ARF1 prevents aberrant type I interferon induction by regulating STING activation and recycling.

Type I interferon (IFN) signalling is tightly controlled. Upon recognition of DNA by cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) translocates along the endoplasmic reticulum (ER)-Golgi axis to induce IFN signalling. Termination is achieved through autophagic degradation or recycling of STING by retrograde Golgi-to-ER transport. Here, we identify the GTPase ADP-ribosylation factor 1 (ARF1) as a crucial negative regulator of cGAS-STING signalling. Heterozygous ARF1 missense mutations cause a previously unrecognized type I interferonopathy associated with enhanced IFN-stimulated gene expression. Disease-associated, GTPase-defective ARF1 increases cGAS-STING dependent type I IFN signalling in cell lines and primary patient cells. Mechanistically, mutated ARF1 perturbs mitochondrial morphology, causing cGAS activation by aberrant mitochondrial DNA release, and leads to accumulation of active STING at the Golgi/ERGIC due to defective retrograde transport. Our data show an unexpected dual role of ARF1 in maintaining cGAS-STING homeostasis, through promotion of mitochondrial integrity and STING recycling.

A noncanonical enzymatic function of PIWIL4 maintains genomic integrity and leukemic growth in AML.

RNA-binding proteins (RBPs) form a large and diverse class of factors, many members of which are overexpressed in hematologic malignancies. RBPs participate in various processes of messenger RNA (mRNA) metabolism and prevent harmful DNA:RNA hybrids or R-loops. Here, we report that PIWIL4, a germ stem cell-associated RBP belonging to the RNase H-like superfamily, is overexpressed in patients with acute myeloid leukemia (AML) and is essential for leukemic stem cell function and AML growth, but dispensable for healthy human hematopoietic stem cells. In AML cells, PIWIL4 binds to a small number of known piwi-interacting RNA. Instead, it largely interacts with mRNA annotated to protein-coding genic regions and enhancers that are enriched for genes associated with cancer and human myeloid progenitor gene signatures. PIWIL4 depletion in AML cells downregulates the human myeloid progenitor signature and leukemia stem cell (LSC)-associated genes and upregulates DNA damage signaling. We demonstrate that PIWIL4 is an R-loop resolving enzyme that prevents R-loop accumulation on a subset of AML and LSC-associated genes and maintains their expression. It also prevents DNA damage, replication stress, and activation of the ATR pathway in AML cells. PIWIL4 depletion potentiates sensitivity to pharmacological inhibition of the ATR pathway and creates a pharmacologically actionable dependency in AML cells.

Process- and product-related impurities in the ChAdOx1 nCov-19 vaccine.

ChAdOx1 nCov-19 and Ad26.COV2.S are approved vaccines inducing protective immunity against SARS-CoV-2 infection in humans by expressing the Spike protein of SARS-CoV-2. We analyzed protein content and protein composition of ChAdOx1 nCov-19 and Ad26.COV2.S by biochemical methods and by mass spectrometry. Four out of four tested lots of ChAdOx1 nCoV-19 contained significantly higher than expected levels of host cell proteins (HCPs) and of free viral proteins. The most abundant contaminating HCPs belonged to the heat-shock protein and cytoskeletal protein families. The HCP content exceeded the 400 ng specification limit per vaccine dose, as set by the European Medicines Agency (EMA) for this vaccine, by at least 25-fold and the manufacturer's batch-release data in some of the lots by several hundred-fold. In contrast, three tested lots of the Ad26.COV2.S vaccine contained only very low amounts of HCPs. As shown for Ad26.COV2.S production of clinical grade adenovirus vaccines of high purity is feasible at an industrial scale. Correspondingly, purification procedures of the ChAdOx1 nCov-19 vaccine should be modified to remove protein impurities as good as possible. Our data also indicate that standard quality assays, as they are used in the manufacturing of proteins, have to be adapted for vectored vaccines.

Biochemical Characterization of a Human Septin Octamer.

