Bicc1 ribonucleoprotein complexes specifying organ laterality are licensed by ANKS6-induced structural remodeling of associated ANKS3.

Organ laterality of vertebrates is specified by accelerated asymmetric decay of Dand5 mRNA mediated by Bicaudal-C1 (Bicc1) on the left side, but whether binding of this or any other mRNA to Bicc1 can be regulated is unknown. Here, we found that a CRISPR-engineered truncation in ankyrin and sterile alpha motif (SAM)-containing 3 (ANKS3) leads to symmetric mRNA decay mediated by the Bicc1-interacting Dand5 3' UTR. AlphaFold structure predictions of protein complexes and their biochemical validation by in vitro reconstitution reveal a novel interaction of the C-terminal coiled coil domain of ANKS3 with Bicc1 that inhibits binding of target mRNAs, depending on the conformation of ANKS3 and its regulation by ANKS6. The dual regulation of RNA binding by mutually opposing structured protein domains in this multivalent protein network emerges as a novel mechanism linking associated laterality defects and possibly other ciliopathies to perturbed dynamics in Bicc1 ribonucleoparticle (RNP) formation.

Role of Bicaudal C1 in renal gluconeogenesis and its novel interaction with the CTLH complex.

Altered glucose and lipid metabolism fuel cystic growth in polycystic kidneys, but the cause of these perturbations is unclear. Renal cysts also associate with mutations in Bicaudal C1 (Bicc1) or in its self-polymerizing sterile alpha motif (SAM). Here, we found that Bicc1 maintains normoglycemia and the expression of the gluconeogenic enzymes FBP1 and PEPCK in kidneys. A proteomic screen revealed that Bicc1 interacts with the C-Terminal to Lis-Homology domain (CTLH) complex. Since the orthologous Gid complex in S. cerevisae targets FBP1 and PEPCK for degradation, we mapped the topology among CTLH subunits and found that SAM-mediated binding controls Bicc1 protein levels, whereas Bicc1 inhibited the accumulation of several CTLH subunits. Under the conditions analyzed, Bicc1 increased FBP1 protein levels independently of the CTLH complex. Besides linking Bicc1 to cell metabolism, our findings reveal new layers of complexity in the regulation of renal gluconeogenesis compared to lower eukaryotes.

The microenvironment patterns the pluripotent mouse epiblast through paracrine Furin and Pace4 proteolytic activities.

The fate of pluripotent cells in early mouse embryos is controlled by graded Nodal signals that are activated by the endoproteases Furin and Pace4. Soluble forms of Furin and Pace4 cleave proNodal in vitro and after secretion in transfected cells, but direct evidence for paracrine activity in vivo is elusive. Here, we show that Furin and Pace4 are released by the extraembryonic microenvironment, and that they cleave a membrane-bound reporter substrate in adjacent epiblast cells and activate Nodal to maintain pluripotency. Secreted Pace4 and Furin also stimulated mesoderm formation, whereas endoderm was only induced by Pace4, correlating with a difference in the spatiotemporal distribution of these proteolytic activities. Our analysis of paracrine Furin and Pace4 activities and their in vivo functions significantly advances our understanding of how the epiblast is patterned by its microenvironment. Adding cell-cell communication to the pleiotropic portfolio of these proteases provides a new framework to study proprotein processing also in other relevant contexts.

Bicaudal C1 promotes pancreatic NEUROG3+ endocrine progenitor differentiation and ductal morphogenesis.

In human, mutations in bicaudal C1 (BICC1), an RNA binding protein, have been identified in patients with kidney dysplasia. Deletion of Bicc1 in mouse leads to left-right asymmetry randomization and renal cysts. Here, we show that BICC1 is also expressed in both the pancreatic progenitor cells that line the ducts during development, and in the ducts after birth, but not in differentiated endocrine or acinar cells. Genetic inactivation of Bicc1 leads to ductal cell over-proliferation and cyst formation. Transcriptome comparison between WT and Bicc1 KO pancreata, before the phenotype onset, reveals that PKD2 functions downstream of BICC1 in preventing cyst formation in the pancreas. Moreover, the analysis highlights immune cell infiltration and stromal reaction developing early in the pancreas of Bicc1 knockout mice. In addition to these functions in duct morphogenesis, BICC1 regulates NEUROG3(+) endocrine progenitor production. Its deletion leads to a late but sustained endocrine progenitor decrease, resulting in a 50% reduction of endocrine cells. We show that BICC1 functions downstream of ONECUT1 in the pathway controlling both NEUROG3(+) endocrine cell production and ductal morphogenesis, and suggest a new candidate gene for syndromes associating kidney dysplasia with pancreatic disorders, including diabetes.

