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E-cadherin is a central molecule in the process of gastric carcinogenesis and its posttranslational modifications by N -glycosylation have been described to induce a deleterious effect on cell adhesion associated with tumor cell invasion. However, the role that site-specific glycosylation of E-cadherin has in its defective function in gastric cancer cells needs to be determined. Using transgenic mice models and human clinical samples, we demonstrated that N -acetylglucosaminyltransferase V (GnT-V)-mediated glycosylation causes an abnormal pattern of E-cadherin expression in the gastric mucosa. In vitro models further indicated that, among the four potential N -glycosylation sites of E-cadherin, Asn-554 is the key site that is selectively modified with β1,6 GlcNAc-branched N -glycans catalyzed by GnT-V. This aberrant glycan modification on this specific asparagine site of E-cadherin was demonstrated to affect its critical functions in gastric cancer cells by affecting E-cadherin cellular localization, cis -dimer formation, molecular assembly and stability of the adherens junctions and cell–cell aggregation, which was further observed in human gastric carcinomas. Interestingly, manipulating this site-specific glycosylation, by preventing Asn-554 from receiving the deleterious branched structures, either by a mutation or by silencing GnT-V, resulted in a protective effect on E-cadherin, precluding its functional dysregulation and contributing to tumor suppression.

Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in cold-adapted and mesophilic chitinases may have acted as a “weak link” from where unfolding was initiated. MD simulations confirmed that the prevalence and flexibility of the loops adjacent to the active site were greater in low-temperature-adapted chitinases and may have led to the occlusion of the active site at higher temperatures compared to their thermostable homologs. Following this, loop “hot spots” for stabilizing and destabilizing mutations were also identified. This information is not only useful for the elucidation of the structure–stability relationship, but will be crucial for designing and engineering chitinases to have enhanced thermoactivity and to withstand harsh industrial processing conditions

Murat Günel and colleagues identify recurrent mutations in POLR2A , which encodes the catalytic subunit of RNA polymerase II, in a subset of meningiomas. They find that POLR2A -mutant tumors can be distinguished on the basis of their super-enhancer and gene expression profiles, which show dysregulation of key meningeal identity genes. RNA polymerase II mediates the transcription of all protein-coding genes in eukaryotic cells, a process that is fundamental to life. Genomic mutations altering this enzyme have not previously been linked to any pathology in humans, which is a testament to its indispensable role in cell biology. On the basis of a combination of next-generation genomic analyses of 775 meningiomas, we report that recurrent somatic p.Gln403Lys or p.Leu438_His439del mutations in POLR2A , which encodes the catalytic subunit of RNA polymerase II (ref. 1 ), hijack this essential enzyme and drive neoplasia. POLR2A mutant tumors show dysregulation of key meningeal identity genes 2 , 3 , including WNT6 and ZIC1 / ZIC4 . In addition to mutations in POLR2A , NF2 , SMARCB1, TRAF7 , KLF4 , AKT1 , PIK3CA , and SMO 4 , 5 , 6 , 7 , 8 , we also report somatic mutations in AKT3 , PIK3R1 , PRKAR1A , and SUFU in meningiomas. Our results identify a role for essential transcriptional machinery in driving tumorigenesis and define mutually exclusive meningioma subgroups with distinct clinical and pathological features.

Cushing's syndrome is caused by excess cortisol production from the adrenocortical gland. In corticotropin-independent Cushing's syndrome, the excess cortisol production is primarily attributed to an adrenocortical adenoma, in which the underlying molecular pathogenesis has been poorly understood. We report a hotspot mutation (L206R) in PRKACA, which encodes the catalytic subunit of cyclic adenosine monophosphate (cAMP)–dependent protein kinase (PKA), in more than 50% of cases with adrenocortical adenomas associated with corticotropin-independent Cushing's syndrome. The L206R PRKACA mutant abolished its binding to the regulatory subunit of PKA (PRKAR1A) that inhibits catalytic activity of PRKACA, leading to constitutive, cAMP-independent PKA activation. These results highlight the major role of cAMP-independent activation of cAMP/PKA signaling by somatic mutations in corticotropin-independent Cushing's syndrome, providing insights into the diagnosis and therapeutics of this syndrome.