Septins are part of the cytoskeleton and polymerize into non-polar filaments of heteromeric hexamers or octamers. They belong to the class of P-loop GTPases but the roles of GTP binding and hydrolysis on filament formation and dynamics are not well understood. The basic human septin building block is the septin rod, a hetero-octamer composed of SEPT2, SEPT6, SEPT7, and SEPT9 with a stoichiometry of 2:2:2:2 (2-6-7-9-9-7-6-2). Septin rods polymerize by end-to-end and lateral joining into linear filaments and higher ordered structures such as rings, sheets, and gauzes. We purified a recombinant human septin octamer from E. coli for in vitro experimentation that is able to polymerize into filaments. We could show that the C-terminal region of the central SEPT9 subunit contributes to filament formation and that the human septin rod decreases the rate of in vitro actin polymerization. We provide further first kinetic data on the nucleotide uptake- and exchange properties of human hexameric and octameric septin rods. We could show that nucleotide uptake prior to hydrolysis is a dynamic process and that a bound nucleotide is exchangeable. However, the hydrolyzed γ-phosphate is not released from the native protein complex. We consequently propose that GTP hydrolysis in human septins does not follow the typical mechanism known from other small GTPases.

Pancreatic cancer-derived organoids - a disease modeling tool to predict drug response.

BACKGROUND: Organotypic cultures derived from pancreatic ductal adenocarcinoma (PDAC) termed pancreatic ductal cancer organoids (PDOs) recapitulate the primary cancer and can be derived from primary or metastatic biopsies. Although isolation and culture of patient-derived pancreatic organoids were established several years ago, pros and cons for individualized medicine have not been comprehensively investigated to date. METHODS: We conducted a feasibility study, systematically comparing head-to-head patient-derived xenograft tumor (PDX) and PDX-derived organoids by rigorous immunohistochemical and molecular characterization. Subsequently, a drug testing platform was set up and validated in vivo. Patient-derived organoids were investigated as well. RESULTS: First, PDOs faithfully recapitulated the morphology and marker protein expression patterns of the PDXs. Second, quantitative proteomes from the PDX as well as from corresponding organoid cultures showed high concordance. Third, genomic alterations, as assessed by array-based comparative genomic hybridization, revealed similar results in both groups. Fourth, we established a small-scale pharmacotyping platform adjusted to operate in parallel considering potential obstacles such as culture conditions, timing, drug dosing, and interpretation of the results. In vitro predictions were successfully validated in an in vivo xenograft trial. Translational proof-of-concept is exemplified in a patient with PDAC receiving palliative chemotherapy. CONCLUSION: Small-scale drug screening in organoids appears to be a feasible, robust and easy-to-handle disease modeling method to allow response predictions in parallel to daily clinical routine. Therefore, our fast and cost-efficient assay is a reasonable approach in a predictive clinical setting.

Noncatalytic Bruton's tyrosine kinase activates PLCγ2 variants mediating ibrutinib resistance in human chronic lymphocytic leukemia cells.

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+] i ), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+] i Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.

MicroRNA-708 is a novel regulator of the Hoxa9 program in myeloid cells.

MicroRNAs (miRNAs) are commonly deregulated in acute myeloid leukemia (AML), affecting critical genes not only through direct targeting, but also through modulation of downstream effectors. Homeobox (Hox) genes balance self-renewal, proliferation, cell death, and differentiation in many tissues and aberrant Hox gene expression can create a predisposition to leukemogenesis in hematopoietic cells. However, possible linkages between the regulatory pathways of Hox genes and miRNAs are not yet fully resolved. We identified miR-708 to be upregulated in Hoxa9/Meis1 AML inducing cell lines as well as in AML patients. We further showed Meis1 directly targeting miR-708 and modulating its expression through epigenetic transcriptional regulation. CRISPR/Cas9 mediated knockout of miR-708 in Hoxa9/Meis1 cells delayed disease onset in vivo, demonstrating for the first time a pro-leukemic contribution of miR-708 in this context. Overexpression of miR-708 however strongly impeded Hoxa9 mediated transformation and homing capacity in vivo through modulation of adhesion factors and induction of myeloid differentiation. Taken together, we reveal miR-708, a putative tumor suppressor miRNA and direct target of Meis1, as a potent antagonist of the Hoxa9 phenotype but an effector of transformation in Hoxa9/Meis1. This unexpected finding highlights the yet unexplored role of miRNAs as indirect regulators of the Hox program during normal and aberrant hematopoiesis.

