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Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3' untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3' UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The tripartite motif-containing protein 66 (TRIM66, also known as TIF1-delta) is a PHD-Bromo-containing protein primarily expressed in post-meiotic male germ cells known as spermatids. Biophysical assays showed that the TRIM66 PHD-Bromodomain binds to H3 N-terminus only when lysine 4 is unmethylated. We addressed TRIM66's role in reproduction by loss-of-function genetics in the mouse. Males homozygous for Trim66-null mutations produced functional spermatozoa. Round spermatids lacking TRIM66 up-regulated a network of genes involved in histone acetylation and H3K4 methylation. Profiling of H3K4me3 patterns in the sperm produced by the Trim66 -null mutant showed minor alterations below statistical significance. Unexpectedly, Trim66 -null males, but not females, sired pups overweight at birth, hence revealing that Trim66 mutations cause a paternal effect phenotype., (© 2024 Mielnicka et al.)

Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo bidirectional transport by binding to both motors simultaneously. However, it is not known how motor activities are coordinated in such circumstances. In the Drosophila female germline, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar messenger RNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. We show through in vitro reconstitution that Tm1-I/C, a tropomyosin-1 isoform, links kinesin-1 in a strongly inhibited state to DDBE-associated oskar mRNA. Nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and structural modeling indicate that Tm1-I/C suppresses kinesin-1 activity by stabilizing its autoinhibited conformation, thus preventing competition with dynein until kinesin-1 is activated in the oocyte. Our work reveals a new strategy for ensuring sequential activity of microtubule motors., (© 2024. The Author(s).)

Baculovirus-mediated gene expression in mammalian cells, BacMam, is a useful alternative to transient transfection for recombinant protein production in various types of mammalian cell lines. We decided to establish BacMam in our lab in order to streamline our workflows for gene expression in insect and mammalian cells, as it is straightforward to parallelize the baculovirus generation for both types of eukaryotic cells. This chapter provides a step-by-step description of the protocols we use for the generation of the recombinant BacMam viruses, the transduction of mammalian cell cultures, and optimization of the protein production conditions through small-scale expression and purification tests., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This overview guides both novices and experienced researchers facing challenging targets to select the most appropriate gene expression system for producing a particular protein. By answering four key questions, readers can determine the most suitable gene expression system following a decision scheme. This guide addresses the most commonly used and accessible systems and provides brief descriptions of the main gene expression systems' key characteristics to assist decision making. Additionally, information has been included for selected less frequently used "exotic" gene expression systems., Competing Interests: Declaration of interests Kim Remans is a member of the STAR Protocols advisory board., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We explored the interplay between the RecA and auxiliary domains of the RNA helicase maleless (MLE) from Drosophila using structural and functional studies. We discovered that MLE exists in a dsRNA-bound open conformation and that the auxiliary dsRBD2 domain aligns the substrate RNA with the accessible helicase tunnel. In an ATP-dependent manner, dsRBD2 associates with the helicase module, leading to tunnel closure around ssRNA. Furthermore, our structures provide a rationale for blunt-ended dsRNA unwinding and 3'-5' translocation by MLE. Structure-based MLE mutations confirm the functional relevance of our model for RNA unwinding. Our findings contribute to our understanding of the fundamental mechanics of auxiliary domains in DExH helicase MLE, which serves as a model for its human ortholog and potential therapeutic target, DHX9/RHA., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The African trypanosome survives the immune response of its mammalian host by antigenic variation of its major surface antigen (the variant surface glycoprotein or VSG). Here we describe the antibody repertoires elicited by different VSGs. We show that the repertoires are highly restricted and are directed predominantly to distinct epitopes on the surface of the VSGs. They are also highly discriminatory; minor alterations within these exposed epitopes confer antigenically distinct properties to these VSGs and elicit different repertoires. We propose that the patterned and repetitive nature of the VSG coat focuses host immunity to a restricted set of immunodominant epitopes per VSG, eliciting a highly stereotyped response, minimizing cross-reactivity between different VSGs and facilitating prolonged immune evasion through epitope variation., Competing Interests: Declaration of interests F.N.P., C.E.S., and J.P.V. report being shareholders of Panosome GmbH. F.N.P. and C.E.S. report being the managing directors of Panosome GmbH., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Primary tumors and distant site metastases form a bidirectionally communicating system. Yet, the molecular mechanisms of this crosstalk are poorly understood. Here, we identified the proteolytically cleaved fragments of angiopoietin-like 4 (ANGPTL4) as contextually active protumorigenic and antitumorigenic contributors in this communication ecosystem. Preclinical studies in multiple tumor models revealed that the C-terminal fragment (cANGPTL4) promoted tumor growth and metastasis. In contrast, the N-terminal fragment of ANGPTL4 (nANGPTL4) inhibited metastasis and enhanced overall survival in a postsurgical metastasis model by inhibiting WNT signaling and reducing vascularity at the metastatic site. Tracing ANGPTL4 and its fragments in tumor patients detected full-length ANGPTL4 primarily in tumor tissues, whereas nANGPTL4 predominated in systemic circulation and correlated inversely with disease progression. The study highlights the spatial context of the proteolytic cleavage-dependent pro- and antitumorigenic functions of ANGPTL4 and identifies and validates nANGPTL4 as a novel biomarker of tumor progression and antimetastatic therapeutic agent., (© 2022 Hübers et al.)