Automated design of ligands to polypharmacological profiles.

The clinical efficacy and safety of a drug is determined by its activity profile across many proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to design drugs rationally a priori against profiles of several proteins would have immense value in drug discovery. Here we describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain-penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein-coupled receptors. Overall, 800 ligand-target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed to be correct. We also demonstrate target engagement in vivo. The approach can be a useful source of drug leads when multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology.

Epiblast-specific loss of HCF-1 leads to failure in anterior-posterior axis specification.

Mammalian Host-Cell Factor 1 (HCF-1), a transcriptional co-regulator, plays important roles during the cell-division cycle in cell culture, embryogenesis as well as adult tissue. In mice, HCF-1 is encoded by the X-chromosome-linked Hcfc1 gene. Induced Hcfc1(cKO/+) heterozygosity with a conditional knockout (cKO) allele in the epiblast of female embryos leads to a mixture of HCF-1-positive and -deficient cells owing to random X-chromosome inactivation. These embryos survive owing to the replacement of all HCF-1-deficient cells by HCF-1-positive cells during E5.5 to E8.5 of development. In contrast, complete epiblast-specific loss of HCF-1 in male embryos, Hcfc1(epiKO/Y), leads to embryonic lethality. Here, we characterize this lethality. We show that male epiblast-specific loss of Hcfc1 leads to a developmental arrest at E6.5 with a rapid progressive cell-cycle exit and an associated failure of anterior visceral endoderm migration and primitive streak formation. Subsequently, gastrulation does not take place. We note that the pattern of Hcfc1(epiKO/Y) lethality displays many similarities to loss of ß-catenin function. These results reveal essential new roles for HCF-1 in early embryonic cell proliferation and development.

Regulation of TGFß and related signals by precursor processing.

Secreted cytokines of the TGFß family are found in all multicellular organisms and implicated in regulating fundamental cell behaviors such as proliferation, differentiation, migration and survival. Signal transduction involves complexes of specific type I and II receptor kinases that induce the nuclear translocation of Smad transcription factors to regulate target genes. Ligands of the BMP and Nodal subgroups act at a distance to specify distinct cell fates in a concentration-dependent manner. These signaling gradients are shaped by multiple factors, including proteases of the proprotein convertase (PC) family that hydrolyze one or several peptide bonds between an N-terminal prodomain and the C-terminal domain that forms the mature ligand. This review summarizes information on the proteolytic processing of TGFß and related precursors, and its spatiotemporal regulation by PCs during development and various diseases, including cancer. Available evidence suggests that the unmasking of receptor binding epitopes of TGFß is only one (and in some cases a non-essential) function of precursor processing. Future studies should consider the impact of proteolytic maturation on protein localization, trafficking and turnover in cells and in the extracellular space.

KAP1 controls endogenous retroviruses in embryonic stem cells.

More than forty per cent of the mammalian genome is derived from retroelements, of which about one-quarter are endogenous retroviruses (ERVs). Some are still active, notably in mice the highly polymorphic early transposon (ETn)/MusD and intracisternal A-type particles (IAP). ERVs are transcriptionally silenced during early embryogenesis by histone and DNA methylation (and reviewed in ref. 7), although the initiators of this process, which is essential to protect genome integrity, remain largely unknown. KAP1 (KRAB-associated protein 1, also known as tripartite motif-containing protein 28, TRIM28) represses genes by recruiting the histone methyltransferase SETDB1, heterochromatin protein 1 (HP1) and the NuRD histone deacetylase complex, but few of its physiological targets are known. Two lines of evidence suggest that KAP1-mediated repression could contribute to the control of ERVs: first, KAP1 can trigger permanent gene silencing during early embryogenesis, and second, a KAP1 complex silences the retrovirus murine leukaemia virus in embryonic cells. Consistent with this hypothesis, here we show that KAP1 deletion leads to a marked upregulation of a range of ERVs, in particular IAP elements, in mouse embryonic stem (ES) cells and in early embryos. We further demonstrate that KAP1 acts synergistically with DNA methylation to silence IAP elements, and that it is enriched at the 5' untranslated region (5'UTR) of IAP genomes, where KAP1 deletion leads to the loss of histone 3 lysine 9 trimethylation (H3K9me3), a hallmark of KAP1-mediated repression. Correspondingly, IAP 5'UTR sequences can impose in cis KAP1-dependent repression on a heterologous promoter in ES cells. Our results establish that KAP1 controls endogenous retroelements during early embryonic development.

