Identification of sequence changes in myosin II that adjust muscle contraction velocity.

The speed of muscle contraction is related to body size; muscles in larger species contract at slower rates. Since contraction speed is a property of the myosin isoform expressed in a muscle, we investigated how sequence changes in a range of muscle myosin II isoforms enable this slower rate of muscle contraction. We considered 798 sequences from 13 mammalian myosin II isoforms to identify any adaptation to increasing body mass. We identified a correlation between body mass and sequence divergence for the motor domain of the 4 major adult myosin II isoforms (ß/Type I, IIa, IIb, and IIx), suggesting that these isoforms have adapted to increasing body mass. In contrast, the non-muscle and developmental isoforms show no correlation of sequence divergence with body mass. Analysis of the motor domain sequence of ß-myosin (predominant myosin in Type I/slow and cardiac muscle) from 67 mammals from 2 distinct clades identifies 16 sites, out of 800, associated with body mass (padj < 0.05) but not with the clade (padj > 0.05). Both clades change the same small set of amino acids, in the same order from small to large mammals, suggesting a limited number of ways in which contraction velocity can be successfully manipulated. To test this relationship, the 9 sites that differ between human and rat were mutated in the human ß-myosin to match the rat sequence. Biochemical analysis revealed that the rat-human ß-myosin chimera functioned like the native rat myosin with a 2-fold increase in both motility and in the rate of ADP release from the actin-myosin crossbridge (the step that limits contraction velocity). Thus, these sequence changes indicate adaptation of ß-myosin as species mass increased to enable a reduced contraction velocity and heart rate.

αI-spectrin represents evolutionary optimization of spectrin for red blood cell deformability.

Spectrin tetramers of the membranes of enucleated mammalian erythrocytes play a critical role in red blood cell survival in circulation. One of the spectrins, αI, emerged in mammals with enucleated red cells after duplication of the ancestral α-spectrin gene common to all animals. The neofunctionalized αI-spectrin has moderate affinity for ßI-spectrin, whereas αII-spectrin, expressed in nonerythroid cells, retains ancestral characteristics and has a 10-fold higher affinity for ßI-spectrin. It has been hypothesized that this adaptation allows for rapid make and break of tetramers to accommodate membrane deformation. We have tested this hypothesis by generating mice with high-affinity spectrin tetramers formed by exchanging the site of tetramer formation in αI-spectrin (segments R0 and R1) for that of αII-spectrin. Erythrocytes with αIIßI presented normal hematologic parameters yet showed increased thermostability, and their membranes were significantly less deformable; under low shear forces, they displayed tumbling behavior rather than tank treading. The membrane skeleton is more stable with αIIßI and shows significantly less remodeling under deformation than red cell membranes of wild-type mice. These data demonstrate that spectrin tetramers undergo remodeling in intact erythrocytes and that this is required for the normal deformability of the erythrocyte membrane. We conclude that αI-spectrin represents evolutionary optimization of tetramer formation: neither higher-affinity tetramers (as shown here) nor lower affinity (as seen in hemolytic disease) can support the membrane properties required for effective tissue oxygenation in circulation.

Protein 4.1G Regulates Cell Adhesion, Spreading, and Migration of Mouse Embryonic Fibroblasts through the ß1 Integrin Pathway.

Protein 4.1G is a membrane skeletal protein that can serve as an adapter between transmembrane proteins and the underlying membrane skeleton. The function of 4.1G remains largely unexplored. Here, using 4.1G knockout mouse embryonic fibroblasts (MEFs) as a model system, we explored the function of 4.1G in motile cells. We show that the adhesion, spreading, and migration of 4.1G(-/-) MEF cells are impaired significantly. We further show that, although the total cellular expression of ß1 integrin is unchanged, the surface expression of ß1 integrin and its active form are decreased significantly in 4.1G(-/-) MEF cells. Moreover, the phosphorylation of focal adhesion kinase, a downstream component of the integrin-mediated signal transduction pathway, is suppressed in 4.1G(-/-) MEF cells. Co-immunoprecipitation experiments and in vitro binding assays showed that 4.1G binds directly to ß1 integrin via its membrane-binding domain. These findings identified a novel role of 4.1G in cell adhesion, spreading, and migration in MEF cells by modulating the surface expression of ß1 integrin and subsequent downstream signal transduction.

