PIK3R1 fusion drives chemoresistance in ovarian cancer by activating ERK1/2 and inducing rod and ring-like structures.

Gene fusions are common in high-grade serous ovarian cancer (HGSC). Such genetic lesions may promote tumorigenesis, but the pathogenic mechanisms are currently poorly understood. Here, we investigated the role of a PIK3R1-CCDC178 fusion identified from a patient with advanced HGSC. We show that the fusion induces HGSC cell migration by regulating ERK1/2 and increases resistance to platinum treatment. Platinum resistance was associated with rod and ring-like cellular structure formation. These structures contained, in addition to the fusion protein, CIN85, a key regulator of PI3K-AKT-mTOR signaling. Our data suggest that the fusion-driven structure formation induces a previously unrecognized cell survival and resistance mechanism, which depends on ERK1/2-activation.

Intermediate filaments in the heart: The dynamic duo of desmin and lamins orchestrates mechanical force transmission.

The intermediate filament (IF) cytoskeleton supports cellular structural integrity, particularly in response to mechanical stress. The most abundant IF proteins in mature cardiomyocytes are desmin and lamins. The desmin network tethers the contractile apparatus and organelles to the nuclear envelope and the sarcolemma, while lamins, as components of the nuclear lamina, provide structural stability to the nucleus and the genome. Mutations in desmin or A-type lamins typically result in cardiomyopathies and recent studies emphasized the synergistic roles of desmin and lamins in the maintenance of nuclear integrity in cardiac myocytes. Here we explore the emerging roles of the interdependent relationship between desmin and lamins in providing resilience to nuclear structure while transducing extracellular mechanical cues into the nucleus.

Lamin A/C phosphorylation at serine 22 is a conserved heat shock response to regulate nuclear adaptation during stress.

The heat shock (HS) response is crucial for cell survival in harmful environments. Nuclear lamin A/C, encoded by the LMNA gene, contributes towards altered gene expression during HS, but the underlying mechanisms are poorly understood. Here, we show that upon HS, lamin A/C was reversibly phosphorylated at serine 22 in concert with HSF1 activation in human cells, mouse cells and Drosophila melanogaster in vivo. Consequently, the phosphorylation facilitated nucleoplasmic localization of lamin A/C and nuclear sphericity in response to HS. Interestingly, lamin A/C knock-out cells showed deformed nuclei after HS and were rescued by ectopic expression of wild-type lamin A, but not by a phosphomimetic (S22D) lamin A mutant. Furthermore, HS triggered concurrent downregulation of lamina-associated protein 2α (Lap2α, encoded by TMPO) in wild-type lamin A/C-expressing cells, but a similar response was perturbed in lamin A/C knock-out cells and in LMNA mutant patient fibroblasts, which showed impaired cell cycle arrest under HS and compromised survival at recovery. Taken together, our results suggest that the altered phosphorylation stoichiometry of lamin A/C provides an evolutionarily conserved mechanism to regulate lamina structure and serve nuclear adaptation and cell survival during HS.

A heterozygous p.S143P mutation in LMNA associates with proteasome dysfunction and enhanced autophagy-mediated degradation of mutant lamins A and C.

Lamins A and C are nuclear intermediate filament proteins that form a proteinaceous meshwork called lamina beneath the inner nuclear membrane. Mutations in the LMNA gene encoding lamins A and C cause a heterogenous group of inherited degenerative diseases known as laminopathies. Previous studies have revealed altered cell signaling pathways in lamin-mutant patient cells, but little is known about the fate of mutant lamins A and C within the cells. Here, we analyzed the turnover of lamins A and C in cells derived from a dilated cardiomyopathy patient with a heterozygous p.S143P mutation in LMNA. We found that transcriptional activation and mRNA levels of LMNA are increased in the primary patient fibroblasts, but the protein levels of lamins A and C remain equal in control and patient cells because of a meticulous interplay between autophagy and the ubiquitin-proteasome system (UPS). Both endogenous and ectopic expression of p.S143P lamins A and C cause significantly reduced activity of UPS and an accumulation of K48-ubiquitin chains in the nucleus. Furthermore, K48-ubiquitinated lamins A and C are degraded by compensatory enhanced autophagy, as shown by increased autophagosome formation and binding of lamins A and C to microtubule-associated protein 1A/1B-light chain 3. Finally, chaperone 4-PBA augmented protein degradation by restoring UPS activity as well as autophagy in the patient cells. In summary, our results suggest that the p.S143P-mutant lamins A and C have overloading and deleterious effects on protein degradation machinery and pharmacological interventions with compounds enhancing protein degradation may be beneficial for cell homeostasis.

Personalized Drug Sensitivity Screening for Bladder Cancer Using Conditionally Reprogrammed Patient-derived Cells.

