Growth Hormone-induced STAT5B Regulates Star Gene Expression Through a Cooperation With cJUN in Mouse MA-10 Leydig Cells.

Leydig cells produce androgens that are essential for male sex differentiation and reproductive function. Leydig cell function is regulated by several hormones and signaling molecules, including growth hormone (GH). Although GH is known to upregulate Star gene expression in Leydig cells, its molecular mechanism of action remains unknown. The STAT5B transcription factor is a downstream effector of GH signaling in other systems. While STAT5B is present in both primary and Leydig cell lines, its function in these cells has yet to be ascertained. Here we report that treatment of MA-10 Leydig cells with GH or overexpression of STAT5B induces Star messenger RNA levels and increases steroid hormone output. The mouse Star promoter contains a consensus STAT5B element (TTCnnnGAA) at -756 bp to which STAT5B binds in vitro (electrophoretic mobility shift assay and supershift) and in vivo (chromatin immunoprecipitation) in a GH-induced manner. In functional promoter assays, STAT5B was found to activate a -980 bp mouse Star reporter. Mutating the -756 bp element prevented STAT5B binding but did not abrogate STAT5B-responsiveness. STAT5B was found to functionally cooperate with DNA-bound cJUN. The STAT5B/cJUN cooperation was only observed in Leydig cells and not in Sertoli or fibroblast cells, indicating that additional Leydig cell-enriched transcription factors are required. The STAT5B/cJUN cooperation was lost only when both STAT5B and cJUN elements were mutated. In addition to identifying the Star gene as a novel target for STAT5B in Leydig cells, our data provide important new insights into the mechanism of GH and STAT5B action in the regulation of Leydig cell function.

Tegument Protein pp150 Sequence-Specific Peptide Blocks Cytomegalovirus Infection.

Human cytomegalovirus (HCMV) tegument protein pp150 is essential for the completion of the final steps in virion maturation. Earlier studies indicated that three pp150nt (N-terminal one-third of pp150) conformers cluster on each triplex (Tri1, Tri2A and Tri2B), and extend towards small capsid proteins atop nearby major capsid proteins, forming a net-like layer of tegument densities that enmesh and stabilize HCMV capsids. Based on this atomic detail, we designed several peptides targeting pp150nt. Our data show significant reduction in virus growth upon treatment with one of these peptides (pep-CR2) with an IC50 of 1.33 µM and no significant impact on cell viability. Based on 3D modeling, pep-CR2 specifically interferes with the pp150-capsid binding interface. Cells pre-treated with pep-CR2 and infected with HCMV sequester pp150 in the nucleus, indicating a mechanistic disruption of pp150 loading onto capsids and subsequent nuclear egress. Furthermore, pep-CR2 effectively inhibits mouse cytomegalovirus (MCMV) infection in cell culture, paving the way for future animal testing. Combined, these results indicate that CR2 of pp150 is amenable to targeting by a peptide inhibitor, and can be developed into an effective antiviral.

C23 gene regulates the nucleolin structure and biosynthesis of ribosomes in bovine intraspecific and interspecific somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) can reprogram differentiated somatic cells to produce individual animals, thus having advantages in animal breeding and chromatin reprogramming. Interspecies SCNT (iSCNT) provides extreme cases of reprogramming failure that can be used to understand the basic biological mechanism of genome reprogramming. It is important to understand the possible mechanisms for the failure of zygotic genome activation (ZGA) in iSCNT embryos in order to improve the efficiency of SCNT embryos. In the present study, we compared the development of bovine-bovine (B-B), ovine-ovine (O-O) SCNT, and ovine-bovine (O-B) iSCNT embryos and found that a developmental block existed in the 8-cell stage in O-B iSCNT embryos. RNA sequencing and q-PCR analysis revealed that the large ribosomal subunit genes (RPL) or the small ribosomal subunit genes (RPS) were expressed at lower levels in the O-B iSCNT embryos. The nucleolin (C23) gene that regulates the ribosomal subunit generation was transcribed at a lower level during embryonic development in O-B iSCNT embryos. In addition, the nucleolin exhibited a clear circular-ring structure in B-B 8-cell stage embryos, whereas this was shell-like or dot-like in the O-B embryos. Furthermore, overexpression of C23 could increase the blastocyst rate of both SCNT and iSCNT embryos and partly rectify the ring-like nucleolin structure and the expression of ribosomal subunit related genes were upregulation, while knockdown of C23 increased the shell-like nucleolin-structure in B-B cloned embryos and downregulated the expression of ribosomal subunit related genes. These results implied that abnormal C23 and ribosome subunit gene expression would lead to the developmental block of iSCNT embryos and ZGA failure. Overexpression of the C23 gene could partly improve the blastocyst development and facilitate the nucleolin structure in bovine preimplantation SCNT embryos.

