Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that induce mitochondrial outer membrane permeabilization, leading to cytochrome c release and activation of apoptotic caspases. In the absence of active caspases, mitochondrial DNA (mtDNA) triggers the innate immune cGAS/STING pathway, causing dying cells to secrete type I interferon. How cGAS gains access to mtDNA remains unclear. We used live-cell lattice light-sheet microscopy to examine the mitochondrial network in mouse embryonic fibroblasts. We found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner mitochondrial membrane to herniate into the cytosol, carrying with it mitochondrial matrix components, including the mitochondrial genome. Apoptotic caspases did not prevent herniation but dismantled the dying cell to suppress mtDNA-induced innate immune signaling.

MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuB's poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB. We demonstrate that MuB is an ATPase associated with diverse cellular activities (AAA+ ATPase) and forms ATP-dependent filaments with or without DNA. We also identify critical residues for MuB's ATPase, DNA binding, protein polymerization, and MuA interaction activities. Using single-particle electron microscopy, we show that MuB assembles into a helical filament, which binds the DNA in the axial channel. The helical parameters of the MuB filament do not match those of the coated DNA. Despite this protein-DNA symmetry mismatch, MuB does not deform the DNA duplex. These findings, together with the influence of MuB filament size on strand-transfer efficiency, lead to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored by MuA for transposition.Phage Mu | nucleoprotein filament D NA transposons are ubiquitous in the genomes of all forms of life and play important evolutionary roles in generating gene diversity and in shaping genomic landscapes (1). Although typically transposons exhibit no strong sequence selectivity for the target DNA site, certain transposons avoid self-destructive insertion (reviewed in ref.2), a phenomenon called "target immunity" because the presence of a copy of the transposon renders nearby DNA sites "immune" to additional insertion by the same transposon (3-8). MuB plays critical roles in this selfimmunity in the bacteriophage Mu transposition process.Phage Mu is one of the most complex and efficient transposable elements (reviewed in refs. 3 and 4). Two phage-coded proteins, MuA and MuB, are essential for efficient Mu transposition. MuA is the transposase responsible for synapsing the two Mu end sequences and for all of the DNA cutting and joining steps in the initial stages of transposition. However, transposition is inefficient in the absence of MuB, and the residual Mu insertion that takes place uses only DNA target sites near or within the transposing element, often leading to self-destruction (9-11). MuB is a small (35-kDa) ATP-dependent nonspecific DNA-binding protein with relatively low ATPase activity (10,12,13). Upon ATP binding, MuB polymerizes preferentially on DNA, but in the absence of DNA it still can form polymers of variable sizes (14, 15). When observed by total internal reflection fluorescence microscopy, GFP-MuB-ATP binds along the DNA molecule forming many short separate segments of polymers, and, as more GFP-MuB is added, the protein-covered segments elongate to form an apparently continuous polymer that fully coats the DNA. Hydrolysis of ATP reverses this process, triggering disassembly of the MuB polymer (16-18)...

Fucosylation of the inner-most Nacetylglucosamine (GlcNAc) of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, the development of monoclonal antibody therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well characterized and provide a framework for understanding N-glycan maturation in the Golgi; however the structural basis for these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here, we describe several structures of mouse and human FUT8 in the apo state and in complex with guanosine diphosphate (GDP), a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80-2.50 Å resolutions. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process. https://www.jbc.org/cgi/

No abstract

Transposition target immunity is a phenomenon observed in some DNA transposons that are able to distinguish the host chromosome from their own DNA sequence, thus avoiding self-destructive insertions. The first molecular insight into target selection and immunity mechanisms came from the study of phage Mu transposition, which uses the protein MuB as a barrier to self-insertion. MuB is an ATP-dependent non-specific DNA binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. However, a detailed mechanistic understanding of MuB functioning was hindered by the poor solubility of the MuB-ATP complexes. Here we comment on the recent discovery that MuB is an AAA+ ATPase that upon ATP binding assembles into helical filaments that coat the DNA. Remarkably, the helical parameters of the MuB filament do not match those of the bound DNA. This intriguing mismatch symmetry led us to propose a model on how MuB targets DNA for transposition, favoring DNA bending and recognition by the transposase at the filament edge. We also speculate on a different protective role of MuB during immunity, where filament stickiness could favor the condensation of the DNA into a compact state that occludes it from the transposase.

