Mutant PTEN in Cancer: Worse Than Nothing.

Tumor suppressors block the development of cancer and are often lost during tumor development. Papa et al. show that partial loss of normal PTEN tumor suppressor function can be compounded by additional disruption caused by the expression of inactive mutant PTEN protein. This has significant implications for patients with PTEN gene mutations.

Expanding the molecular spectrum of pathogenic SHOC2 variants underlying Mazzanti syndrome.

We previously molecularly and clinically characterized Mazzanti syndrome, a RASopathy related to Noonan syndrome that is mostly caused by a single recurrent missense variant (c.4A > G, p.Ser2Gly) in SHOC2, which encodes a leucine-rich repeat-containing protein facilitating signal flow through the RAS-mitogen-associated protein kinase (MAPK) pathway. We also documented that the pathogenic p.Ser2Gly substitution causes upregulation of MAPK signaling and constitutive targeting of SHOC2 to the plasma membrane due to the introduction of an N-myristoylation recognition motif. The almost invariant occurrence of the pathogenic c.4A > G missense change in SHOC2 is mirrored by a relatively homogeneous clinical phenotype of Mazzanti syndrome. Here, we provide new data on the clinical spectrum and molecular diversity of this disorder and functionally characterize new pathogenic variants. The clinical phenotype of six unrelated individuals carrying novel disease-causing SHOC2 variants is delineated, and public and newly collected clinical data are utilized to profile the disorder. In silico, in vitro and in vivo characterization of the newly identified variants provides evidence that the consequences of these missense changes on SHOC2 functional behavior differ from what had been observed for the canonical p.Ser2Gly change but converge toward an enhanced activation of the RAS-MAPK pathway. Our findings expand the molecular spectrum of pathogenic SHOC2 variants, provide a more accurate picture of the phenotypic expression associated with variants in this gene and definitively establish a gain-of-function behavior as the mechanism of disease.

Thermal Proteome Profiling in Zebrafish Reveals Effects of Napabucasin on Retinoic Acid Metabolism.

Thermal proteome profiling (TPP) allows for the unbiased detection of drug-target protein engagements in vivo. Traditionally, 1 cell type is used for TPP studies, with the risk of missing important differentially expressed target proteins. The use of whole organisms would circumvent this problem. Zebrafish embryos are amenable to such an approach. Here, we used TPP on whole zebrafish embryo lysate to identify protein targets of napabucasin, a compound that may affect signal transducer and activator of transcription 3 (Stat3) signaling through an ill-understood mechanism. In zebrafish embryos, napabucasin induced developmental defects consistent with inhibition of Stat3 signaling. TPP profiling showed no distinct shift in Stat3 upon napabucasin treatment, but effects were detected on the oxidoreductase, Pora, which might explain effects on Stat3 signaling. Interestingly, thermal stability of several aldehyde dehydrogenases was affected. Moreover, napabucasin activated aldehyde dehydrogenase enzymatic activity in vitro. Aldehyde dehydrogenases have crucial roles in retinoic acid metabolism, and functionally, we validated napabucasin-mediated activation of the retinoic acid pathway in zebrafish in vivo. We conclude that TPP profiling in whole zebrafish embryo lysate is feasible and facilitates direct correlation of in vivo effects of small molecule drugs with their protein targets.

The seventh international RASopathies symposium: Pathways to a cure-expanding knowledge, enhancing research, and therapeutic discovery.

RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed.

Protein tyrosine phosphatase alpha inhibits hypothalamic leptin receptor signaling and regulates body weight in vivo.