Most eukaryotic proteins are N-terminally acetylated by a set of Nα acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi Chaetomium thermophilum and Neurospora crassa with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that Ct Naa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution.

Interferons (IFN) are cytokines which, upon binding to cell surface receptors, trigger a series of downstream biochemical events including Janus Kinase (JAK) activation, phosphorylation of Signal Transducer and Activator of Transcription protein (STAT), translocation of pSTAT to the nucleus and transcriptional activation. Dysregulated IFN signalling has been linked to cancer progression and auto-immune diseases. Here, we report the serendipitous discovery of a small molecule that blocks IFNγ activation of JAK-STAT signalling. Further lead optimisation gave rise to a potent and more selective analogue that exerts its activity by a mechanism consistent with direct IFNγ targeting in vitro, which reduces bleeding in model of haemorrhagic colitis in vivo. This first-in-class small molecule also inhibits type I and III IFN-induced STAT phosphorylation in vitro. Our work provides the basis for the development of pan-IFN inhibitory drugs., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Decreased susceptibilities of the human malaria parasite Plasmodium falciparum towards the endoperoxide antimalarial artemisinin are linked to mutations of residue C580 of PfKelch13, a homologue of the redox sensor Keap1 and other vertebrate BTB-Kelch proteins. Here, we addressed whether mutations alter the artemisinin susceptibility by modifying the redox properties of PfKelch13 or by compromising its native fold or abundance. Using selection-linked integration and the glmS ribozyme, efficient down-regulation of PfKelch13 resulted in ring-stage survival rates around 40%. While the loss of the thiol group of C469 or of the potential disulfide bond between residues C580 and C532 had no effect on the artemisinin susceptibility, the thiol group of C473 could not be replaced. Furthermore, we detected two different forms of PfKelch13 with distinct electrophoretic mobilities around 85 and 95 kDa, suggesting an unidentified post-translational modification. We also established a protocol for the production of recombinant PfKelch13 and produced an antibody against the protein. Recombinant PfKelch13 adopted alternative oligomeric states and only two of its seven cysteine residues, C469 and C473, reacted with Ellman's reagent. While common field mutations resulted in misfolded and completely insoluble recombinant PfKelch13, cysteine-to-serine replacements had no effect on the solubility except for residue C473. In summary, in contrast to residues C469, C532, and C580, the surface-exposed thiol group of residue C473 appears to be essential. However, not the redox properties but impaired folding of PfKelch13, resulting in a decreased PfKelch13 abundance, alters the artemisinin susceptibility and is the central parameter for mutant selection., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. This suppression is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins during RNA suppression remains a long-standing open question. Here, we characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex comprising the RNA binding domains is flexible with unoccupied nucleotides functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, the presence of the Pum-HD/Nanos-ZnF complex has no effect on the equilibrium RNA binding affinity of the Brat RNA binding domain. This is in accordance with previous studies, which showed that Brat can suppress mRNA independently and is distributed uniformly throughout the embryo., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