Many patients with muscle-invasive bladder cancer (BC) are either ineligible for or do not benefit from cisplatin-based chemotherapy, and there is an unmet need to estimate individuals' drug sensitivities. We investigated the suitability of conditionally reprogrammed (CR) cells for the characterization of BC properties and their feasibility for personalized drug sensitivity screening. The CR cultures were established from six BC tumors with varying histology and stage. Four cultures were successfully propagated for genomic, transcriptomic, and protein expression profiling and compared to the parental tumors. Two out of four CR cultures (urothelial carcinoma and small cell neuroendocrine carcinoma [SmCC]) corresponded well to their parental tumors and underwent drug sensitivity screening to identify novel drugs for the respective tumors. Both cultures were sensitive to standard BC chemotherapy agents (eg cisplatin and gemcitabine) and to conventional drugs such as taxanes and inhibitors of topoisomerase and proteasome. The SmCC cells were also sensitive to statins (eg, atorvastatin and pitavastatin). In summary, after confirming their representativeness and origin, we conclude that CR cells are a feasible platform for personalized drug sensitivity testing and might thus add to the approaches used to personalize BC treatment strategies. PATIENT SUMMARY: We investigated the conditional reprogramming method for generating patient-derived bladder cancer cell cultures and studied their feasibility for planning personalized treatment strategies.

Modeling of LMNA-Related Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cells.

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure and heart transplantation. A portion of familial DCM is due to mutations in the LMNA gene encoding the nuclear lamina proteins lamin A and C and without adequate treatment these patients have a poor prognosis. To get better insights into pathobiology behind this disease, we focused on modeling LMNA-related DCM using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM). Primary skin fibroblasts from DCM patients carrying the most prevalent Finnish founder mutation (p.S143P) in LMNA were reprogrammed into hiPSCs and further differentiated into cardiomyocytes (CMs). The cellular structure, functionality as well as gene and protein expression were assessed in detail. While mutant hiPSC-CMs presented virtually normal sarcomere structure under normoxia, dramatic sarcomere damage and an increased sensitivity to cellular stress was observed after hypoxia. A detailed electrophysiological evaluation revealed bradyarrhythmia and increased occurrence of arrhythmias in mutant hiPSC-CMs on ß-adrenergic stimulation. Mutant hiPSC-CMs also showed increased sensitivity to hypoxia on microelectrode array and altered Ca2+ dynamics. Taken together, p.S143P hiPSC-CM model mimics hallmarks of LMNA-related DCM and provides a useful tool to study the underlying cellular mechanisms of accelerated cardiac degeneration in this disease.

Deleterious assembly of the lamin A/C mutant p.S143P causes ER stress in familial dilated cardiomyopathy.

Mutation of the LMNA gene, encoding nuclear lamin A and lamin C (hereafter lamin A/C), is a common cause of familial dilated cardiomyopathy (DCM). Among Finnish DCM patients, the founder mutation c.427T>C (p.S143P) is the most frequently reported genetic variant. Here, we show that p.S143P lamin A/C is more nucleoplasmic and soluble than wild-type lamin A/C and accumulates into large intranuclear aggregates in a fraction of cultured patient fibroblasts as well as in cells ectopically expressing either FLAG- or GFP-tagged p.S143P lamin A. In fluorescence loss in photobleaching (FLIP) experiments, non-aggregated EGFP-tagged p.S143P lamin A was significantly more dynamic. In in vitro association studies, p.S143P lamin A failed to form appropriate filament structures but instead assembled into disorganized aggregates similar to those observed in patient cell nuclei. A whole-genome expression analysis revealed an elevated unfolded protein response (UPR) in cells expressing p.S143P lamin A/C. Additional endoplasmic reticulum (ER) stress induced by tunicamycin reduced the viability of cells expressing mutant lamin further. In summary, p.S143P lamin A/C affects normal lamina structure and influences the cellular stress response, homeostasis and viability.

LMNA Mutation c.917T>G (p.L306R) Leads to Deleterious Hyper-Assembly of Lamin A/C and Associates with Severe Right Ventricular Cardiomyopathy and Premature Aging.

Mutations in the LMNA gene coding for the nuclear lamina proteins lamin A and its smaller splice form lamin C associate with a heterogeneous group of diseases collectively called laminopathies. Here, we describe a 2-year-old patient with a previously undescribed phenotype including right ventricular cardiomyopathy, progeroid features, and premature death. Sequencing of LMNA revealed a novel heterozygous de novo mutation p.L306R located in the α-helical rod domain of A-type lamins. Fibroblasts from the patient showed reduced proliferation and early premature replicative senescence, as characterized by progressive hyperlobulation of the nuclei, abnormally clustered centromeres, loss of lamin B1, and reorganization of promyelocytic leukemia nuclear bodies. Furthermore, the patient cells were more sensitive to double-strand DNA breaks. Similar structural and phenotypic defects were observed in normal fibroblasts transfected with FLAG-tagged p.L306R lamin A. Correspondingly, in vitro assembly studies revealed that the p.L306R generates a "hyper-assembly" mutant of lamin A that forms extensive fiber arrays under physiological conditions where wild-type lamin A is still largely soluble. In summary, we report a novel LMNA p.L306R mutation that leads to previously undescribed hyper-assembly of lamin A, heavy distortion of nuclear shape and that manifests as right ventricular cardiomyopathy and premature aging.

