Tobias Langenberg

From Vessel Sprouting to Normalization: Role of the Prolyl Hydroxylase Domain Protein/Hypoxia-Inducible Factor Oxygen-Sensing Machinery

The accepted model of vessel branching distinguishes several endothelial cell fates. At the forefront of a vessel sprout, “tip cells” guide the sprouting vessel toward an angiogenic stimulus. Behind the tip, “stalk cells” proliferate to elongate the vessel branch and create a lumen. In mature vessels, endothelial cells acquire a streamlined shape to optimally conduct blood flow. For this purpose, endothelial cells switch to the “phalanx” cell fate, which is characterized by quiescent and nonproliferating cells aligned in a tight cobblestonelike layer. Vessel maturation also requires the recruitment of mural cells (ie, smooth muscle cells and pericytes). These cell fates are often altered in pathological conditions, most prominently during the formation of tumor vasculature. Given the essential role of hypoxia as the driving force for initiating angiogenesis, it is not surprising that the hypoxia-sensing machinery controls key steps in physiological and pathological angiogenesis.

FAF1, a Gene that Is Disrupted in Cleft Palate and Has Conserved Function in Zebrafish

Cranial neural crest (CNC) is a multipotent migratory cell population that gives rise to most of the craniofacial bones. An intricate network mediates CNC formation, epithelial-mesenchymal transition, migration along distinct paths, and differentiation. Errors in these processes lead to craniofacial abnormalities, including cleft lip and palate. Clefts are the most common congenital craniofacial defects. Patients have complications with feeding, speech, hearing, and dental and psychological development. Affected by both genetic predisposition and environmental factors, the complex etiology of clefts remains largely unknown. Here we show that Fas-associated factor-1 (FAF1) is disrupted and that its expression is decreased in a Pierre Robin family with an inherited translocation. Furthermore, the locus is strongly associated with cleft palate and shows an increased relative risk. Expression studies show that faf1 is highly expressed in zebrafish cartilages during embryogenesis. Knockdown of zebrafish faf1 leads to pharyngeal cartilage defects and jaw abnormality as a result of a failure of CNC to differentiate into and express cartilage-specific markers, such as sox9a and col2a1. Administration of faf1 mRNA rescues this phenotype. Our findings therefore identify FAF1 as a regulator of CNC differentiation and show that it predisposes humans to cleft palate and is necessary for lower jaw development in zebrafish.

Design of novel artemisinin‐like derivatives with cytotoxic and anti‐angiogenic properties

Artemisinins are plant products with a wide range of medicinal applications. Most prominently, artesunate is a well tolerated and effective drug for treating malaria, but is also active against several protozoal and schistosomal infections, and additionally exhibits anti‐angiogenic, anti‐tumorigenic and anti‐viral properties. The array of activities of the artemisinins, and the recent emergence of malaria resistance to artesunate, prompted us to synthesize and evaluate several novel artemisinin‐like derivatives. Sixteen distinct derivatives were therefore synthesized and the in vitro cytotoxic effects of each were tested with different cell lines. The in vivo anti‐angiogenic properties were evaluated using a zebrafish embryo model. We herein report the identification of several novel artemisinin‐like compounds that are easily synthesized, stable at room temperature, may overcome drug‐resistance pathways and are more active in vitro and in vivo than the commonly used artesunate. These promising findings raise the hopes of identifying safer and more effective strategies to treat a range of infections and cancer.

Claudin-like protein 24 interacts with the VEGFR-2 and VEGFR-3 pathways and regulates lymphatic vessel development

The Claudin-like protein of 24 kDa (CLP24) is a hypoxia-regulated transmembrane protein of unknown function. We show here that clp24 knockdown in Danio rerio and Xenopus laevis results in defective lymphatic development. Targeted disruption of Clp24 in mice led to enlarged lymphatic vessels having an abnormal smooth muscle cell coating. We also show that the Clp24(-/-) phenotype was further aggravated in the Vegfr2(+/LacZ) or Vegfr3(+/LacZ) backgrounds and that CLP24 interacts with vascular endothelial growth factor receptor-2 (VEGFR-2) and VEGFR-3 and attenuates the transcription factor CREB phosphorylation via these receptors. Our results indicate that CLP24 is a novel regulator of VEGFR-2 and VEGFR-3 signaling pathways and of normal lymphatic vessel structure.