A 130-kDa protein 4.1B regulates cell adhesion, spreading, and migration of mouse embryo fibroblasts by influencing actin cytoskeleton organization.

Protein 4.1B is a member of protein 4.1 family, adaptor proteins at the interface of membranes and the cytoskeleton. It is expressed in most mammalian tissues and is known to be required in formation of nervous and cardiac systems; it is also a tumor suppressor with a role in metastasis. Here, we explore functions of 4.1B using primary mouse embryonic fibroblasts (MEF) derived from wild type and 4.1B knock-out mice. MEF cells express two 4.1B isoforms: 130 and 60-kDa. 130-kDa 4.1B was absent from 4.1B knock-out MEF cells, but 60-kDa 4.1B remained, suggesting incomplete knock-out. Although the 130-kDa isoform was predominantly located at the plasma membrane, the 60-kDa isoform was enriched in nuclei. 130-kDa-deficient 4.1B MEF cells exhibited impaired cell adhesion, spreading, and migration; they also failed to form actin stress fibers. Impaired cell spreading and stress fiber formation were rescued by re-expression of the 130-kDa 4.1B but not the 60-kDa 4.1B. Our findings document novel, isoform-selective roles for 130-kDa 4.1B in adhesion, spreading, and migration of MEF cells by affecting actin organization, giving new insight into 4.1B functions in normal tissues as well as its role in cancer.

The Protein 4.1 family: hub proteins in animals for organizing membrane proteins.

Proteins of the 4.1 family are characteristic of eumetazoan organisms. Invertebrates contain single 4.1 genes and the Drosophila model suggests that 4.1 is essential for animal life. Vertebrates have four paralogues, known as 4.1R, 4.1N, 4.1G and 4.1B, which are additionally duplicated in the ray-finned fish. Protein 4.1R was the first to be discovered: it is a major mammalian erythrocyte cytoskeletal protein, essential to the mechanochemical properties of red cell membranes because it promotes the interaction between spectrin and actin in the membrane cytoskeleton. 4.1R also binds certain phospholipids and is required for the stable cell surface accumulation of a number of erythrocyte transmembrane proteins that span multiple functional classes; these include cell adhesion molecules, transporters and a chemokine receptor. The vertebrate 4.1 proteins are expressed in most tissues, and they are required for the correct cell surface accumulation of a very wide variety of membrane proteins including G-Protein coupled receptors, voltage-gated and ligand-gated channels, as well as the classes identified in erythrocytes. Indeed, such large numbers of protein interactions have been mapped for mammalian 4.1 proteins, most especially 4.1R, that it appears that they can act as hubs for membrane protein organization. The range of critical interactions of 4.1 proteins is reflected in disease relationships that include hereditary anaemias, tumour suppression, control of heartbeat and nervous system function. The 4.1 proteins are defined by their domain structure: apart from the spectrin/actin-binding domain they have FERM and FERM-adjacent domains and a unique C-terminal domain. Both the FERM and C-terminal domains can bind transmembrane proteins, thus they have the potential to be cross-linkers for membrane proteins. The activity of the FERM domain is subject to multiple modes of regulation via binding of regulatory ligands, phosphorylation of the FERM associated domain and differential mRNA splicing. Finally, the spectrum of interactions of the 4.1 proteins overlaps with that of another membrane-cytoskeleton linker, ankyrin. Both ankyrin and 4.1 link to the actin cytoskeleton via spectrin, and we hypothesize that differential regulation of 4.1 proteins and ankyrins allows highly selective control of cell surface protein accumulation and, hence, function. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé

A conserved sequence in calmodulin regulated spectrin-associated protein 1 links its interaction with spectrin and calmodulin to neurite outgrowth.

