Phospholamban (PLN) is a type II membrane protein that inhibits the sarcoplasmic reticulum Ca 2þ -ATPase (SERCA), thereby regulating calcium homeostasis in cardiac muscle. In membranes, PLN forms pentamers that have been proposed to function either as a storage for active monomers or as ion channels. Here, we report the T-state structure of pentameric PLN solved by a hybrid solution and solid-state NMR method. In lipid bilayers, PLN adopts a pinwheel topology with a narrow hydrophobic pore, which excludes ion transport. In the T state, the cytoplasmic amphipathic helices (domains Ia) are absorbed into the lipid bilayer with the transmembrane domains arranged in a left-handed coiled-coil configuration, crossing the bilayer with a tilt angle of approximately 11°with respect to the membrane normal. The tilt angle difference between the monomer and pentamer is approximately 13°, showing that intramembrane helix-helix association forces dominate over the hydrophobic mismatch, driving the overall topology of the transmembrane assembly. Our data reveal that both topology and function of PLN are shaped by the interactions with lipids, which fine-tune the regulation of SERCA.hybrid NMR method | PISEMA | calcium regulation | oligomeric protein | dipolar assisted rotational resonance recoupling T he membrane protein complex formed by Ca 2þ -ATPase (SERCA) and phospholamban (PLN) regulates Ca 2þ concentration within the sarcoplasmic reticulum (SR), thereby controlling muscle excitation-contraction coupling (1, 2). PLN is a 52-residue transmembrane (TM) protein highly conserved across mammals (2). Its helix-loop-helix secondary structure is further subdivided into four dynamic domains: domain Ia (1-16), loop (17)(18)(19)(20)(21)(22), and domain II (31-52) (3, 4). The hydrophobic TM domain II is the most conserved and responsible for SERCA inhibition, whereas the cytoplasmic domain harbors two phosphorylation sites that reverse PLN inhibitory function (2). PLN has a direct role in the pathophysiology of the heart muscle, with three lethal mutations linked to dilated cardiomyopathy in humans (R9C-PLN, R14del, and L39-truncated-PLN) (5). In both synthetic and cell membranes, PLN forms pentamers that dissociate into monomers upon interacting with SERCA (1, 6). Although the stoichiometry of the SERCA/PLN complex has been assessed (1, 6), both the role and the structure of the PLN pentamer remain a matter of active debate. Because PLN expression in both atria and ventricles is higher than SERCA, it is likely that oligomerization may participate in SERCA regulation (7). Insights into PLN organization in the membrane have come from biochemical and biophysical data (2,6,8). Initial electrophysiological measurements indicated that PLN formed Ca 2þ channels (9). However, more recent electrochemical studies concluded that PLN does not conduct Cl − or Ca 2þ ions (10).Divergent structural models for the PLN pentamer have been proposed in the literature (8). Although very similar in the secondary structure content, these models differ in t...

The regulatory interaction of phospholamban (PLN) with Ca 2þ -ATPase controls the uptake of calcium into the sarcoplasmic reticulum, modulating heart muscle contractility. A missense mutation in PLN cytoplasmic domain (R9C) triggers dilated cardiomyopathy in humans, leading to premature death. Using a combination of biochemical and biophysical techniques both in vitro and in live cells, we show that the R9C mutation increases the stability of the PLN pentameric assembly via disulfide bridge formation, preventing its binding to Ca 2þ -ATPase as well as phosphorylation by protein kinase A. These effects are enhanced under oxidizing conditions, suggesting that oxidative stress may exacerbate the cardiotoxic effects of the PLN R9C mutant. These results reveal a regulatory role of the PLN pentamer in calcium homeostasis, going beyond the previously hypothesized role of passive storage for active monomers.SERCA | ventricular dilatation | calcium regulation | heart failure | membrane proteins H eart failure (HF) is the leading cause of morbidity and mortality worldwide (1, 2). The most prominent disorder leading to HF is dilated cardiomyopathy (DCM), a disease characterized by left ventricular dilatation and impaired systolic function (1, 2). DCM has both acquired and genetic etiologies (1, 2). Recent genome sequencing has revealed a high incidence of DCM-associated mutations in cytoskeletal, nuclear, as well as sarcomeric proteins (3). A number of mutations have been indentified in calcium handling proteins, which play a central role in the mechanics of heart muscle contractility (3-6).Cardiac muscle contraction (systole) begins when an action potential causes membrane depolarization, activating the sarcolemmal L-type calcium (Ca 2þ ) channels. Ca 2þ flows through the L-type Ca 2þ -channels into the cytosol. This increase in Ca 2þ concentration induces a large-scale release of Ca 2þ into the cytosol from intracellular stores by the sarcoplasmic reticulum (SR) Ca 2þ -release channels (or ryanodine receptors). Ca 2þ then moves toward the contractile apparatus, where it binds the troponin complex and initiates contraction. Muscle relaxation (diastole) occurs when Ca 2þ is sequestered into the SR by the SR Ca 2þ -ATPase (SERCA) (7) a membrane-embedded Ca 2þ pump (8). SERCA is regulated by phospholamban (PLN), which reduces its apparent Ca 2þ affinity (9, 10). PLN's inhibition is reversed by cAMP-dependent protein kinase A (PKA), which phosphorylates PLN at Ser16, enhancing cardiac contractility and reestablishing Ca 2þ flux (11).PLN is a single-pass membrane protein, which comprises three structural domains (12-14), further subdivided into four dynamic domains [cytoplasm: domain Ia (residues 1-16), loop (residues 17-22), domain Ib (residues 23-30); transmembrane: domain II (residues 31-52)] (15) (Fig. S1). In membranes, PLN forms homopentamers arranged in a pinwheel topology that are in equilibrium with monomers (16, 17) that bind SERCA with 1∶1 stoichiometry (6, 18-21). Also, it has been proposed that the PLN monomer-pen...