The ParaHox gene Cdx4 induces acute erythroid leukemia in mice.

Acute erythroid leukemia (AEL) is a rare and aggressive form of acute leukemia, the biology of which remains poorly understood. Here we demonstrate that the ParaHox gene CDX4 is expressed in patients with acute erythroid leukemia, and that aberrant expression of Cdx4 induced homogenously a transplantable acute erythroid leukemia in mice. Gene expression analyses demonstrated upregulation of genes involved in stemness and leukemogenesis, with parallel downregulation of target genes of Gata1 and Gata2 responsible for erythroid differentiation. Cdx4 induced a proteomic profile that overlapped with a cluster of proteins previously defined to represent the most primitive human erythroid progenitors. Whole-exome sequencing of diseased mice identified recurrent mutations significantly enriched for transcription factors involved in erythroid lineage specification, as well as TP53 target genes partly identical to the ones reported in patients with AEL. In summary, our data indicate that Cdx4 is able to induce stemness and inhibit terminal erythroid differentiation, leading to the development of AEL in association with co-occurring mutations.

Morphological and primary structural consistency of fibrils from different AA patients (common variant).

Aims: To test the hypothesis that the fibril morphology and the fibril protein primary structure are conserved across different patients suffering from the common variant of systemic Amyloid A (AA) amyloidosis. Methods: Amyloid fibrils were extracted from the renal tissue of four patients. The fibril morphology was analysed in negatively stained samples with transmission electron microscopy (TEM). The fibril protein identity and fragment length were determined by using mass spectrometry. Results: The fibrils show a consistent morphology in all four patients and exhibit an average width of ∼9.6 nm and an average pitch of ∼112 nm. All fibrils are composed of polypeptide chains that can be assigned to human serum amyloid A (SAA) 1.1 protein. All fragments lack the N-terminal arginine residue and are C-terminally truncated. Differences exist concerning the exact C-terminal cleavage site. The most prominent cleavage site occurs at residues 64-67. Conclusions: Our data demonstrate that AA amyloid fibrils are consistent at the level of the protein primary structure and fibril morphology in the four analysed patients.

An Interaction Network of the Human SEPT9 Established by Quantitative Mass Spectrometry.

Septins regulate the organization of the actin cytoskeleton, vesicle transport and fusion, chromosome alignment and segregation, and cytokinesis in mammalian cells. SEPT9 is part of the core septin hetero-octamer in human cells which is composed of SEPT2, SEPT6, SEPT7, and SEPT9. SEPT9 has been linked to a variety of intracellular functions as well as to diseases and diverse types of cancer. A targeted high-throughput approach to systematically identify the interaction partners of SEPT9 has not yet been performed. We applied a quantitative proteomics approach to establish an interactome of SEPT9 in human fibroblast cells. Among the newly identified interaction partners were members of the myosin family and LIM domain containing proteins. Fluorescence microscopy of SEPT9 and its interaction partners provides additional evidence that SEPT9 might participate in vesicle transport from and to the plasma membrane as well as in the attachment of actin stress fibers to cellular adhesions.

Dissecting the nucleotide binding properties of the septins from S. cerevisiae.

Septins are a conserved family of guanosine triphosphate (GTP)-binding proteins that assemble into an ordered array of filaments at the mother bud neck in Saccharomyces cerevisiae cells. They are present in all higher eukaryotes except plants. Septins belong structurally to the P-Loop nucleoside triphosphatase (NTPases) like Rab and Ras. However, unlike other small guanosine triphosphatase (GTPases) septins are supposed to act as scaffolds rather than signalling mediators. This is why they are considered as the fourth class of cytoskeletal proteins. It is assumed that septins fulfil their functions independently of the bound nucleotide. The role of guanosine diphosphosphate (GDP) and GTP binding and subsequent hydrolysis was controversial debated in the last couple of years. Lack of crystal structures of yeast septin subunits or rods and difficulties to isolate single monomeric septin subunits often hindered the correlation of results obtained from in vivo studies with biochemical data. Recently, nucleotide binding and hydrolysis was connected to the formation of septin rods from its subunits. However, the evidence was only indirectly obtained through the use of septin mutants in the context of intact cells. We provide here mechanistic insight into the nucleotide binding of the yeast septins by in vitro assays using purified septin rods and building blocks, thereby adding further insights to the already available models on septin filament formation.