Calmodulin regulated spectrin-associated protein 1 (CAMSAP1) is a vertebrate microtubule-binding protein, and a representative of a family of cytoskeletal proteins that arose with animals. We reported previously that the central region of the protein, which contains no recognized functional domain, inhibited neurite outgrowth when over-expressed in PC12 cells [Baines et al., Mol. Biol. Evol. 26 (2009), p. 2005]. The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins (Baines et al. 2009). In the central region, three short well-conserved regions are characteristic of CAMSAP-family members. One of these, CAMSAP-conserved region 1 (CC1), bound to both ßIIΣ1-spectrin and Ca(2+)/calmodulin in vitro. The binding of Ca(2+)/calmodulin inhibited spectrin binding. Transient expression of CC1 in PC12 cells inhibited neurite outgrowth. siRNA knockdown of CAMSAP1 inhibited neurite outgrowth in PC12 cells or primary cerebellar granule cells: this could be rescued in PC12 cells by wild-type CAMSAP1-enhanced green fluorescent protein, but not by a CC1 mutant. We conclude that CC1 represents a functional region of CAMSAP1, which links spectrin-binding to neurite outgrowth.

Comprehensive characterization of protein 4.1 expression in epithelium of large intestine.

The protein 4.1 family consists of four members, 4.1R, 4.1N, 4.1B and 4.1G, each encoded by a distinct gene. All 4.1 mRNAs undergo extensive alternative splicing. Functionally, they usually serve as adapters that link actin-based cytoskeleton to plasma membrane proteins. It has been reported that 4.1 proteins are expressed in most animal cell types and tissues including epithelial cells and epithelial tissues. However, the expression of 4.1 proteins in large intestine has not been well characterized. In the present study, we performed RT-PCR, western blot and immunohistochemistry analysis to characterize the transcripts, the protein expression and cellular localization of 4.1 proteins in the epithelia of mouse large intestine. We show that multiple transcripts derive from each gene, including eight 4.1R isoforms, four 4.1N isoforms, four 4.1B isoforms and six 4.1G isoforms. However, at the protein level, only one or two major bands were detected, implying that not all transcripts are translated and/or the proteins do not accumulate at detectable levels. Immunohistochemistry revealed that 4.1R, 4.1N and 4.1B are all expressed at the lateral membrane as well as cytoplasm of epithelial cells, suggesting a potentially redundant role of these proteins. Our findings not only provide new insights into the structure of protein 4.1 genes but also lay the foundation for future functional studies.

Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of beta1 integrin.

Protein 4.1R is a membrane-cytoskeleton adaptor protein that has diverse roles in controlling the cell surface expression and/or function of transmembrane proteins, and in organizing F-actin. 4.1R is expressed in keratinocytes, but its role in these cells has not been explored. Here, we have investigated the role of 4.1R in skin using 4.1R(-/-) mice. Cell adhesion, spreading, migration and motility were significantly impaired in 4.1R(-/-) keratinocytes, and 4.1R(-/-) mice exhibited defective epidermal wound healing. Cultured 4.1R(-/-) keratinocytes on fibronectin failed to form actin stress fibres and focal adhesions. Furthermore, in the absence of 4.1R, the surface expression, and consequently the activity of ß1 integrin were reduced. These data enabled the identification of a functional role for protein 4.1R in keratinocytes by modulating the surface expression of ß1 integrin, possibly through a direct association between 4.1R and ß1 integrin.

Isoforms of protein 4.1 are differentially distributed in heart muscle cells: relation of 4.1R and 4.1G to components of the Ca2+ homeostasis system.