African trypanosomes cause disease in humans and livestock, avoiding host immunity by changing the expression of variant surface glycoproteins (VSGs); the major glycosylphosphatidylinositol (GPI) anchored antigens coating the surface of the bloodstream stage. Proper trafficking of VSGs is therefore critical to pathogen survival. The valence model argues that GPI anchors regulate progression and fate in the secretory pathway and that, specifically, a valence of two (VSGs are dimers) is critical for stable cell surface association. However, recent reports that the MITat1.3 (M1.3) VSG N-terminal domain (NTD) behaves as a monomer in solution and in a crystal structure challenge this model. We now show that the behavior of intact M1.3 VSG in standard in vivo trafficking assays is consistent with an oligomer. Nevertheless, Blue Native Gel electrophoresis and size exclusion chromatography-multiangle light scattering chromatography of purified full length M1.3 VSG indicates a monomer in vitro. However, studies with additional VSGs show that multiple oligomeric states are possible, and that for some VSGs oligomerization is concentration dependent. These data argue that individual VSG monomers possess different propensities to self-oligomerize, but that when constrained at high density to the cell surface, oligomeric species predominate. These results resolve the apparent conflict between the valence hypothesis and the M1.3 NTD VSG crystal structure., (© 2021 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances within cells. The mechanochemical cycle of kinesins is well described, but how they establish cargo specificity is not fully understood. Transport of oskar mRNA to the posterior pole of the Drosophila oocyte is mediated by Drosophila kinesin-1, also called kinesin heavy chain (Khc), and a putative cargo adaptor, the atypical tropomyosin, a Tm1. How the proteins cooperate in mRNA transport is unknown. Here, we present the high-resolution crystal structure of a Khc- a Tm1 complex. The proteins form a tripartite coiled coil comprising two in-register Khc chains and one a Tm1 chain, in antiparallel orientation. We show that a Tm1 binds to an evolutionarily conserved cargo binding site on Khc, and mutational analysis confirms the importance of this interaction for mRNA transport in vivo. Furthermore, we demonstrate that Khc binds RNA directly and that it does so via its alternative cargo binding domain, which forms a positively charged joint surface with a Tm1, as well as through its adjacent auxiliary microtubule binding domain. Finally, we show that a Tm1 plays a stabilizing role in the interaction of Khc with RNA, which distinguishes a Tm1 from classical motor adaptors., (© 2021 Dimitrova-Paternoga et al.; Published by Cold Spring Harbor Laboratory Press.)

Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding proteins. Here, we benchmark the application of PROSS as a stand-alone tool for protein scientists with no or limited experience in modeling. Twelve laboratories from the Protein Production and Purification Partnership in Europe (P4EU) challenged the PROSS algorithm with 14 unrelated protein targets without support from the PROSS developers. For each target, up to six designs were evaluated for expression levels and in some cases, for thermal stability and activity. In nine targets, designs exhibited increased heterologous expression levels either in prokaryotic and/or eukaryotic expression systems under experimental conditions that were tailored for each target protein. Furthermore, we observed increased thermal stability in nine of ten tested targets. In two prime examples, the human Stem Cell Factor (hSCF) and human Cadherin-Like Domain (CLD12) from the RET receptor, the wild type proteins were not expressible as soluble proteins in E. coli, yet the PROSS designs exhibited high expression levels in E. coli and HEK293 cells, respectively, and improved thermal stability. We conclude that PROSS may improve stability and expressibility in diverse cases, and that improvement typically requires target-specific expression conditions. This study demonstrates the strengths of community-wide efforts to probe the generality of new methods and recommends areas for future research to advance practically useful algorithms for protein science., Competing Interests: Declaration of Competing Interest AG and SJF are named inventors on patents relating to the PROSS method and various designs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique has been extensively used within the scientific community due to its low sample consumption, ease of use, and ubiquitous applicability, MST/TRIC has not enjoyed the unambiguous acceptance from biophysicists afforded to other biophysical techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR). This might be attributed to several facts, e.g., that various (not fully understood) effects are contributing to the signal, that the technique is licensed to only a single instrument developer, NanoTemper Technology, and that its reliability and reproducibility have never been tested independently and systematically. Thus, a working group of ARBRE-MOBIEU has set up a benchmark study on MST/TRIC to assess this technique as a method to characterize biomolecular interactions. Here we present the results of this study involving 32 scientific groups within Europe and two groups from the US, carrying out experiments on 40 Monolith instruments, employing a standard operation procedure and centrally prepared samples. A protein-small molecule interaction, a newly developed protein-protein interaction system and a pure dye were used as test systems. We characterized the instrument properties and evaluated instrument performance, reproducibility, the effect of different analysis tools, the influence of the experimenter during data analysis, and thus the overall reliability of this method.

