Formins are proteins involved in regulation and construction of actin filaments of eucaryotic organism. They parcipitate in regulating cytokinesis, polar tip growth, and thus participate in development of whole organisms. There are 2 classes of formins in Arabidopsis thaliana. Both classes include FH1 and FH2 domains (formin homology 1 a 2). Class I formins have N-terminal transmembrane domain, unlike class II formins. Some formins of class II have a N-terminal PTEN domain (Phosphatase and Tensin Homolog). Sequence analyses predicted membrane binding via phosphatase or C2 subdomain of PTEN. This thesis was focused on the formin AtFH14, specifically its PTEN domain. Based on predicted sequence, a DNA fragment encoding the PTEN domain was amplified, sequenced and cloned to destination vectors for YFP and EOS phusions. Marked protein was visualized by transient expression in Nicotiana benthamiana. Stably transformed Arabidopsis lines were prepared for stably expression of protein. The tagged protein was localized in cortical cytoplasm, cytoplasmatical strands, probably in nuclear membrane or perinuclear cytoplasm, as well as in peculiar "folicle-like" structures that might be due to binding of PTEN at the periphery of some membrane organelles. Also were seen filament structures, maybe caused by PTEN binding...

Adipose tissue tumors (lipomas) frequently develop in patients with heterozygous germ line phosphatase and tensin homolog (PTEN) mutations. simvastatin has been demonstrated to exhibit antitumor effects, and so the aim of the present study was to assess the effects of simvastatin on the growth of human PTEN haploinsufficient lipoma cells. Whether the effects of simvastatin in lipomas are mediated via PTEN upregulation was also assessed. The results of the present study revealed that simvastatin treatment reduced cell viability and induced apoptosis in human lipoma cells. Furthermore, it was demonstrated that the expression of cellular PTEN mRNA and protein was increased following simvastatin stimulation. In addition, the phosphorylation of protein kinase B and downstream targets of mammalian target of rapamycin and 4E‑binding protein (4E‑BP)‑1 was attenuated. It was also demonstrated that simvastatin induced PTEN transcriptional upregulation by increasing peroxisome proliferator‑activated receptor (PPAR)γ expression. The small interfering RNA‑mediated knockdown of PPARγ abrogated the stimulatory effect of simvastatin on the PTEN protein, but did not influence apoptosis. The results of the present study suggest that simvastatin may be beneficial for patients with inoperable PTEN haploinsufficient lipomas.

Patients with phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome and germline mutations in PTEN frequently develop lipomatosis, for which there is no standard treatment. Rapamycin was shown to reduce the growth of lipoma cells with heterozygous PTEN deficiency in vitro, but concomitantly induced an upregulation of AKT phosphorylation. Since it was shown that resveratrol stabilizes PTEN, we asked whether co-incubation with resveratrol could suppress the rapamycin-induced AKT phosphorylation in PTEN-deficient lipoma cells. Resveratrol incubation resulted in decreased lipoma cell viability by inducing G1-phase cell cycle arrest and apoptosis. PTEN expression and AKT phosphorylation were not significantly changed, whereas p70S6 kinase (p70S6K) phosphorylation was reduced in PTEN-deficient lipoma cells after resveratrol incubation. Rapamycin/resveratrol co-incubation significantly decreased viability further at lower doses of resveratrol and resulted in decreased p70S6K phosphorylation compared to rapamycin incubation alone, suggesting that resveratrol potentiated the growth inhibitory effects of rapamycin by reducing p70S6K activation. Both viability and p70S6K phosphorylation of primary PTEN wild-type preadipocytes were less affected compared to PTEN-deficient lipoma cells by equimolar concentrations of resveratrol. These results support the concept of combining chemopreventive natural compounds with mammalian target of rapamycin (mTOR) inhibitors to increase the efficacy of chemotherapeutic drugs for patients suffering from overgrowth syndromes.