Identification of a glycosphingolipid transfer protein GLTP1 in Arabidopsis thaliana.

Arabidopsis thaliana At2g33470 encodes a glycolipid transfer protein (GLTP) that enhances the intervesicular trafficking of glycosphingolipids in vitro. GLTPs have previously been identified in animals and fungi but not in plants. Thus, At2g33470 is the first identified plant GLTP and we have designated it AtGTLP1. AtGLTP1 transferred BODIPY-glucosylceramide at a rate of 0.7 pmol x s(-1), but BODIPY-galactosylceramide and BODIPY-lactosylceramide were transferred slowly, with rates below 0.1 pmol x s(-1). AtGLTP1 did not transfer BODIPY-sphingomyelin, monogalactosyldiacylglycerol or digalactosyldiacylglycerol. The human GLTP transfers BODIPY-glucosylceramide, BODIPY-galactosylceramide and BODIPY-lactosylceramide with rates greater than 0.8 pmol.s(-1). Structural models showed that the residues that are most critical for glycosphingolipid binding in human GLTP are conserved in AtGLTP1, but some of the sugar-binding residues are unique, and this provides an explanation for the distinctly different transfer preferences of AtGLTP1 and human GLTP. The AtGLTP1 variant Arg59Lys/Asn95Leu showed low BODIPY-glucosylceramide transfer activity, indicating that Arg59 and/or Asn95 are important for the specific binding of glucosylceramide to AtGLTP1. We also show that, in A. thaliana, AtGLTP1 together with At1g21360 and At3g21260 constitute a small gene family orthologous to the mammalian GLTPs. However, At1g21360 and At3g21260 did not transfer any of the tested lipids in vitro.

Glycosphingolipid synthesis requires FAPP2 transfer of glucosylceramide.

The molecular machinery responsible for the generation of transport carriers moving from the Golgi complex to the plasma membrane relies on a tight interplay between proteins and lipids. Among the lipid-binding proteins of this machinery, we previously identified the four-phosphate adaptor protein FAPP2, the pleckstrin homology domain of which binds phosphatidylinositol 4-phosphate and the small GTPase ARF1. FAPP2 also possesses a glycolipid-transfer-protein homology domain. Here we show that human FAPP2 is a glucosylceramide-transfer protein that has a pivotal role in the synthesis of complex glycosphingolipids, key structural and signalling components of the plasma membrane. The requirement for FAPP2 makes the whole glycosphingolipid synthetic pathway sensitive to regulation by phosphatidylinositol 4-phosphate and ARF1. Thus, by coupling the synthesis of glycosphingolipids with their export to the cell surface, FAPP2 emerges as crucial in determining the lipid identity and composition of the plasma membrane.

Membrane interaction and activity of the glycolipid transfer protein.

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.

Structural evidence for adaptive ligand binding of glycolipid transfer protein.

Glycolipids participate in many important cellular processes and they are bound and transferred with high specificity by glycolipid transfer protein (GLTP). We have solved three different X-ray structures of bovine GLTP at 1.4 angstroms, 1.6 angstroms and 1.8 angstroms resolution, all with a bound fatty acid or glycolipid. The 1.4 angstroms structure resembles the recently characterized apo-form of the human GLTP but the other two structures represent an intermediate conformation of the apo-GLTPs and the human lactosylceramide-bound GLTP structure. These novel structures give insight into the mechanism of lipid binding and how GLTP may conformationally adapt to different lipids. Furthermore, based on the structural comparison of the GLTP structures and the three-dimensional models of the related Podospora anserina HET-C2 and Arabidopsis thaliana accelerated cell death protein, ACD11, we give structural explanations for their specific lipid binding properties.

Crystallization and X-ray analysis of bovine glycolipid transfer protein.

Glycolipid-transfer protein (GLTP) is a 24 kDa basic cytosolic protein that facilitates the transfer of glycolipids between bilayer membranes in vitro, but its in vivo function is unknown. Human, bovine, porcine and murine GLTPs have recently been cloned and share high sequence identity to each other. The three-dimensional structure of GLTP has not yet been solved and no structures of any proteins related to GLTP are known. Therefore, the structure of GLTP might reveal a currently unknown fold. Here, the crystallization and preliminary X-ray analysis of bovine GLTP are reported for the first time. Protein prepared by recombinant techniques using an Escherichia coli expression system has been crystallized using the vapour-diffusion method. The crystals belong to space group P2(1), with unit-cell parameters a = 55.4, b = 34.9, c = 58.5 A, alpha = gamma = 90, beta = 116 degrees. The crystals diffract to 1.6 A resolution and a 97.1% complete data set with an R(merge) of 6.7% has been collected from a single crystal at 100 K using synchrotron radiation.