Reciprocal stabilization of transcription factor binding integrates two signaling pathways to regulate fission yeast fbp1 transcription.

Transcriptional regulation, a pivotal biological process by which cells adapt to environmental fluctuations, is achieved by the binding of transcription factors to target sequences in a sequence-specific manner. However, how transcription factors recognize the correct target from amongst the numerous candidates in a genome has not been fully elucidated. We here show that, in the fission-yeast fbp1 gene, when transcription factors bind to target sequences in close proximity, their binding is reciprocally stabilized, thereby integrating distinct signal transduction pathways. The fbp1 gene is massively induced upon glucose starvation by the activation of two transcription factors, Atf1 and Rst2, mediated via distinct signal transduction pathways. Atf1 and Rst2 bind to the upstream-activating sequence 1 region, carrying two binding sites located 45 bp apart. Their binding is reciprocally stabilized due to the close proximity of the two target sites, which destabilizes the independent binding of Atf1 or Rst2. Tup11/12 (Tup-family co-repressors) suppress independent binding. These data demonstrate a previously unappreciated mechanism by which two transcription-factor binding sites, in close proximity, integrate two independent-signal pathways, thereby behaving as a hub for signal integration.

The role of StAR2 gene in testicular differentiation and spermatogenesis in Nile tilapia (Oreochromis niloticus).

Sex steroids play critical roles in sex differentiation and gonadal development in teleosts. Steroidogenic acute regulatory protein (StAR), transporting cholesterol (the substrate for steroidogenesis) from the outer mitochondrial membrane to the inner membrane, is the first rate-limiting factor of steroidogenesis. Interestingly, two StAR genes (named as StAR1 and StAR2) have been isolated from non-mammalian vertebrates. To characterize the functions of the novel StAR2 gene in the gonadal differentiation and fertility, we generated a StAR2 homozygous mutant line in Nile tilapia (Oreochromis niloticus). StAR2 gene knockout in male tilapia impeded meiotic initiation, associate with the down-regulation of meiosis related gene expressions of vasa, sycp3 and dazl at 90 days after hatching (dah). Meanwhile, cyp11b2 expression and serum 11-KT production significantly declined in StAR2-/- XY fish at 90 dah. From 120-300 dah, spermatogenesis gradually recovered, and so did the expressions of vasa, sycp3 and dazl in StAR2-/- XY fish testes. However, seminiferous lobules arranged disorderly in StAR2-/- XY fish testes at 300 dah. The number of Leydig cells and expressions of downstream steroidogenesis enzymes including cyp11a1, 3ß-HSD-I, 3ß-HSD-II, cyp17a1 and cyp17a2 decreased in StAR2-/- XY fish testes at 300 dah. Serum testosterone and 11-KT levels were significantly lower in StAR2-/- XY fish than that of their control counterparts. Furthermore, significantly elevated ar, fsh and lh expressions in StAR2-deficient XY fish testes and pituitaries were found when compared with the control XY fish. Testes degeneration and spermatogenic cell apoptosis were observed, while no sperm were squeezed out in StAR2-/- XY fish testes at 540 dah. Taken together, our results suggest that StAR2 has a role in testicular development, spermatogenesis and spermiation by regulating androgen production in tilapia, but may not be essential and could be compensated.

The Role of DMP1 in CKD-MBD.

PURPOSE OF REVIEW: Chronic kidney disease-mineral and bone disorder (CKD-MBD) has become a global health crisis with very limited therapeutic options. Dentin matrix protein 1 (DMP1) is a matrix extracellular protein secreted by osteocytes that has generated recent interest for its possible involvement in CKD-MBD pathogenesis. This is a review of DMP1 established regulation and function, and early studies implicating DMP1 in CKD-MBD. RECENT FINDINGS: Patients and mice with CKD show perturbations of DMP1 expression in bone, associated with impaired osteocyte maturation, mineralization, and increased fibroblast growth factor 23 (FGF23) production. In humans with CKD, low circulating DMP1 levels are independently associated with increased cardiovascular events. We recently showed that DMP1 supplementation lowers circulating FGF23 levels and improves bone mineralization and cardiac outcomes in mice with CKD. Mortality rates are extremely high among patients with CKD and have only marginally improved over decades. Bone disease and FGF23 excess contribute to mortality in CKD by increasing the risk of bone fractures and cardiovascular disease, respectively. Previous studies focused on DMP1 loss-of-function mutations have established its role in the regulation of FGF23 and bone mineralization. Recent studies show that DMP1 supplementation may fill a crucial therapeutic gap by improving bone and cardiac health in CKD.