Understanding how body weight is regulated at the molecular level is essential for treating obesity. We show that female mice genetically lacking protein tyrosine phosphatase (PTP) receptor type α (PTPRA) exhibit reduced weight and adiposity and increased energy expenditure, and are more resistant to diet-induced obesity than matched wild-type control mice. These mice also exhibit reduced levels of circulating leptin and are leptin hypersensitive, suggesting that PTPRA inhibits leptin signaling in the hypothalamus. Male and female PTPRA-deficient mice fed a high-fat diet were leaner and displayed increased metabolic rates and lower circulating leptin levels, indicating that the effects of loss of PTPRA persist in the obese state. Molecularly, PTPRA down-regulates leptin receptor signaling by dephosphorylating the receptor-associated kinase JAK2, with which the phosphatase associates constitutively. In contrast to the closely related tyrosine phosphatase ε, leptin induces only weak phosphorylation of PTPRA at its C-terminal regulatory site Y789, and this does not affect the activity of PTPRA toward JAK2. PTPRA is therefore an inhibitor of hypothalamic leptin signaling in vivo and may prevent premature activation of leptin signaling, as well as return signaling to baseline after exposure to leptin.-Cohen-Sharir, Y., Kuperman, Y., Apelblat, D., den Hertog, J., Spiegel, I., Knobler, H., Elson, A. Protein tyrosine phosphatase alpha inhibits hypothalamic leptin receptor signaling and regulates body weight in vivo.

Hubrecht Institute Centennial - From embryos to stem cells.

During the last 100 years, the Hubrecht Institute evolved from a small laboratory aimed at providing research material to the scientific community to a modern, fully equipped research institute with state-of-the-art infrastructure, performing research at the highest standard. The past 100 years have been eventful for the Hubrecht Institute with many glorious moments, but also threats to be shut down on several occasions. Here, we will briefly review the rich history of the Hubrecht Institute.

Recent advances in understanding the role of protein-tyrosine phosphatases in development and disease.

Protein-tyrosine phosphatases (PTPs) remove phosphate groups from tyrosine residues, and thereby propagate or inhibit signal transduction, and hence influence cellular processes such as cell proliferation and differentiation. The importance of tightly controlled PTP activity is reflected by the numerous mechanisms employed by the cell to control PTP activity, including a variety of post-translational modifications, and restricted subcellular localization. This review highlights the strides made in the last decade and discusses the important role of PTPs in key aspects of embryonic development: the regulation of stem cell self-renewal and differentiation, gastrulation and somitogenesis during early embryonic development, osteogenesis, and angiogenesis. The tentative importance of PTPs in these processes is highlighted by the diseases that present upon aberrant activity.

Noonan and LEOPARD syndrome Shp2 variants induce heart displacement defects in zebrafish.

Germline mutations in PTPN11, encoding Shp2, cause Noonan syndrome (NS) and LEOPARD syndrome (LS), two developmental disorders that are characterized by multiple overlapping symptoms. Interestingly, Shp2 catalytic activity is enhanced by NS mutations and reduced by LS mutations. Defective cardiac development is a prominent symptom of both NS and LS, but how the Shp2 variants affect cardiac development is unclear. Here, we have expressed the most common NS and LS Shp2-variants in zebrafish embryos to investigate their role in cardiac development in vivo. Heart function was impaired in embryos expressing NS and LS variants of Shp2. The cardiac anomalies first occurred during elongation of the heart tube and consisted of reduced cardiomyocyte migration, coupled with impaired leftward heart displacement. Expression of specific laterality markers was randomized in embryos expressing NS and LS variants of Shp2. Ciliogenesis and cilia function in Kupffer's vesicle was impaired, likely accounting for the left/right asymmetry defects. Mitogen-activated protein kinase (MAPK) signaling was activated to a similar extent in embryos expressing NS and LS Shp2 variants. Interestingly, inhibition of MAPK signaling prior to gastrulation rescued cilia length and heart laterality defects. These results suggest that NS and LS Shp2 variant-mediated hyperactivation of MAPK signaling leads to impaired cilia function in Kupffer's vesicle, causing left-right asymmetry defects and defective early cardiac development.

Pivotal role of Pten in the balance between proliferation and differentiation of hematopoietic stem cells in zebrafish.