Keywords:Hop/STIP1/STI1 (Hsp70/Hsp90-organising protein) Pancreatic cancer Immunohistochemistry In vitro invasion MMP-2 a b s t r a c tWe previously identified Hop as over expressed in invasive pancreatic cancer cell lines and malignant tissues of pancreatic cancer patients, suggesting an important role for Hop in the biology of invasive pancreatic cancer. Hop is a co-chaperone protein that binds to both Hsp70/Hsp90. We hypothesised that by targeting Hop, signalling pathways modulating invasion and client protein stabilisation involving Hsp90-dependent complexes may be altered.In this study, we show that Hop knockdown by small interfering (si)RNA reduces the invasion of pancreatic cancer cells, resulting in decreased expression of the downstream target gene, matrix metalloproteinases-2 (MMP-2). Hop in conditioned media co-immunoprecipitates with MMP-2, implicating a possible extracellular function for Hop. Knockdown of Hop expression also reduced expression levels of Hsp90 client proteins, HER2, Bcr-Abl, c-MET and v-Src. Furthermore, Hop is strongly expressed in high grade PanINs compared to lower PanIN grades, displaying differential localisation in invasive ductal pancreatic cancer, indicating that the localisation of Hop is an important factor in pancreatic tumours.Our data suggests that the attenuation of Hop expression inactivates key signal transduction proteins which may decrease the invasiveness of pancreatic cancer cells possibly through the modulation of Hsp90 activity. Therefore, targeting Hop in pancreatic cancer may constitute a viable strategy for targeted cancer therapy.

Background: Renal cell carcinoma patients respond poorly to conventional chemotherapy, this unresponsiveness may be attributable to multidrug resistance (MDR). The mechanisms of MDR in renal cancer are not fully understood and the specific contribution of ABC transporter proteins which have been implicated in the chemoresistance of various cancers has not been fully defined in this disease.

Background : Markers of pancreatic cancer invasion were investigated in two clonal populations of the cell line, MiaPaCa-2, Clone #3 (high invasion) and Clone #8 (low invasion) using proteomic profiling of an in vitro model of pancreatic cancer.

The current treatment of choice for metastatic pancreatic cancer involves single agent gemcitabine or combination of gemcitabine with capecitabine and erlotinib (tyrosine kinase inhibitor). Only 25-30% of patients respond to this treatment and patients who do respond initially ultimately exhibit disease progression. Median survival for pancreatic cancer patients has reached a plateau due to inherent and acquired resistance to these agents. Key molecular factors implicated in this resistance include: deficiencies in drug uptake, alteration of drug targets, activations of DNA repair pathways, resistance to apoptosis, and the contribution of the tumor microenvironment. Moreover, for newer agents including tyrosine kinase inhibitors, over expression of signaling proteins, mutations in kinase domains, activation of alternative pathways, mutations of genes downstream of the target, and/or amplification of the target represent key challenges for treatment efficacy. Here we will review the contribution of known mechanisms and markers of resistance to key pancreatic cancer drug treatments.

With a five-year survival rate of 9%, pancreatic ductal adenocarcinoma (PDAC) is the deadliest of all cancers. The rapid mortality makes PDAC difficult to research, and inspires a resolve to create reliable, tractable cellular models for preclinical cancer research. Organoids are increasingly used to model PDAC as they maintain the differentiation status, molecular and genomic signatures of the original tumour. In this paper, we present novel methodologies and experimental approaches to develop PDAC organoids from PDX tumours, and the simultaneous development of matched primary cell lines. Moreover, we also present a method of recapitulating primary cell line cultures to organoids (CLOs). We highlight the usefulness of CLOs as PDAC organoid models, as they maintain similar transcriptomic signatures as their matched patient-derived organoids and patient derived xenografts (PDX)s. These models provide a manageable, expandable in vitro resource for downstream applications such as high throughput screening, functional genomics, and tumour microenvironment studies. With a rapid progression and fatal outcome, pancreatic cancer is one of the deadliest of all cancers. Long-term survivors are limited to those with resected early stage tumours; however, overall survival rate is a dismal 9%, with a median survival time of 7-11 months 1,2. As pancreatic cancer is notoriously asymptomatic at an early stage, 80% of patients are diagnosed after the cancer has metastasised, making them ineligible for resection, which is the only curative treatment. The number of cases of pancreatic cancer has been steadily rising since 2004 3. It is currently the fourth most common cause of cancer death in the US, and by 2030 it is estimated that it will surpass breast and colorectal cancer to become the second most common cause of death by cancer 4. The majority of pancreatic cancers are in the exocrine pancreas (95%) known as pancreatic ductal adenocarcinoma (PDAC), and 5% are in the endocrine pancreas 5. The leading epidemiological factors include smoking, obesity, type II diabetes mellitus and acute pancreatitis, which account for approximately 25% of PDAC cases 6-9. A limitation in the understanding of the disease progression and development of effective treatments in PDAC may be due the lack of in vitro patient tumour representative models. Established 2D cell lines are the most widely used model for the development and testing of chemotherapeutics for over 50 years 10. The ability of cell lines to grow indefinitely makes them a low-cost, repeatable model, easy to manipulate and are an important base for discovery and proof-of-concept studies. Their importance in cancer research is indisputable, however, their use as a robust clinical model is questionable 11. During passaging, cell lines undergo genetic modifications, such as copy number variation and point mutations 12. Cell lines also have a high level of homogeneity, which does not represent the heterogenetic nature of PDAC tumours, and not all cancer subtypes are well represented as t...

Background: Factors mediating the invasion of pancreatic cancer cells through the extracellular matrix (ECM) are not fully understood.