Disruption of the expression of the proprotein convertase PC7 reduces BDNF production and affects learning and memory in mice.

PC7 belongs to the proprotein convertase family, whose members are implicated in the cleavage of secretory precursors. The in vivo function of PC7 is unknown. Herein, we find that the precursor proBDNF is processed into mature BDNF in COS-1 cells coexpressing proBDNF with either PC7 or Furin. Conversely, the processing of proBDNF into BDNF is markedly reduced in the absence of either Furin or PC7 in mouse primary hepatocytes. In vivo we observe that BDNF and PC7 mRNAs are colocalized in mouse hippocampus and amygdala and that mature BDNF protein levels are reduced in these brain areas in PC7 KO mice but not in the hippocampus of PC1/3 KO mice. Various behavioral tests reveal that in PC7 KO mice spatial memory is intact and plasticity of responding is mildly abnormal. Episodic and emotional memories are severely impaired, but both are rescued with the tyrosine receptor kinase B agonist 7,8-dihydroxyflavone. Altogether, these results support an in vivo role for PC7 in the regulation of certain types of cognitive performance, in part via proBDNF processing. Because polymorphic variants of human PC7 are being characterized, it will be important in future studies to determine their effects on additional physiological and behavioral processes.

Nodal·Gdf1 heterodimers with bound prodomains enable serum-independent nodal signaling and endoderm differentiation.

The TGFß family member Nodal is central to control pluripotent stem cell fate, but its use as a stem cell differentiation factor is limited by low specific activity. During development, Nodal depends on growth and differentiation factor (Gdf)-1 and on the shared co-receptor Cryptic to specify visceral left-right axis asymmetry. We therefore asked whether the functionality of Nodal can be augmented by Gdf1. Because Nodal and Gdf1 coimmunoprecipitate each other, they were predicted to form heterodimers, possibly to facilitate diffusion or to increase the affinity for signaling receptors. Here, we report that Gdf1 suppresses an unexpected dependence of Nodal on serum proteins and that it is critically required for non-autonomous signaling in cells expressing Cryptic. Nodal, Gdf1, and their cleaved propeptides copurified as a heterodimeric low molecular weight complex that stimulated Activin receptor (Acvr) signaling far more potently than Nodal alone. Although heterodimerization with Gdf1 did not increase binding of Nodal to Fc fusions of co-receptors or Acvr extracellular domains, it was essential for soluble Acvr2 to inhibit Nodal signaling. This implies that Gdf1 potentiates Nodal activity by stabilizing a low molecular weight fraction that is susceptible to neutralization by soluble Acvr2. Finally, in differentiating human ES cells, endodermal markers were more efficiently induced by Nodal·Gdf1 than by Nodal, suggesting that Nodal·Gdf1 is an attractive new reagent to direct stem cell differentiation.