Lack of WWC2 Protein Leads to Aberrant Angiogenesis in Postnatal Mice.

The WWC protein family is an upstream regulator of the Hippo signalling pathway that is involved in many cellular processes. We examined the effect of an endothelium-specific WWC1 and/or WWC2 knock-out on ocular angiogenesis. Knock-outs were induced in C57BL/6 mice at the age of one day (P1) and evaluated at P6 (postnatal mice) or induced at the age of five weeks and evaluated at three months of age (adult mice). We analysed morphology of retinal vasculature in retinal flat mounts. In addition, in vivo imaging and functional testing by electroretinography were performed in adult mice. Adult WWC1/2 double knock-out mice differed neither functionally nor morphologically from the control group. In contrast, the retinas of the postnatal WWC knock-out mice showed a hyperproliferative phenotype with significantly enlarged areas of sprouting angiogenesis and a higher number of tip cells. The branching and end points in the peripheral plexus were significantly increased compared to the control group. The deletion of the WWC2 gene was decisive for these effects; while knocking out WWC1 showed no significant differences. The results hint strongly that WWC2 is an essential regulator of ocular angiogenesis in mice. As an activator of the Hippo signalling pathway, it prevents excessive proliferation during physiological angiogenesis. In adult animals, WWC proteins do not seem to be important for the maintenance of the mature vascular plexus.

Fascin1 empowers YAP mechanotransduction and promotes cholangiocarcinoma development.

Mechanical forces control cell behavior, including cancer progression. Cells sense forces through actomyosin to activate YAP. However, the regulators of F-actin dynamics playing relevant roles during mechanostransduction in vitro and in vivo remain poorly characterized. Here we identify the Fascin1 F-actin bundling protein as a factor that sustains YAP activation in response to ECM mechanical cues. This is conserved in the mouse liver, where Fascin1 regulates YAP-dependent phenotypes, and in human cholangiocarcinoma cell lines. Moreover, this is relevant for liver tumorigenesis, because Fascin1 is required in the AKT/NICD cholangiocarcinogenesis model and it is sufficient, together with AKT, to induce cholangiocellular lesions in mice, recapitulating genetic YAP requirements. In support of these findings, Fascin1 expression in human intrahepatic cholangiocarcinomas strongly correlates with poor patient prognosis. We propose that Fascin1 represents a pro-oncogenic mechanism that can be exploited during intrahepatic cholangiocarcinoma development to overcome a mechanical tumor-suppressive environment.

Flexible pivoting of dynamin pleckstrin homology domain catalyzes fission: insights into molecular degrees of freedom.

The neuronal dynamin1 functions in the release of synaptic vesicles by orchestrating the process of GTPase-dependent membrane fission. Dynamin1 associates with the plasma membrane-localized phosphatidylinositol-4,5-bisphosphate (PIP2) through the centrally located pleckstrin homology domain (PHD). The PHD is dispensable as fission (in model membranes) can be managed, even when the PHD-PIP2 interaction is replaced by a generic polyhistidine- or polylysine-lipid interaction. However, the absence of the PHD renders a dramatic dampening of the rate of fission. These observations suggest that the PHD-PIP2-containing membrane interaction could have evolved to expedite fission to fulfill the requirement of rapid kinetics of synaptic vesicle recycling. Here, we use a suite of multiscale modeling approaches to explore PHD-membrane interactions. Our results reveal that 1) the binding of PHD to PIP2-containing membranes modulates the lipids toward fission-favoring conformations and softens the membrane, and 2) PHD associates with membrane in multiple orientations using variable loops as pivots. We identify a new loop (VL4), which acts as an auxiliary pivot and modulates the orientation flexibility of PHD on the membrane-a mechanism that we believe may be important for high-fidelity dynamin collar assembly. Together, these insights provide a molecular-level understanding of the catalytic role of PHD in dynamin-mediated membrane fission.