Construction of a series of genomically recoded organisms whose growth is restricted by the expression of essential genes dependent on exogenously supplied synthetic amino acids introduces novel orthogonal barriers between these engineered organisms and the environment, thereby creating safer genetically modified organisms. Two routes to safer GMOs Two manuscripts published in this issue of Nature describe independent approaches towards generating an organism dependent on unnatural amino acids, a development which could find applications for biocontainment and exploration of previously unsampled fitness landscapes. George Church and colleagues redesigned essential enzymes in an organism ( Escherichia coli ) with an altered genetic code to make it metabolically dependent on non-standard amino acids for survival. The resulting genetically modified organisms (GMOs) cannot metabolically circumvent their biocontainment mechanisms and show unprecedented resistance to evolutionary escape. The few escapees are rapidly outcompeted by unmodified organisms. Using multiplex automated genome engineering, Farren Isaacs and colleagues construct a series of genomically recoded organisms whose growth is restricted by the expression of essential genes that depend on exogenously supplied synthetic amino acids. They constructed synthetic auxotrophs with advanced orthogonal barriers between engineered organisms and the environment, thereby creating safer GMOs. Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals 1 . Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems 2 , 3 , which include bioremediation 4 and probiotics 5 . Although safeguards have been designed to control cell growth by essential gene regulation 6 , inducible toxin switches 7 and engineered auxotrophies 8 , these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations 9 , 10 . Here we describe the construction of a series of genomically recoded organisms (GROs) 11 whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase pair into the chromosome of a GRO derived from Escherichia coli that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering 12 , we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harbouring three TAG codons in conserved functional residues 13 of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼10 11 cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches 2 , 3 , 6 , 7 , 8 , 9 , 10 . We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternative genetic codes that impart genetic isolation by impeding horizontal gene transfer 11 and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.

A20 is a deubiquitinating enzyme that restrict inflammation by various mechanisms. Ma and colleagues show that A20 inhibits necroptosis by inhibiting the ubiquitination of RIPK3 and formation of the RIPK1-RIPK3 complex. A20 is an anti-inflammatory protein linked to multiple human diseases; however, the mechanisms by which A20 prevents inflammatory disease are incompletely defined. We found that A20-deficient T cells and fibroblasts were susceptible to caspase-independent and kinase RIPK3–dependent necroptosis. Global deficiency in RIPK3 significantly restored the survival of A20-deficient mice. A20-deficient cells exhibited exaggerated formation of RIPK1-RIPK3 complexes. RIPK3 underwent physiological ubiquitination at Lys5 (K5), and this ubiquitination event supported the formation of RIPK1-RIPK3 complexes. Both the ubiquitination of RIPK3 and formation of the RIPK1-RIPK3 complex required the catalytic cysteine of A20's deubiquitinating motif. Our studies link A20 and the ubiquitination of RIPK3 to necroptotic cell death and suggest additional mechanisms by which A20 might prevent inflammatory disease.

Adrenal Cushing's syndrome is caused by excess production of glucocorticoid from adrenocortical tumors and hyperplasias, which leads to metabolic disorders. We performed whole-exome sequencing of 49 blood-tumor pairs and RNA sequencing of 44 tumors from cortisol-producing adrenocortical adenomas (ACAs), adrenocorticotropic hormone–independent macronodular adrenocortical hyperplasias (AIMAHs), and adrenocortical oncocytomas (ADOs). We identified a hotspot in the PRKACA gene with a L205R mutation in 69.2% (27 out of 39) of ACAs and validated in 65.5% of a total of 87 ACAs. Our data revealed that the activating L205R mutation, which locates in the P+1 loop of the protein kinase A (PKA) catalytic subunit, promoted PKA substrate phosphorylation and target gene expression. Moreover, we discovered the recurrently mutated gene DOT1L in AIMAHs and CLASP2 in ADOs. Collectively, these data highlight potentially functional mutated genes in adrenal Cushing's syndrome.

Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.

The perception mechanism for the strigolactone (SL) class of plant hormones has been a subject of debate because their receptor, DWARF14 (D14), is an α/β-hydrolase that can cleave SLs. Here we show via time-course analyses of SL binding and hydrolysis by Arabidopsis thaliana D14, that the level of uncleaved SL strongly correlates with the induction of the active signaling state. In addition, we show that an AtD14 D218A catalytic mutant that lacks enzymatic activity is still able to complement the atd14 mutant phenotype in an SL-dependent manner. We conclude that the intact SL molecules trigger the D14 active signaling state, and we also describe that D14 deactivates bioactive SLs by the hydrolytic degradation after signal transmission. Together, these results reveal that D14 is a dual-functional receptor, responsible for both the perception and deactivation of bioactive SLs. Cleavage of strigolactone by the D14 receptor was assumed to produce an active intermediate that promotes signaling. Here the authors show that D14 activity is not dependent on cleavage activity and propose a new model whereby ligand hydrolysis serves to deactivate strigolactone signaling.

The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein–coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here, we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.