Background: Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) is caused by germ line mutations in the PTEN gene. Symptoms include cancer pre- disposition, immune deviations, and lipomas/lipomatosis. No causal standard therapy is available. We describe a therapeutic attempt with the mammalian target of rapamycin (mTOR) inhibitor sirolimus for a PHTS patient suffering from thymus hyperplasia and lipomatosis. We furthermore assessed the in vitro effects of sirolimus and other inhibitors on lipoma cells of the patient. Methods: The patient underwent clinical and blood examinations and whole-body magnetic resonance imaging to assess tumor sizes. Lipoma cells of the patient were incubated with inhibitors of the phosphoinositide3-kinase (PI3K)/AKT/ mTOR signaling pathway to analyze the effects on proliferation, adipocyte differentiation, and survival in vitro. Results: Sirolimus treatment improved somatic growth and reduced thymus volume. These effects diminished over the treatment period of 19 mo. Sirolimus decreased lipoma cell proliferation and adipocyte differentiation in vitro but did not cause apoptosis. PI3K and AKT inhibitors induced apoptosis significantly. Conclusion: Sirolimus treatment led to an improvement of the patient’s clinical status and a transient reduction of the thymus. Our in vitro findings point to PI3K and AKT inhibitors as potential treatment options for patients with severe forms of PHTS.

The focus of this dissertation is to understand the physicochemical factors that affect the spatiotemporal control of phosphoinositide signaling events. Despite their low abundance in cellular membranes ( ~ 1% of total lipids) phosphoinositides are assuming major roles in the spatiotemporal regulation of cellular signaling, therefore making this group of lipids an attractive area of study, especially for identifying drug targets. The main phosphoinositide studied in this dissertation is phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which regulates various intracellular signaling pathways, notably the PI3K/AKT pathway. The PI3K/AKT pathway plays a critical role in regulating diverse cellular functions including metabolism, growth, proliferation, and survival. Thus, dysregulation of the PI3K/AKT pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. PI(4,5)P2 regulates phosphoinositide signaling in the PI3K/AKT pathway through interaction of its highly anionic headgroup with polybasic proteins. The highly specific manner that allows hundreds of structurally diverse proteins to interact with lipid species found in such low supply may require the local formation of PI(4,5)P2 clusters (domains). Although a significant amount of evidence has accumulated over the past decade that supports the notion of PI(4,5)P2-rich clusters, our understanding regarding the structural determinants required for cluster formation remains limited. Studies have shown that PI(4,5)P2 clustering is induced by cellular cations interacting with PI(4,5)P2 via electrostatic interactions, suggesting that non-clustering/clustering transitions are particularly sensitive to ionic conditions. However, why some ions are more effectively cluster PI(4,5)P2 than others remains to be understood. For our first research aim, we investigated the effects of divalent (Ca2+) and monovalent cations (Na+, K+ ) on PI(4,5)P2 clustering to understand the ionic environment required for electrostatic PI(4,5)P2 cluster formation. We used monolayers at the air/water interface (Langmuir films) to monitor PI(4,5)P2 molecular packing in the presence of each cation. Our results indicated that Ca2+ individually and Ca2+ along with K+ had a greater effects on PI(4,5)P2 cluster formation than Na+ and K+, individually and combined. We hypothesize that the cations shield the negatively charged headgroups, allowing adjacent PI(4,5)P2 molecules to interact via H- bonding networks. The analysis of the electrostatic environment required for stable PI(4,5)P2 clustering will help us understand important aspects of PI(4,5)P2 mediated signaling events, such as the temporal control of protein binding to PI(4,5)P2 clusters to enhance their function. Another important spatiotemporal modulator that affects the local concentration of PI(4,5)P2 clusters is cholesterol, a steroid present in large quantities (30-40 mole%) in the plasma membrane. Cholesterol has been shown to induce the formation of liquid-ordered domains when interacting with an otherwise gel phase forming lipid, however, the interaction of cholesterol with an inner leaflet lipid species that favors more of a disordered environment to form clusters is poorly understood. We hypothesize that cations along with cholesterol work synergistically to induce PI(4,5)P2 clustering. Thus, our second research aim was to investigate the role of cholesterol on PI(4,5)P2 clustering by monitoring the molecular packing of PI(4,5)P2 in the presence of both cholesterol and cations. This aim was investigated similarly to the first aim with Langmuir trough monolayer film experiments. Our results showed that cholesterol in the presence of Ca2+ had an additive effect leading to the strongest condensation of the monolayer (increase in PI(4,5)P2 packing). Our hypothesis is that Ca2+ significantly reduces the negative electron density of the phosphate groups, allowing the cholesterol hydroxyl group to interact with PI(4,5)P2 headgroup through hydrogen-bond formation. To confirm our hypothesis, we collaborated with a computational group at the NIH that performed all-atom molecular dynamics (MD) simulations that closely agreed with our experimental data. Thus we were able to determine that the cholesterol hydroxyl group directly interacts via hydrogen-bonding with the phosphodiester group as well as the PI(4,5)P2 hydroxyl groups in the 2- and 6-position. The insight into the structural positioning of cholesterol moving closer to the PI(4,5)P2 headgroup region suggests this unique interaction is important for PI(4,5)P2 cluster formation. Other anionic lipid species are suspected to interact with PI(4,5)P2 and strengthen PI(4,5)P2 clustering. We were particularly interested in the interaction of PI(4,5)P2 with phosphatidylinositol (PI) and phosphatidylserine (PS) because both are abundant in the plasma membrane, ~6-10% and ~10-20% respectively, and both electrostatically bind to peripheral proteins. Therefore, the third research aim analyzed the capacity of PI and PS to form stable clusters with PI(4,5)P2. We hypothesize that a mixed PI/PI(4,5)P2 or PS/PI(4,5)P2 domains are ideal for protein binding, since in combination PI or PS with PI(4,5)P2 would provide the necessary negative electrostatic environment, while PI(4,5)P2 would provide the high specificity and additional electrostatics for protein binding. Langmuir trough monolayer films were used to investigate the stabilization of PI/PI(4,5)P2 and PS/PI(4,5)P2 monolayers in the presence of Ca2+. Our results showed a condensation of the monolayer for both PI/PI(4,5)P2 and PS/PI(4,5)P2 with an increase in Ca2+concentrations, which suggests that Ca2+ shields the highly negatively charged phosphomonoester groups of PI(4,5)P2 allowing PI and PS to participate in PI(4,5)P2’s hydrogen-bond network. Interestingly, both PI and PS equally stabilized PI(4,5)P2 cluster formation, therefore it is highly likely that these lipids interact in vivo to form large stable electrostatic domains required for protein binding. The first three aims provided us with information about the physiological relevant environments required for PI(4,5)P2 cluster formation, while the last aim was geared towards understanding the temporal control of protein association with phosphoinositides in the plasma membrane. Specifically, we analyzed the plasma membrane association of PTEN-L, a translation variant protein of PTEN, that has the ability to exit and enter back into cells, unlike classical PTEN. The ability of PTEN-L to facilitate entry across the anionic and hydrophobic layers of the plasma membrane (in the case of direct transport of PTEN-L across the membrane) or into phospholipid transport vesicles (in the case of vesicular transport of PTEN-L across cells) is likely due to the addition of the 173 N-terminal amino acids, the alternative translated region (ATR-domain). Thus, our fourth research aim focused on the biophysical role of the ATR-domain to associate with inner leaflet plasma membrane lipids. Using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to monitor secondary structural changes of the ATR-domain upon lipid binding, it was revealed that both PS and PI(4,5)P2 induced conformational change towards a slight increase in β-sheet content in an otherwise unstructured domain suggesting these lipids are required for ATR-domain interaction with the PM. Further studies revealed that the ATR-domain affects the integrity of PS lipid vesicles, further indicating the presence of PS is required to drive ATR-domain across the membrane. This aim provides information on ATR-domain lipid binding preferences aiding in our understanding of the biological and functional role of PTEN-L as a deliverable tumor suppressor protein. The overall goal of the research in this dissertation is to understand factors that fine-tune PI(4,5)P2 cluster formation in space and time. Our first three research aims were designed to understand the synergistic effects of spatiotemporal modulators (cations, cholesterol, and anionic lipids) on local concentration of PI(4,5)P2 clusters. Our results indicate that Ca2+, cholesterol, and the presence of anionic lipids PI and PS all induce stable domains, thus it is highly likely this is part of the biological environment required in vivo for cationic proteins to bind. The last aim, the association of the ATR-domain with phospholipids in the plasma membrane, provided evidence that PS is likely required to drive the ATR-domain across the plasma membrane. This dissertation unifies nearly two decades worth of research by shedding light on synergistic modulators of PI(4,5)P2 cluster formation (Figure 1). Thus, this work has potentially far reaching consequences for understanding temporal control of the spatially resolved protein activity.