As the scientific community strives to make published results more transparent and reliable, it has become obvious that poor data reproducibility can often be attributed to insufficient quality control of experimental reagents. In this context, proteins and peptides reagents require much stricter quality controls than those routinely performed on them in a significant proportion of research laboratories. Members of the ARBRE-MOBIEU and the P4EU networks have combined their expertise to generate guidelines for the evaluation of purified proteins used in life sciences and medical trials. These networks, representing more than 150 laboratories specialized in protein production and/or protein molecular biophysics, have implemented such guidelines in their respective laboratories. Over a one-year period, the network members evaluated the contribution these guidelines made toward obtaining more productive, robust and reproducible research by correlating the applied quality controls to given samples with the reliability and reproducibility of the scientific data obtained using these samples in follow-up experiments. The results indicate that QC guideline implementation facilitates the optimization of the protein purification process and improves the reliability of downstream experiments. It seems, therefore, that investing in protein QC might be advantageous to all the stakeholders in life sciences (researchers, editors, and funding agencies alike), because this practice improves data veracity and minimizes loss of valuable time and resources. In the light of these conclusions, the network members suggest that the implementation of these simple QC guidelines should become minimal reporting practice in the publication of data derived from the use of protein and peptide reagents.

Suramin has been a primary early-stage treatment for African trypanosomiasis for nearly 100 yr. Recent studies revealed that trypanosome strains that express the variant surface glycoprotein (VSG) VSGsur possess heightened resistance to suramin. Here, we show that VSGsur binds tightly to suramin but other VSGs do not. By solving high-resolution crystal structures of VSGsur and VSG13, we also demonstrate that these VSGs define a structurally divergent subgroup of the coat proteins. The co-crystal structure of VSGsur with suramin reveals that the chemically symmetric drug binds within a large cavity in the VSG homodimer asymmetrically, primarily through contacts of its central benzene rings. Structure-based, loss-of-contact mutations in VSGsur significantly decrease the affinity to suramin and lead to a loss of the resistance phenotype. Altogether, these data show that the resistance phenotype is dependent on the binding of suramin to VSGsur, establishing that the VSG proteins can possess functionality beyond their role in antigenic variation.

Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease., Competing Interests: Declaration of Interests I.M., J.J.B., and T.C. are inventors on EU patent applications PCT/EP2018/078592 and PCT/EP2019/074885 filled by the Max Planck Society and related to the technology for site-specific generation of Ser-ADP-ribosylated peptides., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC 50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the 'up' ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD + concentration. We therefore hypothesized that defects of the NAD + salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD + salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD + levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.

Protein-protein and protein-ligand interactions often involve conformational changes or structural rearrangements that can be quantified by solution small-angle X-ray scattering (SAXS). These scattering intensity measurements reveal structural details of the bound complex, the number of species involved and, additionally, the strength of interactions if carried out as a titration. Although a core part of structural biology workflows, SAXS-based titrations are not commonly used in drug discovery contexts. This is because prior knowledge of expected sample requirements, throughput and prediction accuracy is needed to develop reliable ligand screens. This study presents the use of the histidine-binding protein (26 kDa) and other periplasmic binding proteins to benchmark ligand screen performance. Sample concentrations and exposure times were varied across multiple screening trials at four beamlines to investigate the accuracy and precision of affinity prediction. The volatility ratio between titrated scattering curves and a common apo reference is found to most reliably capture the extent of structural and population changes. This obviates the need to explicitly model scattering intensities of bound complexes, which can be strongly ligand-dependent. Where the dissociation constant is within 10 2 of the protein concentration and the total exposure times exceed 20 s, the titration protocol presented at 0.5 mg ml -1 yields affinities comparable to isothermal titration calorimetry measurements. Estimated throughput ranges between 20 and 100 ligand titrations per day at current synchrotron beamlines, with the limiting step imposed by sample handling and cleaning procedures., (© Po-chia Chen et al. 2020.)

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis. We identify a conserved phosphorylation site at T207 of BRCA2 that constitutes a bona fide docking site for PLK1 and is phosphorylated in mitotic cells. We show that BRCA2 bound to PLK1 forms a complex with the phosphatase PP2A and phosphorylated-BUBR1. Reducing BRCA2 binding to PLK1, as observed in BRCA2 breast cancer variants S206C and T207A, alters the tetrameric complex resulting in unstable kinetochore-microtubule interactions, misaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-mutated tumors.