SPLIFF: A Single-Cell Method to Map Protein-Protein Interactions in Time and Space.

Protein interactions occur at certain times and at specific cellular places. The past years have seen a massive accumulation of binary protein-protein interaction data. The rapid increase of this context-free information necessitates robust methods to monitor protein interactions with temporal and spatial resolution in single cells. We have developed a simple split-ubiquitin-based method (SPLIFF) that uses the ratio of two fluorescent reporters as a signal for protein-protein interactions. One protein of the pair of interest is attached to the linear fusion of mCherry, the C-terminal half of ubiquitin, and GFP (mCherry-Cub-GFP). The other potential binding partner is expressed as a C-terminal fusion to the N-terminal half of ubiquitin (Nub). Upon co-expression the interaction between the two proteins of interest induces the reassociation of Nub and Cub to the native-like ubiquitin. GFP is subsequently cleaved from the C-terminus of Cub and degraded whereas the red-fluorescent mCherry stays attached to the Cub-fusion protein. We first implemented this method in the model yeast Saccharomyces cerevisiae. One fusion protein is expressed in cells of the a-mating type and the complementary fusion protein in cells of the α-mating type. Upon mixing, both cell types fuse and the Nub- and Cub-fusion proteins are free to interact. The red and green fluorescence is monitored by two-channel fluorescence time-lapse microcopy. The moment of cell fusion defines the start of the analysis. The calculated ratio of green to red fluorescence allows mapping the spatiotemporal interaction profiles of the investigated proteins in single cells.

A protein complex containing Epo1p anchors the cortical endoplasmic reticulum to the yeast bud tip.

The cortical endoplasmic reticulum (cER) of yeast underlies the plasma membrane (PM) at specific contact sites to enable a direct transfer of information and material between both organelles. During budding, directed movement of cER to the young bud followed by subsequent anchorage at its tip ensures the faithful inheritance of this organelle. The ER membrane protein Scs2p tethers the cER to the PM and to the bud tip through so far unknown receptors. We characterize Epo1p as a novel member of the polarisome that interacts with Scs2p exclusively at the cell tip during bud growth and show that Epo1p binds simultaneously to the Cdc42p guanosine triphosphatase-activating protein Bem3p. Deletion of EPO1 or deletion of BEM3 in a polarisome-deficient strain reduces the amount of cER at the tip. This analysis therefore identifies Epo1p as a novel and important component of the polarisome that promotes cER tethering at sites of polarized growth.

Detection of antibodies against paramyxoviruses in tortoises.

Sera from a total of 202 tortoises from six countries and nine species were tested for antibodies against four different reptilian paramyxoviruses (ferlaviruses, ferlaVs) by hemagglutination inhibition (HI) test. The viruses used were a tortoise PMV (tPMV) and three squamatid PMV isolates, each belonging to a different subgroup of ferlaV within the genus Ferlavirus. HI tests revealed that antibodies against ferlaVs occurred regularly in the tested samples (5.5%). One and a half percent of the tested samples have measurable antibody titers against the group A isolate, 3% had antibodies against the group B isolate, and 1% had antibodies against the group C isolate. The significantly highest number of positive reactions was detected against the tortoise isolate (5%). Most of the animals that tested positive for one of the snake isolates also tested positive in HI assays with the tortoise isolate. Of the samples from different origins, the sera from Great Britain showed the highest percentage of positive tested animals (10.3%, n = 39), followed by those from Spain (10%, n = 10), while none of the samples from Madagascar or Italy scored positive. Since in most cases animals from one country came from the same collection, this does not represent the real prevalence of ferlaV in tortoises in these countries but rather indicates that ferlaVs occur in a number of different countries and tortoise species.