Phosphatase and tensin homolog (PTEN) Hamartoma Tumor Syndrome (PHTS) is linked to heterozygous germline mutations in the tumor suppressor gene PTEN. While clinical features of PHTS are broad, the scientific focus of this work lay on the development of aberrant adipose tissue growth in the form of lipomas in pediatric PHTS patients. Although lipomas are generally of benign nature, obstruction of other organs due to their size can lead to life threatening complications. PTEN antagonizes the growth promoting PI3K/AKT pathway, leading to a hyper activation in PHTS patients. Therefore, inhibitors of this pathway might be considered for pharmacological therapy. Treatment attempts with the mTOR inhibitor rapamycin showed beneficial effects, but reports of adaption to the drug indicated the need for further research. In the first part of the project, published in Cancers 2019, we tested effects of the PI3Kα inhibitor alpelisib on growth, apoptosis, senescence and adipogenesis of lipoma cells from PHTS patients. Alpelisib was previously used to successfully treat patients with PI3K related overgrowth syndrome (PROS). PROS is caused by mosaic somatic activating mutations in the PI3Kα catalytic subunit and shares an increased PI3K signaling and a predisposition for lipoma development as a common feature with PHTS. We compared the effects of alpelisib on PHTS patients’ lipoma cells (LipPD1-3) and PROS patients’ lipoma cells (Lip3 and Lip4) and found a similar dose and time dependent reduction of cell viability. We also tested a combined treatment of alpelisib and rapamycin and observed a synergistic activity of the drug combination on cell viability. Proliferation was reduced in PHTS lipoma cells in a concentration dependent manner. In contrast, alpelisib did not induce cell death as measured via annexin V/PI apoptosis assay and LDH cytotoxicity assay. The reduction in cell number was found to be facilitated via induction of senescence in the lipoma cells as determined via senescence associated β-galactosidase (SA β-gal) assay and detection of senescence marker CDKN2A (p16) expression. Interestingly, alpelisib did not only inhibit cell growth but also adipogenesis of the lipoma cells. We established lipoma cell spheroids in 3D culture as lipoma models. While size of control spheroids continuously increased during 10 days in adipogenic culture medium, the size of spheroids in medium containing 10 µM alpelisib decreased, indicating reduced lipid accumulation. An important advantage of alpelisib compared to rapamycin treatment, was observed on deactivation of the downstream PI3K target AKT. While both drugs inhibited phosphorylation of mTORC1 and ribosomal protein S6, only alpelisib reduced AKT phosphorylation. The observed effects on cell viability were similar in PHTS and PROS lipoma cells. PROS patients, including 15 children and adolescents, were successfully treated with alpelisib and exhibited only mild side effects. The in vivo safety and efficacy in pediatric PROS patients studied Vernot et al. 2018, together with our results on cell viability in PROS and PHTS patients’ lipoma cells, provide hope for a beneficial effect of alpelisib in treatment of PHTS related lipoma formation. Primary cells from the stromal vascular fraction (SVF) of adipose tissue can be used as human in vitro models for adipogenesis. Disadvantages include their limited availability, donor variability, limited proliferation and especially adipogenic potential that declines during continuous culture. Therefore, new cell models with enhanced or prolonged adipogenic potential are a useful tool for adipose tissue biology research. In this respect, we established and characterized a lipoma cell strain termed LipPD1 and published the findings in Adipocyte 2020. LipPD1 cells were isolated from abdominal lipoma tissue of an 11-month old male PHTS patient with a heterozygous deletion of exons 2-9 of the PTEN gene and suffering from a severe lipomatosis. SVF cells from the lipoma tissue and control SVF cells from visceral adipose tissue of obese, but otherwise healthy donors undergoing bariatric surgery were isolated via collagenase digest and selected for plastic adherence. LipPD1 cells were compared to the control SVF cells and SGBS