Neurodegenerative phosphoprotein signaling landscape in models of SCA3.

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disorder resulting from an aberrant expansion of a polyglutamine stretch in the ataxin-3 protein and subsequent neuronal death. The underlying intracellular signaling pathways are currently unknown. We applied the Reverse-phase Protein MicroArray (RPMA) technology to assess the levels of 50 signaling proteins (in phosphorylated and total forms) using three in vitro and in vivo models expressing expanded ataxin-3: (i) human embryonic kidney (HEK293T) cells stably transfected with human ataxin-3 constructs, (ii) mouse embryonic fibroblasts (MEF) from SCA3 transgenic mice, and (iii) whole brains from SCA3 transgenic mice. All three models demonstrated a high degree of similarity sharing a subset of phosphorylated proteins involved in the PI3K/AKT/GSK3/mTOR pathway. Expanded ataxin-3 strongly interfered (by stimulation or suppression) with normal ataxin-3 signaling consistent with the pathogenic role of the polyglutamine expansion. In comparison with normal ataxin-3, expanded ataxin-3 caused a pro-survival stimulation of the ERK pathway along with reduced pro-apoptotic and transcriptional responses.

Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1.

Richter's transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase-associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.

MEX3A contributes to development and progression of glioma through regulating cell proliferation and cell migration and targeting CCL2.

Glioma is one of the most commonly diagnosed intracranial malignant tumors with extremely high morbidity and mortality, whose treatment was seriously limited because of the unclear molecular mechanism. In this study, in order to identify a novel therapeutic target for glioma treatment, we explored the functions and mechanism of MEX3A in regulating glioma. The immunohistochemical staining of MEX3A in glioma and normal tissues revealed the upregulation of MEX3A and further indicated the relationship between high MEX3A expression and higher malignancy as well as poorer prognosis of glioma. In vitro loss-of-function and gain-of-function experiments comprehensively demonstrated that MEX3A may promote glioma development through regulating cell proliferation, cell apoptosis, cell cycle, and cell migration. In vivo experiments also suggested the inhibition of glioma growth by MEX3A knockdown. Moreover, our mechanistic study identifies CCL2 as a potential downstream target of MEX3A, which possesses similar regulatory effects on glioma development with MEX3A and could attenuate the promotion of glioma induced by MEX3A overexpression. Overall, MEX3A was identified as a potential tumor promoter in glioma development and therapeutic target in glioma treatment.

Deletion of Golgi protein 73 delayed hepatocyte proliferation of mouse in the early stages of liver regeneration.

BACKGROUND AND AIM: Golgi protein 73 (GP73) is a transmembrane protein that can promote the proliferation of cancer cells. However, the roles of GP73 in the proliferation of non-malignant hepatocytes have rarely been investigated. METHODS: The wild-type (GP73+/+ ) and GP73 gene knockout mice (GP73-/- ) were subject to 70% partial hepatectomy (PHx) to explore the involvement of GP73 in liver regeneration. RESULTS: After PHx, a significant increase of GP73 expression was observed in GP73+/+ mouse liver. Noticeably, promoted recovery of liver mass was observed in GP73-/- mouse at Day 1 after PHx, as showed by the liver/body weight ratio. RNA sequencing revealed that genes relevant to cell cycle and inflammation response in the residual liver tissues were severely suppressed with the deletion of GP73, particularly the inactivation of NF-κB signal pathway in early phase of liver regeneration. In line with this, we do see the downregulation of cell cycle-related protein including cyclin D1, p-cyclin D1, ß-catenin, as well as interleukin 6, tumor necrosis factor-α, CCl2, and CXCl10. In contrast, activation of mTOR signaling pathway was documented, accompanied with the histological hypertrophy of hepatocytes in GP73-/- mouse. CONCLUSIONS: Golgi protein 73 deletion leads to delayed response of liver regeneration and inflammation in the early stages of liver regeneration after PHx.

Targeting the SARS-CoV2 nucleocapsid protein for potential therapeutics using immuno-informatics and structure-based drug discovery techniques.