De novo design of a biologically active amyloid

Aggregation by design Amyloid aggregation is driven by short sequences within proteins that self-assemble into characteristic amyloid structures. About 30 human proteins are implicated in amyloid-associated diseases, but many more contain short sequences that are potentially amyloidogenic. Gallardo et al. designed a peptide based on an amyloidogenic sequence in the vascular endothelial growth factor receptor VEGFR2. The peptide induced VEGFR2 to form aggregates with features characteristic of amyloids. Amyloids were toxic only in cells that required VEGFR2 activity, suggesting that the toxicity was due to loss of function of VEGFR2, rather than to inherent toxicity of the aggregates. The peptide inhibited VEGFR2-dependent tumor growth in a mouse tumor model. Science, this issue p. 10.1126/science.aah4949 A designed peptide drives a protein that does not usually aggregate to form amyloids. INTRODUCTION It has been shown that most proteins possess amyloidogenic segments. However, only about 30 human proteins are known to be involved in amyloid-associated pathologies, and it is still not clear what determines amyloid toxicity in these diseases. We investigated whether an endogenously expressed protein that contains sequences with known amyloidogenic segments, but is not known to aggregate either under normal or pathological conditions, can be induced to do so by seeding it with a peptide comprising the protein’s own amyloidogenic fragment. We chose to target the protein vascular endothelial growth factor receptor 2 (VEGFR2) because it has well-characterized biological function and so could provide a model system with which to investigate the relationship between protein loss of function and amyloid toxicity in different cellular contexts. RATIONALE The capacity of the amyloid conformation of disease proteins to catalyze their own amyloid conversion demonstrates the sequence specificity of amyloid assembly. Because the core of amyloids consists of short amyloidogenic sequence fragments, we hypothesized that a short amyloidogenic protein sequence of VEGFR2, a protein normally not associated with protein aggregation, should be able to interact with and specifically induce the aggregation of VEGFR2, resulting in its functional knockdown. We used TANGO, an algorithm that predicts aggregation-prone sequences, to identify potential amyloidogenic fragments in VEGFR2. We synthesized these fragments as a tandem repeat in a peptide framework in which each unit is flanked by charged residues and coupled by a short peptide linker. The thinking behind this design was that the tandem repeats would promote the formation of diffusable soluble oligomeric aggregates, whereas the charged residues would kinetically stabilize these oligomers and reduce the rate of insoluble fibril formation. RESULTS By screening for loss of function, we identified one peptide, termed “vascin,” that was highly potent at inhibiting VEGFR2. This sequence was derived from the translocation signaling sequence of VEGFR2. We found that vascin is an amyloidogenic peptide that readily forms small β-structured oligomers, ranging from dimers to nonamers, that slowly convert to amyloid fibrils. When added to cell culture medium, these oligomers are efficiently absorbed by the cell, where they interact with and promote the aggregation and partial degradation of nascent VEGFR2. Vascin aggregation does not induce the aggregation of known disease amyloids. Neither do vascin oligomers affect the function of the related EGF receptor or the surface translocation of other receptors. We found vascin only to be toxic to cells that are dependent on VEGFR2 function, suggesting that toxicity is due to loss of VEGFR2 function and not to vascin aggregation or vascin-induced VEGFR2 aggregation. Consistent with this, we found that vascin is active in vivo and could reduce tumor growth in a VEGFR2-sensitive subcutaneous B16 melanoma syngenic tumor model in mice but is not intrinsically toxic to other tissues. CONCLUSION We found that a short amyloidogenic protein fragment can induce the aggregation of a protein normally not associated with amyloidosis in a manner that recapitulates key biophysical and biochemical characteristics of natural amyloids. In addition, we found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, rather than being generic, amyloid toxicity here appears to be both protein-specific and conditional on a requirement for VEGFR2 protein function. A synthetic amyloid peptide induces aggregation. We designed vascin, a synthetic amyloid peptide based on an amyloidogenic fragment of the signal peptide of VEGFR2. Vascin forms prefibrillar oligomers that penetrate mammalian cells and interacts with the nascent VEGFR2 protein, resulting in its aggregation and functional knockdown. [Composition includes parts of an image from iStock.com/luismmolina.] Most human proteins possess amyloidogenic segments, but only about 30 are associated with amyloid-associated pathologies, and it remains unclear what determines amyloid toxicity. We designed vascin, a synthetic amyloid peptide, based on an amyloidogenic fragment of vascular endothelial growth factor receptor 2 (VEGFR2), a protein that is not associated to amyloidosis. Vascin recapitulates key biophysical and biochemical characteristics of natural amyloids, penetrates cells, and seeds the aggregation of VEGFR2 through direct interaction. We found that amyloid toxicity is observed only in cells that both express VEGFR2 and are dependent on VEGFR2 activity for survival. Thus, amyloid toxicity here appears to be both protein-specific and conditional—determined by VEGFR2 loss of function in a biological context in which target protein function is essential.

