Clathrin-coated vesicles transport selected integral membrane proteins from the cell surface and the trans-Golgi network to the endosomal system. Before fusing with their target the vesicles must be stripped of their coats. This process is effected by the chaperone protein hsp70c together with a 100K cofactor which we here identify as the coat protein auxilin. Auxilin binds with high affinity to assembled clathrin lattices and, in the presence of ATP, recruits hsp70c. Dissociation of the lattice does not depend as previously supposed on clathrin light chains or on the amino-terminal domain of the heavy chain. The presence of a J-domain at its carboxy terminus now defines auxilin as a member of the DnaJ protein family. In conjunction with hsp70, DnaJ proteins catalyse protein folding, protein transport across membranes and the selective disruption of protein-protein interactions. We show that deletion of the J-domain of auxilin results in the loss of cofactor activity.

Key Points• IDH1 promotes leukemogenesis in vivo in cooperation with HoxA9.• Pharmacologic inhibition of mutant IDH1 efficiently inhibits AML cells of IDH1-mutated patients but not of normal CD34 1 bone marrow cells.Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, and chondrosarcoma patients. Mutant IDH produces 2-hydroxyglutarate (2HG), which induces histone-and DNA-hypermethylation through inhibition of epigenetic regulators. We investigated the role of mutant IDH1 using the mouse transplantation assay. Mutant IDH1 alone did not transform hematopoietic cells during 5 months of observation. However, mutant IDH1 greatly accelerated onset of myeloproliferative disease-like myeloid leukemia in mice in cooperation with HoxA9 with a mean latency of 83 days compared with cells expressing HoxA9 and wild-type IDH1 or a control vector (167 and 210 days, respectively, P 5 .001). Mutant IDH1 accelerated cell-cycle transition through repression of cyclindependent kinase inhibitors Cdkn2a and Cdkn2b, and activated mitogen-activated protein kinase signaling. By computational screening, we identified an inhibitor of mutant IDH1, which inhibited mutant IDH1 cells and lowered 2HG levels in vitro, and efficiently blocked colony formation of AML cells from IDH1-mutated patients but not of normal CD341 bone marrow cells. These data demonstrate that mutant IDH1 has oncogenic activity in vivo and suggest that it is a promising therapeutic target in human AML cells. (Blood.

On-surface synthesis constitutes a rapidly growing field of research due to its promising application for creating stable molecular structures on surfaces. While self-assembled structures rely on reversible interactions, on-surface synthesis provides the potential for creating long-term stable structures with well-controlled properties, for example superior electron transport for future molecular electronic devices. On-surface synthesis holds the promise for preparing insoluble compounds that cannot be produced in solution. Another highly exciting aspect of on-surface synthesis is the chance to discover new reaction pathways due to the two-dimensional confinement of the reaction educts. In this review, we discuss the current state-of-the-art and classify the reactions that have been successfully performed so far. Special emphasis is put on electrically insulating surfaces, as these substrates pose particular challenges for on-surface synthesis while at the same time bearing high potential for future use, for example, in molecular electronics.

The anoxygenic phototrophic bacterium Rhodobacter sphaeroides uses different energy sources depending on environmental conditions including aerobic respiration or, in the absence of oxygen, photosynthesis. Photosynthetic genes are repressed at high oxygen tension, but at intermediate levels their partial expression prepares the organism for using light energy. Illumination, however, enhances repression under semi-aerobic conditions. Here, we describe molecular details of two proteins involved in oxygen- and light-control of photosynthesis gene expression, the light-sensing anti-repressor AppA and the transcriptional repressor PpsR. We combine information from crystal structures of both proteins and their complex with hydrogen-deuterium exchange data to show that light-activation of AppA–PpsR2 affects the PpsR effector region within the complex. DNA-binding studies demonstrate the formation of a light-sensitive ternary AppA–PpsR–DNA complex. Implications of these results for light- and oxygen-regulation are discussed, highlighting new insights into blue-light-mediated signal transduction.

Summary Background We previously identified a functional variant in a let-7 microRNA (miRNA) complementary site in the 3′-untranslated region of the KRAS oncogene (rs61764370) which is associated with cancer. We aimed to investigate the association of this KRAS variant with breast cancer and tumour biology. Methods We assessed frequency distributions of the KRAS variant in 415 patients with histologically confirmed breast cancer and 457 controls from Connecticut, USA (study group 1) and association of this variant with breast-cancer subtypes in 690 Irish women with known oestrogen receptor (ER), progesterone receptor (PR), and HER2 statuses, and 360 controls (study group 2). We pooled data for study groups 1 and 2 with a cohort of 140 women with triple-negative breast cancer and 113 controls to assess the association of the KRAS variant with triple-negative breast cancer risk, and genome-wide mRNA and specific miRNA expression in patients with triple-negative breast cancer. Findings Although frequency distributions of the KRAS variant in study group 1 did not differ between all genotyped individuals, eight (33%) of 24 premenopausal women with ER/PR-negative cancer had the KRAS variant, compared with 27 (13%) of 201 premenopausal controls (p=0·015). In study group 2, the KRAS variant was significantly enriched in women with triple-negative breast cancer (19 [21%] of 90 cases) compared with 64 (13%) of 478 for luminal A, 13 (15%) of 87 for luminal B, and two (6%) of 35 for HER2-positive subgroups (p=0·044). Multivariate analysis in the pooled study groups showed that the KRAS variant was associated with triple-negative breast cancer in premenopausal women (odds ratio 2·307, 95% CI 1·261–4·219, p=0·0067). Gene-expression analysis of triple-negative breast-cancer tumours suggested that KRAS-variant positive tumours have significantly altered gene expression, and are enriched for the luminal progenitor and BRCA1 deficiency signatures. miRNA analysis suggested reduced levels of let-7 miRNA species in KRAS-variant tumours. Interpretation The KRAS variant might be a genetic marker for development of triple-negative breast cancer in premenopausal women, and altered gene and miRNA expression signatures should enable molecular and biological stratification of patients with this subgroup of breast cancer. Funding US National Institutes of Health.