The 4.1 proteins are cytoskeletal adaptor proteins that are linked to the control of mechanical stability of certain membranes and to the cellular accumulation and cell surface display of diverse transmembrane proteins. One of the four mammalian 4.1 proteins, 4.1R (80 kDa/120 kDa isoforms), has recently been shown to be required for the normal operation of several ion transporters in the heart (Stagg MA et al. Circ Res, 2008; 103: 855-863). The other three (4.1G, 4.1N and 4.1B) are largely uncharacterised in the heart. Here, we use specific antibodies to characterise their expression, distribution and novel activities in the left ventricle. We detected 4.1R, 4.1G and 4.1N by immunofluorescence and immunoblotting, but not 4.1B. Only one splice variant of 4.1N and 4.1G was seen whereas there are several forms of 4.1R. 4.1N, like 4.1R, was present in intercalated discs, but unlike 4.1R, it was not localised at the lateral plasma membrane. Both 4.1R and 4.1N were in internal structures that, at the level of resolution of the light microscope, were close to the Z-disc (possibly T-tubules). 4.1G was also in intracellular structures, some of which were coincident with sarcoplasmic reticulum. 4.1G existed in an immunoprecipitable complex with spectrin and SERCA2. 80 kDa 4.1R was present in subcellular fractions enriched in intercalated discs, in a complex resistant to solubilization under non-denaturing conditions. At the intercalated disc 4.1R does not colocalise with the adherens junction protein, ß-catenin, but does overlap with the other plasma membrane signalling proteins, the Na/K-ATPase and the Na/Ca exchanger NCX1. We conclude that isoforms of 4.1 proteins are differentially compartmentalised in the heart, and that they form specific complexes with proteins central to cardiomyocyte Ca(2+) metabolism.

Inter-subunit interactions in erythroid and non-erythroid spectrins.

Spectrins comprise α- and ß-subunits made up predominantly of a series of homologous repeating units of about 106 amino acids; the α- and ß-chains form antiparallel dimers by lateral association, and tetramers through head-to-head contacts between the dimers. Here we consider the first of these interactions. (1) We confirm earlier observations, showing that the first two paired repeats (ßIR1 with αIR21, and ßIR2 with αRI20) at one end of the erythroid spectrin (αIßI) dimer are necessary and sufficient to unite the chains; (2) we resolve a conflict in published reports by showing that the strength of the interaction is considerably increased on adding the adjoining pair of repeats (ßIR3-αIR19); (3) in brain (αIIßII) spectrin the first two pairs of repeats are similarly essential and sufficient for heterodimer formation; (4) this interaction is ~60-fold stronger than that in the erythroid counterpart, but no enhancement can be detected on addition of three further pairs of repeats; (5) formation of a tight αIßI dimer probably depends on structural coupling of the first two repeats in each chain; (6) an analysis of the sequences of the strongly interacting repeats, ßIR1, ßIIR1, αIR21 and αIIR20 and repeats in α-actinin, which also interact very strongly in forming an antiparallel dimer, affords a possible explanation for the different properties of the two spectrin isoforms in respect of the stability of the inter-chain interactions, and also suggests the evolutionary path by which the erythroid and non-erythroid sequences diverged.

The CKK domain (DUF1781) binds microtubules and defines the CAMSAP/ssp4 family of animal proteins.

We describe a structural domain common to proteins related to human calmodulin-regulated spectrin-associated protein1 (CAMSAP1). Analysis of the sequence of CAMSAP1 identified a domain near the C-terminus common to CAMSAP1 and two other mammalian proteins KIAA1078 and KIAA1543, which we term a CKK domain. This domain was also present in invertebrate CAMSAP1 homologues and was found in all available eumetazoan genomes (including cnidaria), but not in the placozoan Trichoplax adherens, nor in any nonmetazoan organism. Analysis of codon alignments by the sitewise likelihood ratio method gave evidence for strong purifying selection on all codons of mammalian CKK domains, potentially indicating conserved function. Interestingly, the Drosophila homologue of the CAMSAP family is encoded by the ssp4 gene, which is required for normal formation of mitotic spindles. To investigate function of the CKK domain, human CAMSAP1-enhanced green fluorescent protein (EGFP) and fragments including the CKK domain were expressed in HeLa cells. Both whole CAMSAP1 and the CKK domain showed localization coincident with microtubules. In vitro, both whole CAMSAP1-glutathione-s-transferase (GST) and CKK-GST bound to microtubules. Immunofluorescence using anti-CAMSAP1 antibodies on cerebellar granule neurons revealed a microtubule pattern. Overexpression of the CKK domain in PC12 cells blocked production of neurites, a process that requires microtubule function. We conclude that the CKK domain binds microtubules and represents a domain that evolved with the metazoa.