cells, which are one of the few available human preadipocyte cell strains established by Wabitsch et al. in 2001. To date the underlying genetic cause for the retained adipogenic potential in SGBS cells remains unclear. LipPD1 cells cultured for 40-60 days showed a high adipogenic potential comparable to SGBS cells, while control SVF cells lost their capacity for adipogenesis during this period as shown via Oil red O and Nile red lipid staining. Expression of adipogenic marker genes PPARγ, aP2, FASN, leptin and adiponectin was markedly increased after 8 days of adipogenesis compared to undifferentiated cells in LipPD1 and SGBS but was only slightly elevated in control SVF cells. Adipocyte functional properties were similar in LipPD1 and SGBS cells. Both cell strains formed spheroids in 3D culture, which increased in size during culture in adipogenic medium, reflecting lipid accumulation. A major difference between the cell strains was an increased basal and stimulated PI3K pathway activation in LipPD1 cells, reflecting their PTEN haploinsuffciency. In conclusion, LipPD1 cells are comparable to SGBS cells as a human adipocyte model, with the advantage of knowing the genetic lesion responsible for the enhanced adipogenic potential compared to wild-type SVF cells. A main goal of this project was to investigate the underlying mechanism of lipoma development in PHTS patients and the role of PTEN in adipose tissue. Therefore, PTEN was downregulated in SVF cells transiently via siRNA (PTEN KD) or stable with the CRISPR method (PTEN CR) and the observed effects were published in The Journal of Biological Chemistry in 2021. PTEN KD cells were compared to controls simultaneously transfected with scramble siRNA and PTEN CR cells were compared to cells simultaneously transfected with control guideRNA with no genomic target. An advantage of this approach compared to the previous analysis of PHTS lipoma cells was that controls and PTEN mutant/knockdown cells had the same genetic background. Using both methods we achieved downregulation of PTEN to similar extends as observed in the PHTS lipoma cells and confirmed PI3K pathway activation by detecting enhanced AKT and ribosomal protein S6 phosphorylation. The permanent downregulation of PTEN in PTEN CR cells allowed for the analysis of effects in long-term culture. Both, PTEN KD and PTEN CR cells, showed an increased proliferation rate compared to control cells, reflecting the growth promoting nature of the PI3K pathway. We downregulated PTEN transiently in SVF cells that already lost their capacity for adipogenesis and found that the adipogenic potential was restored partially as shown by Nile red staining and observations of an increased spheroid size in 3D culture. Cultures of PTEN CR and control cells also revealed marked differences in adipogenic potential after 2-6 weeks in culture. Following the notion that PTEN downregulation restored the adipogenic potential lost in long-term culture, we investigated if PTEN protein levels change during continuous culture of PTEN wild-type SVF cells. We found an increase of PTEN levels and downregulation of AKT phosphorylation in high-passage SVF cells. In addition, the senescence marker p21, p16, p15 and HIPK2 were downregulated at the mRNA level. After long-term culture, there was less senescence in PTEN CR cells compared to controls as determined via SA β-gal staining. We performed RNA sequencing of PTEN KD versus control SVF cells as an untargeted approach and identified 1379 genes regulated in conditions of PTEN downregulation. Gene set enrichment analysis identified 18 significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways with Cellular Senescence most significantly enriched. To find genes responsible for the enhanced adipogenesis observed in PTEN KD cells, we compared our gene set with results from two other RNA sequencing studies of lipid accumulation models. We found 36 overlapping genes and chose the downregulated FOXO1 and RNF144B for further analysis. To check whether the effects of PTEN downregulation on adipogenesis could be attenuated by reintroducing these factors into PTEN CR cells, we overexpressed RNF144B and constitutively active FOXO1 in these cells. While RNF144B had no