Self-renewing hematopoietic stem/progenitor cells (HSPCs) produce blood cells of all lineages throughout life. Phosphatase and tensin homolog (PTEN), a tumor suppressor that antagonizes phosphatidylinositol 3-kinase (PI3K) signaling, is frequently mutated in hematologic malignancies such as bone marrow failure and leukemia. We set out to investigate whether Pten is required for hematopoiesis. Analysis of zebrafish mutants lacking functional Pten revealed that HSPCs colonized the caudal hematopoietic tissue normally. There, HSPCs hyperproliferated and engaged in all blood lineages. However, they failed to differentiate into mature blood cells. Hence, Pten mutant zebrafish embryos displayed hallmarks of leukemia in humans. Inhibition of PI3K signaling in mutants lacking functional Pten suppressed hyperproliferation and released the differentiation arrest. We conclude that Pten has an essential role in the balance between proliferation and differentiation of blood cells.

Comparative interactomics analysis of different ALS-associated proteins identifies converging molecular pathways.

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment available. An increasing number of genetic causes of ALS are being identified, but how these genetic defects lead to motor neuron degeneration and to which extent they affect common cellular pathways remains incompletely understood. To address these questions, we performed an interactomic analysis to identify binding partners of wild-type (WT) and ALS-associated mutant versions of ATXN2, C9orf72, FUS, OPTN, TDP-43 and UBQLN2 in neuronal cells. This analysis identified several known but also many novel binding partners of these proteins. Interactomes of WT and mutant ALS proteins were very similar except for OPTN and UBQLN2, in which mutations caused loss or gain of protein interactions. Several of the identified interactomes showed a high degree of overlap: shared binding partners of ATXN2, FUS and TDP-43 had roles in RNA metabolism; OPTN- and UBQLN2-interacting proteins were related to protein degradation and protein transport, and C9orf72 interactors function in mitochondria. To confirm that this overlap is important for ALS pathogenesis, we studied fragile X mental retardation protein (FMRP), one of the common interactors of ATXN2, FUS and TDP-43, in more detail in in vitro and in vivo model systems for FUS ALS. FMRP localized to mutant FUS-containing aggregates in spinal motor neurons and bound endogenous FUS in a direct and RNA-sensitive manner. Furthermore, defects in synaptic FMRP mRNA target expression, neuromuscular junction integrity, and motor behavior caused by mutant FUS in zebrafish embryos, could be rescued by exogenous FMRP expression. Together, these results show that interactomics analysis can provide crucial insight into ALS disease mechanisms and they link FMRP to motor neuron dysfunction caused by FUS mutations.

Pten function in zebrafish: anything but a fish story.

Zebrafish is an excellent model system for the analysis of gene function. We and others use zebrafish to investigate the function of the tumor suppressor, Pten, in tumorigenesis and embryonic development. Zebrafish have two pten genes, ptena and ptenb. The recently identified N-terminal extension of human PTEN that may facilitate cell membrane transfer, appears not to be conserved in zebrafish Ptena or Ptenb. Mutants that retain a single wild type pten allele develop tumors, predominantly hemangiosarcomas. Homozygous double mutants are embryonic lethal. Zebrafish embryos lacking functional Pten display enhanced proliferation of endothelial cells, resulting in hyperbranching of blood vessels. In addition, ptena-/-ptenb-/- mutant embryos display enhanced proliferation of hematopoietic stem and progenitor cells and concomitant arrest of differentiation, although Pten-deficient cells commit to all blood cell lineages. Zebrafish is an ideal model for intravital imaging and future work using ptena-/-ptenb-/- mutants will enhance our understanding of the function of Pten in vivo.

Tumor Suppressors in Zebrafish: From TP53 to PTEN and Beyond.

Zebrafish are increasingly being used to study cancer. Almost all tumor types have been found in zebrafish. However, tumor incidence is relatively low and tumors develop late in life. Functional inactivation of tumor suppressors is a crucial step in cancer progression and more and more tumor suppressor genes are being studied in zebrafish. Most often tumor suppressors have been inactivated by reverse genetics approaches using targeted disruption. However, some tumor suppressor mutants were identified by forward genetic screens for mutants with a particular phenotype. Some of the latter genes had not been recognized as tumor suppressors yet. Similarly, a screen for genes that suppress tumor formation in zebrafish in vivo led to the identification of a novel tumor suppressor gene. In this review, I will provide an overview of what the zebrafish has taught us about tumor suppressors.