Urine Fetuin-A is a biomarker of autosomal dominant polycystic kidney disease progression.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by numerous fluid-filled cysts that frequently result in end-stage renal disease. While promising treatment options are in advanced clinical development, early diagnosis and follow-up remain a major challenge. We therefore evaluated the diagnostic value of Fetuin-A as a new biomarker of ADPKD in human urine. RESULTS: We found that renal Fetuin-A levels are upregulated in both Pkd1 and Bicc1 mouse models of ADPKD. Measurement by ELISA revealed that urinary Fetuin-A levels were significantly higher in 66 ADPKD patients (17.5 ± 12.5 µg/mmol creatinine) compared to 17 healthy volunteers (8.5 ± 3.8 µg/mmol creatinine) or 50 control patients with renal diseases of other causes (6.2 ± 2.9 µg/mmol creatinine). Receiver operating characteristics (ROC) analysis of urinary Fetuin-A levels for ADPKD rendered an optimum cut-off value of 12.2 µg/mmol creatinine, corresponding to 94% of sensitivity and 60% of specificity (area under the curve 0.74 ; p = 0.0019). Furthermore, urinary Fetuin-A levels in ADPKD patients correlated with the degree of renal insufficiency and showed a significant increase in patients with preserved renal function followed for two years. CONCLUSIONS: Our findings establish urinary Fetuin-A as a sensitive biomarker of the progression of ADPKD. Further studies are required to examine the pathogenic mechanisms of elevated renal and urinary Fetuin-A in ADPKD.

Stepwise release of Activin-A from its inhibitory prodomain is modulated by cysteines and requires furin coexpression to promote melanoma growth.

The Activin-A precursor dimer can be cleaved by furin, but how this proteolytic maturation is regulated in vivo and how it facilitates access to signaling receptors is unclear. Here, analysis in a syngeneic melanoma grafting model shows that without furin coexpression, Activin-A failed to accelerate tumor growth, correlating with failure of one or both subunits to undergo cleavage in signal-sending cells, even though compensatory processing by host cells nonetheless sustained elevated circulating Activin-A levels. In reporter assays, furin-independent cleavage of one subunit enabled juxtacrine Activin-A signaling, whereas completion of proteolytic maturation by coexpressed furin or by recipient cells stimulated contact-independent activity, crosstalk with BMP receptors, and signal inhibition by follistatin. Mechanistically, Activin-A processing was modulated by allosteric disulfide bonds flanking the furin site. Disruption of these disulfide linkages with the prodomain enabled Activin-A binding to cognate type II receptors independently of proteolytic maturation. Stepwise proteolytic maturation is a novel mechanism to control Activin-A protein interactions and signaling.

Antagonistic interactions among structured domains in the multivalent Bicc1-ANKS3-ANKS6 protein network govern phase transitioning of target mRNAs.

The growing number of diseases linked to aberrant phase transitioning of ribonucleoproteins highlights the need to uncover how the interplay between multivalent protein and RNA interactions is regulated. Cytoplasmic granules of the RNA binding protein Bicaudal-C (Bicc1) are regulated by the ciliopathy proteins ankyrin (ANK) and sterile alpha motif (SAM) domain-containing ANKS3 and ANKS6, but whether and how target mRNAs are affected is unknown. Here, we show that head-to-tail polymers of Bicc1 nucleated by its SAM domain are interconnected by K homology (KH) domains in a protein meshwork that mediates liquid-to-gel transitioning of client transcripts. Moreover, while the dispersion of these granules by ANKS3 concomitantly released bound mRNAs, co-recruitment of ANKS6 by ANKS3 reinstated Bicc1 condensation and ribonucleoparticle assembly. RNA-independent Bicc1 polymerization and its dual regulation by ANKS3 and ANKS6 represent a new mechanism to couple the reversible immobilization of client mRNAs to controlled protein phase transitioning between distinct metastable states.

Inhibition of anti-tumor immunity by melanoma cell-derived Activin-A depends on STING.

The transforming growth factor-ß (TGF-ß) family member activin A (hereafter Activin-A) is overexpressed in many cancer types, often correlating with cancer-associated cachexia and poor prognosis. Activin-A secretion by melanoma cells indirectly impedes CD8+ T cell-mediated anti-tumor immunity and promotes resistance to immunotherapies, even though Activin-A can be proinflammatory in other contexts. To identify underlying mechanisms, we here analyzed the effect of Activin-A on syngeneic grafts of Braf mutant YUMM3.3 mouse melanoma cells and on their microenvironment using single-cell RNA sequencing. We found that the Activin-A-induced immune evasion was accompanied by a proinflammatory interferon signature across multiple cell types, and that the associated increase in tumor growth depended at least in part on pernicious STING activity within the melanoma cells. Besides corroborating a role for proinflammatory signals in facilitating immune evasion, our results suggest that STING holds considerable potential as a therapeutic target to mitigate tumor-promoting Activin-A signaling at least in melanoma.