influence, neither on proliferation nor on adipogenesis of the PTEN CR cells, FOXO1 overexpression reduced adipogenesis in the PTEN CR cells. FOXO1 phosphorylation, which is known to induce adipogenesis, was induced in PTEN KD cells. The lipogenesis inducing factor SREBP1, which is transcriptionally repressed by unphosphorylated FOXO1, was upregulated. In contrast, SREBP1 protein was reduced in the FOXO1 overexpressing PTEN CR cells, explaining the reduced lipid accumulation. These results indicate that the observed adipose tissue overgrowth in PHTS patients is caused by an induction of adipose progenitor growth and adipogenesis, mediated at least partially through repression of FOXO1 transcriptional activity. In summary, these findings provide evidence for a role of PTEN in regulation of adipose tissue expansion and adipogenesis, which could account for the observed lipoma formation in PHTS patients.:Table of Contents 2 Abbreviations 4 Introduction 6 PTEN Hamartoma Tumor Syndrome (PHTS) 6 Phosphoinositide 3-kinase pathway 6 Current PHTS therapy 9 Alpelisib 9 Replicative aging and senescence 10 Adipose tissue and SVF cells 11 Adipose tissue redistribution and PTEN 13 Small interfering RNA (siRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 14 Assays to determine cell viability and cell death 16 Rationale 17 Publications 19 1) The Novel Phosphatidylinositol-3-Kinase (PI3K) Inhibitor Alpelisib Effectively Inhibits Growth of PTEN-Haploinsufficient Lipoma Cells 19 Supplementary figures 36 Supplementary tables 44 2) A new human adipocyte model with PTEN haploinsufficiency 46 Supplementary methods 58 Supplementary figures 61 3) PTEN regulates adipose progenitor cell growth, differentiation, and replicative aging 65 Supplementary figures 78 Supplementary tables 86 Summary 90 Publication bibliography 97 Appendix 103 Darstellung des eigenen Beitrags 103 The Novel Phosphatidylinositol-3-Kinase (PI3K) Inhibitor Alpelisib Effectively Inhibits Growth of PTEN-Haploinsufficient Lipoma Cells. 103 A new human adipocyte model with PTEN haploinsufficiency. 104 PTEN regulates adipose progenitor cell growth, differentiation, and replicative aging 105 Erklärung über die eigenständige Abfassung der Arbeit 106 Lebenslauf 107 Bildungsweg 107 Beruflicher und wissenschaftlicher Werdegang 107 Preise und Förderungen 108 Publikationen 109 Konferenzbeiträge 110 Danksagung 112

Background: Myocardial apoptosis plays an important role in myocardial ischemia/reperfusion (I/R) injury. Activation of PI3K/Akt signaling protects the myocardium from I/R injury. This study investigated the role of miR-214 in hypoxia/ reoxygenation (H/R)-induced cell damage in vitro and myocardial I/R injury in vivo. Methods and Results: H9C2 cardiomyoblasts were transfected with lentivirus expressing miR-214 (LmiR-214) or lentivirus expressing scrambled miR-control (LmiR-control) respectively, to establish cell lines of LmiR-214 and LmiR-control. The cells were subjected to hypoxia for 4 h followed by reoxygenation for 24 h. Transfection of LmiR-214 suppresses PTEN expression, significantly increases the levels of Akt phosphorylation, markedly attenuates LDH release, and enhances the viability of the cells subjected to H/R. In vivo transfection of mouse hearts with LmiR-214 significantly attenuates I/R induced cardiac dysfunction and reduces I/Rinduced myocardial infarct size. LmiR-214 transfection significantly attenuates I/Rinduced myocardial apoptosis and caspase-3/7 and caspase-8 activity. Increased expression of miR-214 by transfection of LmiR-214 suppresses PTEN expression, increases the levels of phosphorylated Akt, represses Bim1 expression and induces Bad phosphorylation in the myocardium. In addition, in vitro data shows transfection of miR-214 mimics to H9C2 cells suppresses the expression and translocation of Bim1 from cytosol to mitochondria and induces Bad phosphorylation. Conclusions: Our in vitro and in vivo data suggests that miR-214 protects cells from H/R induced damage and attenuates I/R induced myocardial injury. The mechanisms involve activation of PI3K/Akt signaling by targeting PTEN expression, induction of Bad phosphorylation, and suppression of Bim1 expression, resulting in decreases in I/R-induced myocardial apoptosis.