Deficiency in hematopoietic phosphatase ptpn6/Shp1 hyperactivates the innate immune system and impairs control of bacterial infections in zebrafish embryos.

Deficiency in Src homology region 2 domain-containing phosphatase 1/protein tyrosine phosphatase nonreceptor type 6 (SHP1/PTPN6) is linked with chronic inflammatory diseases and hematological malignancies in humans. In this study, we exploited the embryonic and larval stages of zebrafish (Danio rerio) as an animal model to study ptpn6 function in the sole context of innate immunity. We show that ptpn6 knockdown induces a spontaneous inflammation-associated phenotype at the late larval stage. Surprisingly, glucocorticoid treatment did not suppress inflammation under ptpn6 knockdown conditions but further enhanced leukocyte infiltration and proinflammatory gene expression. Experiments in a germ-free environment showed that the late larval phenotype was microbe independent. When ptpn6 knockdown embryos were challenged with Salmonella typhimurium or Mycobacterium marinum at earlier stages of development, the innate immune system was hyperactivated to a contraproductive level that impaired the control of these pathogenic bacteria. Transcriptome analysis demonstrated that Kyoto Encyclopedia of Genes and Genomes pathways related to pathogen recognition and cytokine signaling were significantly enriched under these conditions, suggesting that ptpn6 functions as a negative regulator that imposes a tight control over the level of innate immune response activation during infection. In contrast to the hyperinduction of proinflammatory cytokine genes under ptpn6 knockdown conditions, anti-inflammatory il10 expression was not hyperinduced. These results support that ptpn6 has a crucial regulatory function in preventing host-detrimental effects of inflammation and is essential for a successful defense mechanism against invading microbes.

Zebrafish as a model to study PTPs during development.

Protein-tyrosine phosphatases (PTPs) have important roles in signaling, but relatively little is known about their function in vivo. We are using the zebrafish as a model to study the function of PTPs at the organismal, cellular and molecular level. The zebrafish is an excellent experimental model for the analysis of gene function. We have developed methods to quantitatively study effects of PTP knockdown or expression of (mutant) PTPs, particularly with respect to gastrulation cell movements. Moreover, we have studied the phosphoproteome of zebrafish embryos. In this review, we will discuss methods to manipulate the zebrafish genome and techniques that we have developed to assess developmental defects during gastrulation and to assess differences in the phosphoproteome.

A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates.

A versatile assay for protein tyrosine phosphatases (PTP) employing 3-nitrophosphotyrosine containing peptidic substrates is described. These therapeutically important phosphatases feature in signal transduction pathways. The assay involves spectrophotometric detection of 3-nitrotyrosine production from 3-nitrophosphotyrosine containing peptidic substrates, which are accepted by many PTPs. Compared to conventional chromogenic phosphate derivatives, the more realistic peptidic substrates allow evaluating substrate specificity. The assay's applicability is demonstrated by determining kinetic parameters for several PTP-substrate combinations and inhibitor evaluation, as well as detection of PTP activity in lysates. The convenient new assay may assist further adoption of PTPs in drug development.

Protein Tyrosine Phosphatase Studies in Zebrafish.

The zebrafish is an ideal model for functional analysis of genes at the molecular, protein, cell, organ, and organism levels. We have used zebrafish to analyze the function of members of the protein tyrosine phosphatase (PTP) superfamily for more than two decades. The molecular genetic toolbox has significantly improved over the years. Currently, generating mutant lines that lack the function of a PTP gene is relatively straightforward by CRISPR/Cas9 technology-mediated generation of insertions or deletions in the target gene. In addition, generating point mutations using CRISPR/Cas9 technology and homology-directed repair (HDR) is feasible, albeit the success rate could be higher. Here, we describe the methods, including the tips and tricks, that we have used to generate knock-out and knock-in zebrafish lines in PTP genes successfully.