Cripto recruits Furin and PACE4 and controls Nodal trafficking during proteolytic maturation.

The glycosylphosphatidylinositol (GPI)-anchored proteoglycan Cripto binds Nodal and its type I receptor Alk4 to activate Smad2,3 transcription factors, but a role during Nodal precursor processing has not been described. We show that Cripto also binds the proprotein convertases Furin and PACE4 and localizes Nodal processing at the cell surface. When coexpressed as in early embryonic cells, Cripto and uncleaved Nodal already associated during secretion, and a Cripto-interacting region in the Nodal propeptide potentiated the effect of proteolytic maturation on Nodal signalling. Disruption of the trans-Golgi network (TGN) by brefeldin A blocked secretion, but export of Cripto and Nodal to the cell surface was not inhibited, indicating that Nodal is exposed to extracellular convertases before entering the TGN/endosomal system. Density fractionation and antibody uptake experiments showed that Cripto guides the Nodal precursor in detergent-resistant membranes to endocytic microdomains marked by GFP-Flotillin. We conclude that Nodal processing and endocytosis are coupled in signal-receiving cells.

Bicaudal C, a novel regulator of Dvl signaling abutting RNA-processing bodies, controls cilia orientation and leftward flow.

Polycystic diseases and left-right (LR) axis malformations are frequently linked to cilia defects. Renal cysts also arise in mice and frogs lacking Bicaudal C (BicC), a conserved RNA-binding protein containing K-homology (KH) domains and a sterile alpha motif (SAM). However, a role for BicC in cilia function has not been demonstrated. Here, we report that targeted inactivation of BicC randomizes left-right (LR) asymmetry by disrupting the planar alignment of motile cilia required for cilia-driven fluid flow. Furthermore, depending on its SAM domain, BicC can uncouple Dvl2 signaling from the canonical Wnt pathway, which has been implicated in antagonizing planar cell polarity (PCP). The SAM domain concentrates BicC in cytoplasmic structures harboring RNA-processing bodies (P-bodies) and Dvl2. These results suggest a model whereby BicC links the orientation of cilia with PCP, possibly by regulating RNA silencing in P-bodies.

Riding shotgun: a dual role for the epidermal growth factor-Cripto/FRL-1/Cryptic protein Cripto in Nodal trafficking.

Nodal is a secreted protein of the transforming growth factor beta (TGFbeta) family that activates Smad2 and Smad3 transcription factors through complexes of type I and type II activin receptors and glycosyl-phosphatidylinositol-anchored coreceptors of the epidermal growth factor-like Cripto/FRL-1/Cryptic family. During early embryogenesis, it stimulates the proliferation of pluripotent progenitor cells and specifies, in a dosage-dependent manner, their subsequent allocation to distinct germ layers. Available evidence indicates that the signaling strength of Nodal is controlled at the level of endocytic uptake and turnover of activated receptor complexes in early endosomes, but insights into the underlying molecular mechanisms are still limited. In this review, I briefly survey literature on the trafficking of the related TGFbeta receptors, and I discuss recent findings indicating that endocytosis of Nodal is coupled to proteolytic processing of its precursor at the cell surface and that the maturation and internalization of Nodal need to be guided by Cripto to stabilize endosomal signaling platforms.

The nodal precursor acting via activin receptors induces mesoderm by maintaining a source of its convertases and BMP4.

During early mouse development, the subtilisin-like proprotein convertases (SPC) Furin and PACE4 pattern the primitive ectoderm and visceral endoderm, presumably by activating the TGFss-related Nodal precursor. Here, mutation of the SPC motif provides direct evidence that Nodal processing is essential to specify anterior visceral endoderm and mesendoderm. Surprisingly, however, the Nodal precursor binds and activates activin receptors to maintain expression of Furin, PACE4, and Bmp4 in extraembryonic ectoderm at a distance from the Nodal source. In return, Bmp4 induces Wnt3, which amplifies Nodal expression in the epiblast and mediates induction of mesoderm. We conclude that uncleaved Nodal sustains the extraembryonic source of proprotein convertases and Bmp4 to amplify Nodal signaling in two nonredundant feedback loops with dual timescales and to localize primitive streak formation at the posterior pole. Based on mathematical modeling, we discuss how these sequential loops control cell fate.