Comprehensive characterization of expression patterns of protein 4.1 family members in mouse adrenal gland: implications for functions.

The members of the protein 4.1 family, 4.1R, 4.1G, 4.1N, and 4.1B, are encoded by four genes, all of which undergo complex alternative splicing. It is well established that 4.1R, the prototypical member of the family, serves as an adapter that links the spectrin-actin based cytoskeleton to the plasma membrane in red cells. It is required for mechanical resilience of the membrane, and it ensures the cell surface accumulation of selected membrane proteins. However, the function of 4.1 proteins outside erythrocytes remains under-explored, especially in endocrine tissues. Transcripts of all 4.1 homologs have previously been documented to be abundantly expressed in adrenal gland. In order to begin to decipher the function of 4.1 proteins in adrenal gland, we performed a detailed characterization of the expression pattern of various 4.1 proteins and their cellular localization. We show that 4.1R (~80 and ~135 kDa) splice forms are expressed on the membrane of all cells, while a ~160 kDa 4.1G splice form is distributed in the cytoplasm and the membrane of zona glomerulosa and of medullary cells. Two 4.1N splice forms, ~135 and ~95 kDa, are present in the peri-nuclear region of both zona glomerulosa and medullary cells, while a single ~130 kDa 4.1B splice form, is detected in all layers of adrenal gland in both the cytoplasm and the membrane. The characterization of distinct splice forms of various 4.1 proteins with diverse cellular and sub-cellular localization indicates multiple functions for this family of proteins in endocrine functions of adrenal gland.

Cytoskeletal protein 4.1R affects repolarization and regulates calcium handling in the heart.

The 4.1 proteins are a family of multifunctional adaptor proteins. They promote the mechanical stability of plasma membranes by interaction with the cytoskeletal proteins spectrin and actin and are required for the cell surface expression of a number of transmembrane proteins. Protein 4.1R is expressed in heart and upregulated in deteriorating human heart failure, but its functional role in myocardium is unknown. To investigate the role of protein 4.1R on myocardial contractility and electrophysiology, we studied 4.1R-deficient (knockout) mice (4.1R KO). ECG analysis revealed reduced heart rate with prolonged Q-T interval in 4.1R KO. No changes in ejection fraction and fractional shortening, assessed by echocardiography, were found. The action potential duration in isolated ventricular myocytes was prolonged in 4.1R KO. Ca(2+) transients were larger and slower to decay in 4.1R KO. The sarcoplasmic reticulum Ca(2+) content and Ca(2+) sparks frequency were increased. The Na(+)/Ca(2+) exchanger current density was reduced in 4.1R KO. The transient inward current inactivation was faster and the persistent Na(+) current density was increased in the 4.1R KO group, with possible effects on action potential duration. Although no major morphological changes were noted, 4.1R KO hearts showed reduced expression of NaV1.5alpha and increased expression of protein 4.1G. Our data indicate an unexpected and novel role for the cytoskeletal protein 4.1R in modulating the functional properties of several cardiac ion transporters with consequences on cardiac electrophysiology and with possible significant roles during normal cardiac function and disease.

Protein 4.1 and the control of ion channels.