The occurrence of the SARS-CoV2 infection has become a worldwide threat and the urgent need to discover therapeutic interventions remains paramount. The primary roles of the coronavirus nucleocapsid (N) protein are to interact with the viral genome and pack them into ribonucleoprotein complex. It also plays critical roles at many stages of the viral life cycle. Herein, we explore the N protein of SARS-CoV2 to identify promising epitope-based vaccine candidates and target the N-terminal domain of SARS-CoV2 N-protein for potential inhibitors using an integrative bioinformatics approach. We identified B-cell epitopes and T-cell epitopes that are non-toxic, non-allergenic, capable of inducing IFN-γ and structurally stable with high global population coverage of response. The 404SKQLQQSMSSADS416 and 92RRIRGGDGKMKDL104 sequences of N-protein were identified to induce B-cell immunity. We also identified 79SSPDDQIGY87 and 305AQFAPSASAFFGMSR319 as potential T-cell epitopes that form stable structures with human leucocyte antigens. We have also identified zidovudine triphosphate, an anti-HIV agent, as a potential inhibitor of the N-terminal domain of SARS-CoV2 N-protein based on docking and simulation analysis and should be considered for experimental validations. The findings of this study can help fast-track the discovery of therapeutic options to combat COVID-19.

FXYD protein isoforms differentially modulate human Na/K pump function.

Tight regulation of the Na/K pump is essential for cellular function because this heteromeric protein builds and maintains the electrochemical gradients for Na+ and K+ that energize electrical signaling and secondary active transport. We studied the regulation of the ubiquitous human α1ß1 pump isoform by five human FXYD proteins normally located in muscle, kidney, and neurons. The function of Na/K pump α1ß1 expressed in Xenopus oocytes with or without FXYD isoforms was evaluated using two-electrode voltage clamp and patch clamp. Through evaluation of the partial reactions in the absence of K+ but presence of Na+ in the external milieu, we demonstrate that each FXYD subunit alters the equilibrium between E1P(3Na) and E2P, the phosphorylated conformations with Na+ occluded and free from Na+, respectively, thereby altering the apparent affinity for Na+. This modification of Na+ interaction shapes the small effects of FXYD proteins on the apparent affinity for external K+ at physiological Na+. FXYD6 distinctively accelerated both the Na+-deocclusion and the pump-turnover rates. All FXYD isoforms altered the apparent affinity for intracellular Na+ in patches, an effect that was observed only in the presence of intracellular K+. Therefore, FXYD proteins alter the selectivity of the pump for intracellular ions, an effect that could be due to the altered equilibrium between E1 and E2, the two major pump conformations, and/or to small changes in ion affinities that are exacerbated when both ions are present. Lastly, we observed a drastic reduction of Na/K pump surface expression when it was coexpressed with FXYD1 or FXYD6, with the former being relieved by injection of PKA's catalytic subunit into the oocyte. Our results indicate that a prominent effect of FXYD1 and FXYD6, and plausibly other FXYDs, is the regulation of Na/K pump trafficking.

CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress.

While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.

Active sites of human MEPE-ASARM regulating bone matrix mineralization.

The proteolytic fragment ASARM (acidic serine- and aspartate-rich motif) of MEPE (matrix extracellular phosphoglycoprotein) (MEPE-ASARM) may act as an endogenous anti-mineralization factor involved in X-linked hypophosphatemic rickets/osteomalacia (XLH). We synthesized MEPE-ASARM peptides and relevant peptide fragments with or without phosphorylated Ser residues (pSer) to determine the active site(s) of MEPE-ASARM in a rat calvaria cell culture model. None of the synthetic peptides elicited changes in cell death, proliferation or differentiation, but the peptide (pASARM) with three pSer residues inhibited mineralization without causing changes in gene expression of osteoblast markers tested. The anti-mineralization effect was maintained in peptides in which any one of three pSer residues was deleted. Polyclonal antibodies recognizing pASARM but not ASARM abolished the pASARM effect. Deletion of six N-terminal residues but leaving the recognition sites for PHEX (phosphate regulating endopeptidase homolog, X-linked), a membrane endopeptidase responsible for XLH, intact and two C-terminal amino acid residues did not alter the anti-mineralization activity of pASARM. Our results strengthen understanding of the active sites of MEPE-pASARM and allowed us to identify a shorter more stable sequence with fewer pSer residues still exhibiting hypomineralization activity, reducing peptide synthesis cost and increasing reliability for exploring biological and potential therapeutic effects.

Initiation and disassembly of filopodia tip complexes containing VASP and lamellipodin.