The two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anticancer strategies., Competing Interests: GZ, IV, MS, LF, SD, AR, ST, MP, FB, NB, AE, AV, JC, MC, MP No competing interests declared, (© 2018, Zago et al.)

Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204 kDa; human ABL1 wildtype, 126 kDa; human FMRP, 68 kDa; viral vNS1-H1, 76 kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Efficient preparation of high-quality sequencing libraries that well represent the biological sample is a key step for using next-generation sequencing in research. Tn5 enables fast, robust, and highly efficient processing of limited input material while scaling to the parallel processing of hundreds of samples. Here, we present a robust Tn5 transposase purification strategy based on an N-terminal His 6 -Sumo3 tag. We demonstrate that libraries prepared with our in-house Tn5 are of the same quality as those processed with a commercially available kit (Nextera XT), while they dramatically reduce the cost of large-scale experiments. We introduce improved purification strategies for two versions of the Tn5 enzyme. The first version carries the previously reported point mutations E54K and L372P, and stably produces libraries of constant fragment size distribution, even if the Tn5-to-input molecule ratio varies. The second Tn5 construct carries an additional point mutation (R27S) in the DNA-binding domain. This construct allows for adjustment of the fragment size distribution based on enzyme concentration during tagmentation, a feature that opens new opportunities for use of Tn5 in customized experimental designs. We demonstrate the versatility of our Tn5 enzymes in different experimental settings, including a novel single-cell polyadenylation site mapping protocol as well as ultralow input DNA sequencing., (Copyright © 2018 Hennig et al.)

Condensin protein complexes coordinate the formation of mitotic chromosomes and thereby ensure the successful segregation of replicated genomes. Insights into how condensin complexes bind to chromosomes and alter their topology are essential for understanding the molecular principles behind the large-scale chromatin rearrangements that take place during cell divisions. Here, we identify a direct DNA-binding site in the eukaryotic condensin complex, which is formed by its Ycg1 Cnd3 HEAT-repeat and Brn1 Cnd2 kleisin subunits. DNA co-crystal structures reveal a conserved, positively charged groove that accommodates the DNA double helix. A peptide loop of the kleisin subunit encircles the bound DNA and, like a safety belt, prevents its dissociation. Firm closure of the kleisin loop around DNA is essential for the association of condensin complexes with chromosomes and their DNA-stimulated ATPase activity. Our data suggest a sophisticated molecular basis for anchoring condensin complexes to chromosomes that enables the formation of large-sized chromatin loops., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The RNA-chaperone Hfq catalyses the annealing of bacterial small RNAs (sRNAs) with target mRNAs to regulate gene expression in response to environmental stimuli. Hfq acts on a diverse set of sRNA-mRNA pairs using a variety of different molecular mechanisms. Here, we present an unusual crystal structure showing two Hfq-RNA complexes interacting via their bound RNA molecules. The structure contains two Hfq 6 :A 18 RNA assemblies positioned face-to-face, with the RNA molecules turned towards each other and connected via interdigitating base stacking interactions at the center. Biochemical data further confirm the observed interaction, and indicate that RNA-mediated contacts occur between Hfq-RNA complexes with various (ARN) X motif containing RNA sequences in vitro, including the stress response regulator OxyS and its target, fhlA. A systematic computational survey also shows that phylogenetically conserved (ARN) X motifs are present in a subset of sRNAs, some of which share similar modular architectures. We hypothesise that Hfq can co-opt RNA-RNA base stacking, an unanticipated structural trick, to promote the interaction of (ARN) X motif containing sRNAs with target mRNAs on a "speed-dating" fashion, thereby supporting their regulatory function.

The quantity of milk and milk fat and proteins are particularly important traits in dairy livestock. However, little is known about the regions of the genome that influence these traits in goats. We conducted a genome wide association study in French goats and identified 109 regions associated with dairy traits. For a major region on chromosome 14 closely associated with fat content, the Diacylglycerol O-Acyltransferase 1 (DGAT1) gene turned out to be a functional and positional candidate gene. The caprine reference sequence of this gene was completed and 29 polymorphisms were found in the gene sequence, including two novel exonic mutations: R251L and R396W, leading to substitutions in the protein sequence. The R251L mutation was found in the Saanen breed at a frequency of 3.5% and the R396W mutation both in the Saanen and Alpine breeds at a frequencies of 13% and 7% respectively. The R396W mutation explained 46% of the genetic variance of the trait, and the R251L mutation 6%. Both mutations were associated with a notable decrease in milk fat content. Their causality was then demonstrated by a functional test. These results provide new knowledge on the genetic basis of milk synthesis and will help improve the management of the French dairy goat breeding program.