Structural Hot Spots for the Solubility of Globular Proteins

Natural selection shapes protein solubility to physiological requirements and recombinant applications that require higher protein concentrations are often problematic. This raises the question whether the solubility of natural protein sequences can be improved. We here show an anti-correlation between the number of aggregation prone regions (APRs) in a protein sequence and its solubility, suggesting that mutational suppression of APRs provides a simple strategy to increase protein solubility. We show that mutations at specific positions within a protein structure can act as APR suppressors without affecting protein stability. These hot spots for protein solubility are both structure and sequence dependent but can be computationally predicted. We demonstrate this by reducing the aggregation of human α-galactosidase and protective antigen of Bacillus anthracis through mutation. Our results indicate that many proteins possess hot spots allowing to adapt protein solubility independently of structure and function.

Nuclear inclusion bodies of mutant and wild‐type p53 in cancer: a hallmark of p53 inactivation and proteostasis remodeling by p53 aggregation.

Although p53 protein aggregates have been observed in cancer cell lines and tumour tissue, their impact in cancer remains largely unknown. Here, we extensively screened for p53 aggregation phenotypes in tumour biopsies, and identified nuclear inclusion bodies (nIBs) of transcriptionally inactive mutant or wild‐type p53 as the most frequent aggregation‐like phenotype across six different cancer types. p53‐positive nIBs co‐stained with nuclear aggregation markers, and shared molecular hallmarks of nIBs commonly found in neurodegenerative disorders. In cell culture, tumour‐associated stress was a strong inducer of p53 aggregation and nIB formation. This was most prominent for mutant p53, but could also be observed in wild‐type p53 cell lines, for which nIB formation correlated with the loss of p53s transcriptional activity. Importantly, protein aggregation also fuelled the dysregulation of the proteostasis network in the tumour cell by inducing a hyperactivated, oncogenic heat‐shock response, to which tumours are commonly addicted, and by overloading the proteasomal degradation system, an observation that was most pronounced for structurally destabilized mutant p53. Patients showing tumours with p53‐positive nIBs suffered from a poor clinical outcome, similar to those with loss of p53 expression, and tumour biopsies showed a differential proteostatic expression profile associated with p53‐positive nIBs. p53‐positive nIBs therefore highlight a malignant state of the tumour that results from the interplay between (1) the functional inactivation of p53 through mutation and/or aggregation, and (2) microenvironmental stress, a combination that catalyses proteostatic dysregulation. This study highlights several unexpected clinical, biological and therapeutically unexplored parallels between cancer and neurodegeneration. Copyright

A transgenic Xenopus laevis reporter model to study lymphangiogenesis

Summary The importance of the blood- and lymph vessels in the transport of essential fluids, gases, macromolecules and cells in vertebrates warrants optimal insight into the regulatory mechanisms underlying their development. Mouse and zebrafish models of lymphatic development are instrumental for gene discovery and gene characterization but are challenging for certain aspects, e.g. no direct accessibility of embryonic stages, or non-straightforward visualization of early lymphatic sprouting, respectively. We previously demonstrated that the Xenopus tadpole is a valuable model to study the processes of lymphatic development. However, a fluorescent Xenopus reporter directly visualizing the lymph vessels was lacking. Here, we created transgenic Tg(Flk1:eGFP) Xenopus laevis reporter lines expressing green fluorescent protein (GFP) in blood- and lymph vessels driven by the Flk1 (VEGFR-2) promoter. We also established a high-resolution fluorescent dye labeling technique selectively and persistently visualizing lymphatic endothelial cells, even in conditions of impaired lymph vessel formation or drainage function upon silencing of lymphangiogenic factors. Next, we applied the model to dynamically document blood and lymphatic sprouting and patterning of the initially avascular tadpole fin. Furthermore, quantifiable models of spontaneous or induced lymphatic sprouting into the tadpole fin were developed for dynamic analysis of loss-of-function and gain-of-function phenotypes using pharmacologic or genetic manipulation. Together with angiography and lymphangiography to assess functionality, Tg(Flk1:eGFP) reporter tadpoles readily allowed detailed lymphatic phenotyping of live tadpoles by fluorescence microscopy. The Tg(Flk1:eGFP) tadpoles represent a versatile model for functional lymph/angiogenomics and drug screening.

Identifying rescuers of misfolding

High-throughput screening of large libraries of cyclic peptides expressed in bacteria yields rescuers of the pathogenic misfolding of proteins associated with neurodegenerative diseases.