactive. This is associated with an amplified gene response on challenge exposure to stress. Mutant analyses reveal a tight correlation between histone modification patterns and gene priming. The data suggest a histone memory for information storage in the plant stress response. Plants can acquire systemic resistance to stress or infection after a first localized exposure. The paper provides evidence that histone modifications provide a memory that sensitizes defense genes for stronger responses.
Autophagy, an evolutionarily conserved process, has functions both in cytoprotective and programmed cell death mechanisms. Beclin 1, an essential autophagic protein, was recently identified as a BH3‐domain‐only protein that binds to Bcl‐2 anti‐apoptotic family members. The dissociation of beclin 1 from its Bcl‐2 inhibitors is essential for its autophagic activity, and therefore should be tightly controlled. Here, we show that death‐associated protein kinase (DAPK) regulates this process. The activated form of DAPK triggers autophagy in a beclin‐1‐dependent manner. DAPK phosphorylates beclin 1 on Thr 119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl‐X L and the induction of autophagy. These results reveal a substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and they provide a new phosphorylation‐based mechanism that reduces the interaction of beclin 1 with its inhibitors to activate the autophagic machinery.
As a lysosomal/vacuolar degradative pathway that is conserved in eukaryotic organisms, autophagy mediates the turnover of long‐lived proteins and excess or aberrant organelles. The main characteristic of autophagy is the formation of a double‐membrane vesicle, the autophagosome, which envelops part of the cytoplasm and delivers it to the lysosome/vacuole for breakdown and eventual recycling of the degradation products. Among the approximately 30 autophagy‐related (Atg) genes identified so far, there are two ubiquitin‐like proteins, Atg12 and Atg8. Analogous to ubiquitination, Atg12 is conjugated to Atg5 by Atg7—an E1‐like protein—and Atg10—an E2‐like protein. Similarly, Atg7 and Atg3 are the respective E1‐like and E2‐like proteins that mediate the conjugation of Atg8 to phosphatidylethanolamine. Both Atg12–Atg5 and Atg8 localize to the developing autophagosome. The Atg12–Atg5 conjugate facilitates the lipidation of Atg8 and directs its correct subcellular localization. Atg8–phosphatidylethanolamine is probably a scaffold protein that supports membrane expansion and the amount present correlates with the size of autophagosomes.
We report new functions of the cell‐adhesion molecule E‐cadherin in murine pluripotent cells. E‐cadherin is highly expressed in mouse embryonic stem cells, and interference with E‐cadherin causes differentiation. During cellular reprogramming of mouse fibroblasts by OCT4, SOX2, KLF4 and c‐MYC, fully reprogrammed cells were exclusively observed in the E‐cadherin‐positive cell population and could not be obtained in the absence of E‐cadherin. Moreover, reprogrammed cells could be established by viral E‐cadherin in the absence of exogenous OCT4. Thus, reprogramming requires spatial cues that cross‐talk with essential transcription factors. The cell‐adhesion molecule E‐cadherin has important functions in pluripotency and reprogramming. E‐cadherin is required for reprogramming of mouse fibroblasts to iPS cells by Oct4, Sox2, Klf4 and c‐Myc and can also replace Oct4 in this process. This suggests that spatial cues are crucial for reprogramming.
Opposing mitochondrial fission and fusion reactions determine the shape and interconnectivity of mitochondria. Dynamin‐related protein 1 (Drp1) is an ancient mechanoenzyme that uses GTP hydrolysis to power the constriction and division of mitochondria. Although Drp1‐mediated mitochondrial fragmentation is recognized as an early event in the apoptotic programme, acute regulation of Drp1 activity is poorly understood. Here, we identify a crucial phosphorylation site that is conserved in all metazoan Drp1 orthologues. Ser 656 is phosphorylated by cyclic AMP‐dependent protein kinase and dephosphorylated by calcineurin, and its phosphorylation state is controlled by sympathetic tone, calcium levels and cell viability. Pseudophosphorylation of Drp1 by mutation of Ser 656 to aspartic acid leads to the elongation of mitochondria and confers resistance to various pro‐apoptotic insults. Conversely, the constitutively dephosphorylated Ser656Ala mutant Drp1 promotes mitochondrial fragmentation and increases cell vulnerability. Thus, Drp1 phosphorylation at Ser 656 provides a mechanism for the integration of cAMP and calcium signals in the control of mitochondrial shape, apoptosis and other aspects of mitochondrial function.
Several microRNAs (miRNAs) have recently been described as crucial regulators of epithelial‐to‐mesenchymal transition (EMT) and metastasis. By comparing the expression profiles of miRNAs, we found upregulation of miR‐29a in mesenchymal, metastatic RasXT cells relative to epithelial EpRas cells. Overexpression of miR‐29a suppressed the expression of tristetraprolin (TTP), a protein involved in the degradation of messenger RNAs with AU‐rich 3′‐untranslated regions, and led to EMT and metastasis in cooperation with oncogenic Ras signalling. We also observed enhanced miR‐29a and reduced TTP levels in breast cancer patient samples, indicating relevance for human disease. Previously, miR‐29 family members were shown to have tumour‐suppressive effects in haematopoietic, cholangiocytic and lung tumours. Therefore, miRNAs can act as either oncogenes or tumour suppressors, depending on the context.
Natural products have often been the sole means to treat diseases and injuries. in fact, it has only been during the past decades that natural products have taken a secondary role in drug discovery and drug development, after the advent of molecular biology and combinatorial chemistry made possible the rational design of chemical compounds to target specific molecules. The past few years, however, have seen a renewed interest in the use of natural compounds and, more importantly, their role as a basis for drug development.