Recombinant expression of heterologous proteins in E. coli is well established for a wide range of proteins, although in many cases, purifying soluble and properly folded proteins remains challenging (Sorensen and Mortensen, J Biotechnol 115:113-128, 2005; Correa and Oppezzo, Methods Mol Biol 1258:27-44, 2015). Proteins that contain disulfide bonds (e.g., cytokines, growth factors) are often particularly difficult to purify in soluble form and still need optimizing of protocols in almost every step of the process (Berkmen, Protein Expr Purif 82:240-251, 2012; de Marco, Microb Cell Fact 11:129, 2012). Expression of disulfide bonded proteins in the periplasm of E. coli is one approach that can help to obtain soluble protein with the correct disulfide bridges forming in the periplasm. This offers the appropriate conditions for disulfide formation although periplasmic expression can also result in low expression levels and incorrect folding of the target protein (Schlapschy and Skerra, Methods Mol Biol 705:211-224, 2011). Generation of specific antibodies often requires a specific antigenic sequence of a protein in order to get an efficient immune response and minimize cross-reactivity of antibodies. Larger proteins like GST (Glutathione-S-transferase) or MBP (maltose binding protein) as solubilizing fusion partners are frequently used to keep antigens soluble and immunize animals. This approach has the disadvantage that the immune response against the fusion partner leads to additional antibodies that need to be separated from the antigen-specific antibodies. For both classes of proteins mentioned above, a protocol has been developed and optimized using the human version of small ubiquitin-like modifier 3 (SUMO3) protein and its corresponding protease SenP2. This chapter describes the experimental steps for expression, purification, refolding, and cleavage that are applicable to both disulfide-bonded proteins with a defined structure and random protein fragments for antibody generation or larger peptides with defined sequence that are difficult express on their own.

Gene synthesis enables the exploitation of the degeneracy of the genetic code to boost expression of recombinant protein targets for structural studies. This has created new opportunities to obtain structural information on proteins that are normally present in low abundance. Unfortunately, synthetic gene expression occasionally leads to insoluble or misfolded proteins. This could be remedied by recent insights in the effect of codon usage on translation initiation and elongation. In this review, we discuss the interplay between optimal gene and vector design to enhance expression in a particular host and highlight the benefits and potential pitfalls associated with protein expression from synthetic genes., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Many cellular processes involve the recruitment of proteins to specific membranes, which are decorated with distinctive lipids that act as docking sites. The phosphoinositides form signaling hubs, and we examine mechanisms underlying recruitment. We applied a physiological, quantitative, liposome microarray-based assay to measure the membrane-binding properties of 91 pleckstrin homology (PH) domains, the most common phosphoinositide-binding target. 10,514 experiments quantified the role of phosphoinositides in membrane recruitment. For most domains examined, the observed binding specificity implied cooperativity with additional signaling lipids. Analyses of PH domains with similar lipid-binding profiles identified a conserved motif, mutations in which-including some found in human cancers-induced discrete changes in binding affinities in vitro and protein mislocalization in vivo. The data set reveals cooperativity as a key mechanism for membrane recruitment and, by enabling the interpretation of disease-associated mutations, suggests avenues for the design of small molecules targeting PH domains., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.

In many animals, the germ plasm segregates germline from soma during early development. Oskar protein is known for its ability to induce germ plasm formation and germ cells in Drosophila. However, the molecular basis of germ plasm formation remains unclear. Here, we show that Oskar is an RNA-binding protein in vivo, crosslinking to nanos, polar granule component, and germ cell-less mRNAs, each of which has a role in germline formation. Furthermore, we present high-resolution crystal structures of the two Oskar domains. RNA-binding maps in vitro to the C-terminal domain, which shows structural similarity to SGNH hydrolases. The highly conserved N-terminal LOTUS domain forms dimers and mediates Oskar interaction with the germline-specific RNA helicase Vasa in vitro. Our findings suggest a dual function of Oskar in RNA and Vasa binding, providing molecular clues to its germ plasm function., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG) for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284∶26646-26654.) Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19) toward the oncofetal fibronectin (B-FN), a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50-70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG), 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80 mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staining of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compounds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.