The shapes of many eukaryotic cells depends on the actin cytoskeleton, and changes in actin assembly dynamics underlie many changes in cell shape. Ena/VASP-family actin polymerases, for example, modulate cell shape by accelerating actin filament assembly locally and slowing filament capping. When concentrated into discrete foci at the leading edge, VASP promotes filopodia assembly and forms part of a poorly understood molecular complex that remains associated with growing filopodia tips. Here we identify precursors of this filopodia tip complex in migrating B16F1 cells: small leading-edge clusters of the adaptor protein lamellipodin (Lpd) that subsequently recruit VASP and initiate filopodia formation. Dimerization, membrane association, and VASP binding are all required for lamellipodin to incorporate into filopodia tip complexes, and overexpression of monomeric, membrane--targeted lamellipodin mutants disrupts tip complex assembly. Once formed, tip complexes containing VASP and lamellipodin grow by fusing with each other, but their growth is limited by a size-dependent dynamic instability. Our results demonstrate that assembly and disassembly dynamics of filopodia tip complexes are determined, in part, by a network of multivalent interactions between Ena/VASP proteins, EVH1 ligands, and actin filaments.

Tyrosyl phosphorylation of PZR promotes hypertrophic cardiomyopathy in PTPN11-associated Noonan syndrome with multiple lentigines.

Noonan syndrome with multiple lentigines (NSML) is a rare autosomal dominant disorder that presents with cardio-cutaneous-craniofacial defects. Hypertrophic cardiomyopathy (HCM) represents the major life-threatening presentation in NSML. Mutations in the PTPN11 gene that encodes for the protein tyrosine phosphatase (PTP), SHP2, represents the predominant cause of HCM in NSML. NSML-associated PTPN11 mutations render SHP2 catalytically inactive with an "open" conformation. NSML-associated PTPN11 mutations cause hypertyrosyl phosphorylation of the transmembrane glycoprotein, protein zero-related (PZR), resulting in increased SHP2 binding. Here we show that NSML mice harboring a tyrosyl phosphorylation-defective mutant of PZR (NSML/PZRY242F) that is defective for SHP2 binding fail to develop HCM. Enhanced AKT/S6 kinase signaling in heart lysates of NSML mice was reversed in NSML/PZRY242F mice, demonstrating that PZR/SHP2 interactions promote aberrant AKT/S6 kinase activity in NSML. Enhanced PZR tyrosyl phosphorylation in the hearts of NSML mice was found to drive myocardial fibrosis by engaging an Src/NF-κB pathway, resulting in increased activation of IL-6. Increased expression of IL-6 in the hearts of NSML mice was reversed in NSML/PZRY242F mice, and PZRY242F mutant fibroblasts were defective for IL-6 secretion and STAT3-mediated fibrogenesis. These results demonstrate that NSML-associated PTPN11 mutations that induce PZR hypertyrosyl phosphorylation trigger pathophysiological signaling that promotes HCM and cardiac fibrosis.

Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge.

Cells have many types of actin structures, which must assemble from a common monomer pool. Yet, it remains poorly understood how monomers are distributed to and shared between different filament networks. Simplified model systems suggest that monomers are limited and heterogeneous, which alters actin network assembly through biased polymerization and internetwork competition. However, less is known about how monomers influence complex actin structures, where different networks competing for monomers overlap and are functionally interdependent. One example is the leading edge of migrating cells, which contains filament networks generated by multiple assembly factors. The leading edge dynamically switches between the formation of different actin structures, such as lamellipodia or filopodia, by altering the balance of these assembly factors' activities. Here, we sought to determine how the monomer-binding protein profilin 1 (PFN1) controls the assembly and organization of actin in mammalian cells. Actin polymerization in PFN1 knockout cells was severely disrupted, particularly at the leading edge, where both Arp2/3 and Mena/VASP-based filament assembly was inhibited. Further studies showed that in the absence of PFN1, Arp2/3 no longer localizes to the leading edge and Mena/VASP is non-functional. Additionally, we discovered that discrete stages of internetwork competition and collaboration between Arp2/3 and Mena/VASP networks exist at different PFN1 concentrations. Low levels of PFN1 caused filopodia to form exclusively at the leading edge, while higher concentrations inhibited filopodia and favored lamellipodia and pre-filopodia bundles. These results demonstrate that dramatic changes to actin architecture can be made simply by modifying PFN1 availability.