Harzianic acid exerts antimicrobial activity against Gram-positive bacteria and targets the cell membrane.

The thermophilic fungus Oidiodendron flavum is a saprobe that is commonly isolated from soil. Here, we identified a Gram-positive bacteria-selective antimicrobial secondary metabolite from this fungal species, harzianic acid (HA). Using Bacillus subtilis strain 168 combined with dynamic bacterial morphology imaging, we found that HA targeted the cell membrane. To further study the antimicrobial activity of HA, we isolated an HA-resistant strain, Bacillus subtilis strain M9015, and discovered that the mutant had more translucent colonies than the wild type strain, showed cross resistance to rifampin, and harbored five mutations in the coding region of four distinct genes. Further analysis of these genes indicated that the mutation in atpE might be responsible for the translucency of the colonies, and mutation in mdtR for resistance to both HA and rifampin. We conclude that HA is an antimicrobial agent against Gram-positive bacteria that targets the cell membrane.

Loss of Shp1 impairs myeloid cell function and causes lethal inflammation in zebrafish larvae.

PTPN6 encodes SHP1, a protein tyrosine phosphatase with an essential role in immune cell function. SHP1 mutations are associated with neutrophilic dermatoses and emphysema in humans, which resembles the phenotype seen in motheaten mice that lack functional SHP1. To investigate the function of Shp1 in developing zebrafish embryos, we generated a ptpn6 knockout zebrafish line lacking functional Shp1. Shp1 knockout caused severe inflammation and lethality around 17 days post fertilization (dpf). During early development, the myeloid lineage was affected, resulting in a decrease in the number of neutrophils and a concomitant increase in the number of macrophages. The number of emerging hematopoietic stem and progenitor cells (HSPCs) was decreased, but due to hyperproliferation, the number of HSPCs was higher in ptpn6 mutants than in siblings at 5 dpf. Finally, the directional migration of neutrophils and macrophages was decreased in response to wounding, and fewer macrophages were recruited to the wound site. Yet, regeneration of the caudal fin fold was normal. We conclude that loss of Shp1 impaired neutrophil and macrophage function, and caused severe inflammation and lethality at the larval stage.

Paecilomycone Inhibits Quorum Sensing in Gram-Negative Bacteria.

Pseudomonas aeruginosa is an opportunistic pathogen that causes major health care concerns due to its virulence and high intrinsic resistance to antimicrobial agents. Therefore, new treatments are greatly needed. An interesting approach is to target quorum sensing (QS). QS regulates the production of a wide variety of virulence factors and biofilm formation in P. aeruginosa. This study describes the identification of paecilomycone as an inhibitor of QS in both Chromobacterium violaceum and P. aeruginosa. Paecilomycone strongly inhibited the production of virulence factors in P. aeruginosa, including various phenazines, and biofilm formation. In search of the working mechanism, we found that paecilomycone inhibited the production of 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), but not 2'-aminoacetophenone (2-AA). Therefore, we suggest that paecilomycone affects parts of QS in P. aeruginosa by targeting the PqsBC complex and alternative targets or alters processes that influence the enzymatic activity of the PqsBC complex. The toxicity of paecilomycone toward eukaryotic cells and organisms was low, making it an interesting lead for further clinical research. IMPORTANCE Antibiotics are becoming less effective against bacterial infections due to the evolution of resistance among bacteria. Pseudomonas aeruginosa is a Gram-negative pathogen that causes major health care concerns and is difficult to treat due to its high intrinsic resistance to antimicrobial agents. Therefore, new targets are needed, and an interesting approach is to target quorum sensing (QS). QS is the communication system in bacteria that regulates multiple pathways, including the production of virulence factors and biofilm formation, which leads to high toxicity in the host and low sensitivity to antibiotics, respectively. We found a compound, named paecilomycone, that inhibited biofilm formation and the production of various virulence factors in P. aeruginosa. The toxicity of paecilomycone toward eukaryotic cells and organisms was low, making it an interesting lead for further clinical research.