The three-dimensional structure of spinach ferredoxin-NADP+ reductase (NADP+, nicotinamide adenine dinucleotide phosphate) has been determined by x-ray diffraction at 2.6 angstroms (A) resolution and initially refined to an R factor of 0.226 at 2.2 A resolution. The model includes the flavin-adenine dinucleotide (FAD) prosthetic group and the protein chain from residue 19 through the carboxyl terminus at residue 314 and is composed of two domains. The FAD binding domain (residues 19 to 161) has an antiparallel beta barrel core and a single alpha helix for binding the pyrophosphate of FAD. The NADP binding domain (residues 162 to 314) has a central five-strand parallel beta sheet and six surrounding helices. Binding of the competitive inhibitor 2'-phospho-AMP (AMP, adenosine monophosphate) places the NADP binding site at the carboxyl-terminal edge of the sheet in a manner similar to the nucleotide binding of the dehydrogenase family. The structures reveal the key residues that function in cofactor binding and the catalytic center. With these key residues as a guide, conclusive evidence is presented that the ferredoxin reductase structure is a prototype for the nicotinamide dinucleotide and FAD binding domains of the enzymes NADPH-cytochrome P450 reductase, NADPH-sulfite reductase, NADH-cytochrome b5 reductase, and NADH-nitrate reductase. Thus this structure provides a structural framework for the NADH- or NADPH-dependent flavoenzyme parts of five distinct enzymes involved in photosynthesis, in the assimilation of inorganic nitrogen and sulfur, in fatty-acid oxidation, in the reduction of methemoglobin, and in the metabolism of many pesticides, drugs, and carcinogens.

Luminescence imaging has gained attention as a promising bio-imaging modality in situations where fluorescence imaging cannot be applied. However, wider application to multicolour and dynamic imaging is limited by the lack of bright luminescent proteins with emissions across the visible spectrum. Here we report five new spectral variants of the bright luminescent protein, enhanced Nano-lantern (eNL), made by concatenation of the brightest luciferase, NanoLuc, with various colour hues of fluorescent proteins. eNLs allow five-colour live-cell imaging, as well as detection of single protein complexes and even single molecules. We also develop an eNL-based Ca2+ indicator with a 500% signal change, which can image spontaneous Ca2+ dynamics in cardiomyocyte and neural cell models. These eNL probes facilitate not only multicolour imaging in living cells but also sensitive imaging of a wide repertoire of proteins, even at very low expression levels.

The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammationmediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

Germ-line mutations inactivating BRCA2 predispose to cancer. BRCA2-deficient cells exhibit alterations in chromosome number (aneuploidy), as well as structurally aberrant chromosomes. Here, we show that BRCA2 deficiency impairs the completion of cell division by cytokinesis. BRCA2 inactivation in murine embryo fibroblasts (MEFs) and HeLa cells by targeted gene disruption or RNA interference delays and prevents cell cleavage. Impeded cell separation is accompanied by abnormalities in myosin II organization during the late stages in cytokinesis. BRCA2 may have a role in regulating these events, as it localizes to the cytokinetic midbody. Our findings thus link cytokinetic abnormalities to a hereditary cancer syndrome characterized by chromosomal instability and may help to explain why BRCA2-deficient tumors are frequently aneuploid.