Cardiovascular dysfunction is a major complication associated with sepsis induced mortality. Cardiac fibrosis plays a critical role in sepsis induced cardiac dysfunction. The mechanisms of the activation of cardiac fibrosis is unclarified. In this study, we found that microRNA-23b (miR-23b) was up-regulated in heart tissue during cecal ligation and puncture (CLP)-induced sepsis and transfection of miR-23b inhibitor improved survival in late sepsis. Inhibition of miR-23b in the myocardium protected against cardiac output and enhanced left ventricular systolic function. miR-23b inhibitor also alleviated cardiac fibrosis in late sepsis. MiR-23b mediates the activation of TGF-β1/Smad2/3 signaling to promote the differentiation of cardiac fibroblasts through suppression of 5′TG3′-interacting factor 1 (TGIF1). MiR-23b also induces AKT/N-Cadherin signaling to contribute to the deposition of extracellular matrix by inhibiting phosphatase and tensin homologue (PTEN). TGIF1 and PTEN were confirmed as the targets of miR-23b in vitro by Dual-Glo Luciferase assay. miR-23b inhibitor blocked the activation of adhesive molecules and restored the imbalance of pro-fibrotic and anti-fibrotic factors. These data provide direct evidence that miR-23b is a critical contributor to the activation of cardiac fibrosis to mediate the development of myocardial dysfunction in late sepsis. Blockade of miR-23b expression may be an effective approach for prevention sepsis-induced cardiac dysfunction.

The phosphatidylinositol-3-kinase α (PI3Kα) is a heterodimeric enzyme that is composed of a p85α regulatory subunit and a p110α catalytic subunit. PI3Kα plays a critical role in cell survival, growth and differentiation, and is the most frequently mutated pathway in human cancers. The PI3Kα pathway is also targeted by many viruses, such as the human immunodeficiency virus (HIV-1), the herpes simplex virus 1 (HSV-1) or the influenza A virus, to create favourable conditions for viral replication. The regulatory p85α stabilizes the catalytic p110α, but keeps it in an inhibited state. Various ligands can bind to p85α and allosterically activate p110α, but the mechanisms are still ill-defined. Intriguingly, p85α also binds to, and activates, the PTEN phosphatase, which is the antagonist of p110α. Previous studies indicated that only p85α monomers bind to the catalytic p110α subunit, whereas only p85α dimers bind to PTEN. These findings suggest that the balance of p85α monomers and dimers regulates the PI3Kα pathway, and that interrupting this equilibrium could lead to disease development. However, the molecular mechanism for p85α dimerization is controversial, and it is unknown why PTEN only binds to p85α dimers, whereas p110α only binds to p85α monomers. Here we set out to elucidate these questions, and to gain further understanding of how p85α ligands influence p85α dimerization and promote activation of p110α. We first established a comprehensive library of p85α fragments and protocols for their production and purification. By combining biophysical and structural methods such as small angle X-ray scattering, X-ray crystallography, nuclear magnetic resonance, microscale thermophoresis, and chemical crosslinking, we investigated the contributions of all p85α domains to dimerization and ligand binding. Contrarily to the prevailing thought in the field, we find that p85α dimerization and ligand recognition involves multiple domains, including those that directly bind to and inhibit p110α. This finding allows us to suggest a molecular mechanism that links p85α dimerization and allosteric p110α activation through ligands.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatase and tensin homolog, PTEN, is a key tumor suppressor. Mutation of PTEN is associated with both sporadic cancers and a cluster of familial cancer predisposition syndromes called PTEN hamaratoma syndromes. These germline mutations span the length of the PTEN gene with a mutational hot spot localized in exon 5. This exon encodes the catalytic domain of PTEN, which is critical for its tumor suppressor activity. PTEN function is most commonly attributed to lipid phosphatase activity on Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) that leads to inhibition of a cascade with downstream pro-survival effectors including Akt, but PTEN also has phosphatase activity on a small number of proteins. Recently, a mutation, G129E, has been described as a gain of function (GOF) mutation in PTEN knockin mice. This mutant only retains protein phosphatase activity while it completely lacks lipid phosphatase activity. Collectively (in the mouse and in vitro studies), there is no clear mechanism to explain the GOF nature of this mutant. Understanding how mutants of PTEN function in the cells to provide a growth advantage will provide insight into what pathway to therapeutically target. Our central hypothesis is that mutations of PTEN promote tumorigenesis through gain of function activities that result in cell cycle progression. We will determine the signaling pathways that are affected by the gain of function mutant PTEN G129E to better understand the mechanism by which mutants of PTEN confer a growth advantage.