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The peptide binding site of MHC class II molecules is open at both ends and, therefore, does not restrict the length of the bound ligand. Here we show that a partially folded protein antigen (*HEL) spontaneously formed SDS‐unstable complexes with the purified MHC class II molecule I‐Ak (Ak). These complexes were also detected on the surface of antigen‐presenting cells (APCs) where they stimulated T cells. However, they rapidly disappeared after endocytosis. Intracellular processing of *HEL gave rise to SDS‐stable, long‐lived Ak complexes containing *HEL peptides and, unexpectedly, full‐length *HEL. Both SDS‐stable products were formed in low pH compartments and then transported to the plasma membrane. In contrast to *HEL peptides, the stable association of *HEL occurred in an alternative pathway that required mature class II molecules and did not involve HLA‐DM or proteases. SDS‐stable *HEL‐Ak complexes were formed by a reaction of endosomal Ak with endocytosed *HEL, but not by direct conversion of SDS‐unstable complexes derived from the plasma membrane. Our work establishes a fundamental difference between the two MHC class II loading pathways and for the first time demonstrates a full‐length protein as a product of antigen processing.

SIMIBI-class (named after the signal recognition particle, MinD, BioD) nucleotide-binding proteins appeared early in evolution 1 and contain GTPases, as well as ATPases, involved in the correct localization of cellular constituents. The MinD ATPase, as the central part of the Min system, regulates the determination of the cell division site in all bacterial species 2 . SRP-GTPases form a subfamily of the SIMIBI class, with only three members: the signal sequence-binding protein Ffh (SRP54 in Eukarya and Archaea), the SRP receptor FtsY (SR in Eukarya) and FlhF, which is involved in flagella biosynthesis [3][4][5] . They share the conserved NG domain, which contains two major additions to the conserved fold of small G proteins. First, an --element (I-box) is inserted in the effector region; second, the N domain, comprising four -helices, is attached to the N terminus of the G domain. SRP (Ffh together with the SRP RNA) and FtsY constitute the universally conserved co-translational protein-targeting machinery 6,7 . When bound to GTP, Ffh and FtsY form, through interactions between their NG domains 8,9 , a heterodimeric complex that regulates the transfer of a ribosome-nascent chain complex to a vacant translocon in the membrane with a series of conformational rearrangements 10,11 . The two GTPases share a composite active site between their G domains in which GTP hydrolysis is reciprocally activated 12 . The SRP RNA [13][14][15] and membrane lipids 16,17 play fundamental roles in activating the Ffh-FtsY GTPases. The recent structure of the SRP-FtsY complex, together with biochemical implications, suggest that the distal end of the hairpin-like SRP RNA may be involved in this activation 18 . The third SRP-GTPase FlhF, together with the MinD-type protein YlxH (also known as FlhG, FleN, motR or MinD2), is essential for the placement and assembly of flagella 19 in many polar and peritrichous flagellated bacteria [20][21][22][23][24] . FlhF is required for the targeting of the first flagellar protein, FliF, to the cell pole 25 by a mechanism that is so far poorly understood. FlhF is associated with the membrane 25,26 and localizes at the cell pole 20 . The FlhF protein (Fig. 1a) contains an N-terminal B domain that seems to be involved in FliF targeting 25 ; it shares the NG domain fold with the other two members of the SRP-GTPase subfamily. FlhF forms a stable homodimer with GTP and a composite active site that is basically identical to the active site of the Ffh-FtsY heterodimer 5 . In both the homo-and heterodimer, the two nucleotides are bound in a head-to-tail manner, with the -phosphate of one nucleotide interacting with the 3 -OH of the ribose moiety of the other. However, for the homo-and heterodimers formed by the three SRP-GTPases, the molecular mechanism of activation is still unknown. We set out to understand the activation of SRPGTPases by studying FlhF. RESULTS The SRP-GTPase FlhF is activated by YlxHAs FlhF (Fig. 1) forms a stable homodimer, and reciprocal activation has not been observed 5 , we reasoned ...