18Fucosylation of the inner-most N-acetyl-glucosamine (GlcNAc) of N-glycans by fucosyltransferase 8 19 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple 20 modification can have a dramatic impact on the activity and half-life of glycoproteins. These effects 21 are relevant to understanding the invasiveness of some cancers, the development of monoclonal 22 antibody therapeutics, and to a congenital disorder of glycosylation. The acceptor substrate preferences 23 of FUT8 are well characterised and provide a framework for understanding N-glycan maturation in 24 the Golgi, however the structural basis for these substrate preferences and the mechanism through 25 which catalysis is achieved remains unknown. Here, we describe several structures of mouse and 26 human FUT8 in the apo state and in complex with guanosine diphosphate (GDP), a mimic of the donor 27 substrate, and a glycopeptide acceptor substrate. These structures provide insights into: a unique 28 conformational change associated with donor substrate binding; common strategies employed by 29 fucosyltransferases to coordinate GDP; features that define acceptor substrate preferences; and a likely 30 mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also 31 reveal how FUT8 dimerisation plays an important role in defining the acceptor substrate binding site. 32Collectively, this information significantly builds on our understanding of the core-fucosylation 33 process. 34 35 EGFR signalling 1, 14 . These animals also exhibited behavioural abnormalities 15 . Many of these 51 phenotypes are also observed in patients with the recently described FUT8 congenital disorder of 52 glycosylation (CDG-FUT8) 16 . In contrast to CDG-FUT8, which features the ablation of FUT8 activity, 53 many cancers upregulate FUT8 expression and this correlates with a poor 54 prognosis 17 . In melanomas, increased FUT8 activity stabilises L1CAM to promote metastasis 18 . 55Metastasis is also promoted by FUT8 in breast cancers, where increased core-fucosylation of TGFb1R 56 promotes strong constitutive signalling through this receptor and tumour cell migration 19 . The 57 increased core fucosylation of a-fetoprotein is also a well-established biomarker of hepatocellular 58 carcinoma (HCC) 20 . 59Some have speculated that FUT8 antagonists may have therapeutic potential for the treatment 60 of cancer 9,18 , though questions remain around how a hypothetic FUT8 antagonist might impact host 61 immune responses to tumour cells. Regardless, no drug-like small molecule inhibitors have yet been 62 reported for FUT8, or any other human fucosyltransferase (FUT). To some degree, drug discovery 63 efforts are impeded by a limited structural understanding of this enzyme and the mechanism it employs 64to perform core fucosylation. The only reported FUT8 structure possesses no bound ligands, 21 and our 65 only insights into donor and acceptor substrate binding come from STD-NMR, molecular dynamics 66 and docking studies 22, ...

Fluorescent proteins (FPs) have become an essential tool for biological research. Since the isolation and description of green FP, hundreds of FPs have been discovered and created with various characteristics. The excitation of these proteins ranges from ultraviolet (UV) up to near infrared (NIR). Using conventional cytometry, with each detector assigned to a fluorochrome, great care must be taken when selecting the optimal bandpass filters to minimalize the spectral overlap as the emission spectra of FPs are broad. Full-spectrum flow cytometers eliminate the need to change optical filters for analyzing FPs, which simplifies instrument setup. In experiments where more than one FP is used, single-color controls are required. These can be cells expressing each of the proteins separately. In the case of the confetti system, for instance, when four FPs are used, all these proteins will need to be expressed separately so that compensation or spectral unmixing can be performed, and this can be inconvenient and expensive. An appealing alternative is to produce FPs in Escherichia coli, purify them, and covalently couple them to carboxylate polystyrene microspheres. Such microspheres are ready to use and can be stored at 4°C for months or even years without any deterioration in fluorescence. The same procedure can be used to couple antibodies or other proteins to these particles. Here, we describe how to express and purify FPs, how to couple them to microspheres, and how to evaluate the fluorescent properties of the particles.

No abstract