The classical function of 4.1R in red blood cells is to contribute to the mechanochemical properties of the membrane by promoting the interaction between spectrin and actin. More recently, it has been recognized that 4.1R is required for the stable cell surface accumulation of a number of erythrocyte membrane proteins. 4.1R is one member of the mammalian 4.1 family - the others being 4.1N, 4.1G and 4.1B - and is expressed in many cell types other than erythrocytes. Recently we have examined the phenotype of hearts from 4.1R knockout mice. Although they had a generally normal morphology, these hearts exhibited bradycardia, and prolongation of both action potentials and QT intervals. Electrophysiological analysis revealed anomalies in a range of ion channel activities. In addition, the immunoreactivity of voltage-gated Na(+) channel NaV1.5 was reduced, indicating a role for 4.1R in the cellular accumulation of this ion channel. 4.1 proteins also have roles in the accumulation of at least two other classes of ion channel. In epithelia, 4.1 interacts with the store-operated channel TRPC4. In neurons, the ligand-gated channels GluR1 and GluR4 require 4.1 proteins for cell surface accumulation. The spectrum of transmembrane proteins that bind to 4.1 proteins overlaps with that of ankyrin. A hypothesis to investigate in the future is that differential regulation of 4.1 and ankyrins (e.g. by PIP(2)) allows highly selective control of cell surface accumulation and transport activity of a specific range of ion channels.

Evolution of spectrin function in cytoskeletal and membrane networks.

Spectrin is a cytoskeletal protein thought to have descended from an alpha-actinin-like ancestor. It emerged during evolution of animals to promote integration of cells into tissues by assembling signalling and cell adhesion complexes, by enhancing the mechanical stability of membranes and by promoting assembly of specialized membrane domains. Spectrin functions as an (alphabeta([H]))(2) tetramer that cross-links transmembrane proteins, membrane lipids and the actin cytoskeleton, either directly or via adaptor proteins such as ankyrin and 4.1. In the present paper, I review recent findings on the origins and adaptations in this system. (i) The genome of the choanoflagellate Monosiga brevicollis encodes alpha-, beta- and beta(Heavy)-spectrin, indicating that spectrins evolved in the immediate unicellular precursors of animals. (ii) Ankyrin and 4.1 are not encoded in that genome, indicating that spectrin gained function during subsequent animal evolution. (iii) Protein 4.1 gained a spectrin-binding activity in the evolution of vertebrates. (iv) Interaction of chicken or mammal beta-spectrin with PtdInsP(2) can be regulated by differential mRNA splicing, which can eliminate the PH (pleckstrin homology) domain in betaI- or betaII-spectrins; in the case of mammalian betaII-spectrin, the alternative C-terminal region encodes a phosphorylation site that regulates interaction with alpha-spectrin. (v) In mammalian evolution, the single pre-existing alpha-spectrin gene was duplicated, and one of the resulting pair (alphaI) neo-functionalized for rapid make-and-break of tetramers. I hypothesize that the elasticity of mammalian non-nucleated erythrocytes depends on the dynamic rearrangement of spectrin dimers/tetramers under the shearing forces experienced in circulation.

The transitional junction: a new functional subcellular domain at the intercalated disc.

We define here a previously unrecognized structural element close to the heart muscle plasma membrane at the intercalated disc where the myofibrils lead into the adherens junction. At this location, the plasma membrane is extensively folded. Immunofluorescence and immunogold electron microscopy reveal a spectrin-rich domain at the apex of the folds. These domains occur at the axial level of what would be the final Z-disc of the terminal sarcomere in the myofibril, although there is no Z-disc-like structure there. However, a sharp transitional boundary lies between the myofibrillar I-band and intercalated disc thin filaments, identifiable by the presence of Z-disc proteins, alpha-actinin, and N-terminal titin. This allows for the usual elastic positioning of the A-band in the final sarcomere, whereas the transduction of the contractile force normally associated with the Z-disc is transferred to the adherens junctions at the plasma membrane. The axial conjunction of the transitional junction with the spectrin-rich domains suggests a mechanism for direct communication between intercalated disc and contractile apparatus. In particular, it provides a means for sarcomeres to be added to the ends of the cells during growth. This is of particular relevance to understanding myocyte elongation in dilated cardiomyopathy.

Identification and characterization of novel spliced variants of neuregulin 4 in prostate cancer.

PURPOSE: The neuregulin (NRG) 1, 2, and 3 genes undergo extensive alternative mRNA splicing, which results in variants that show structural and functional diversity. The aims of this study were to establish whether the fourth member of this family, NRG4, is expressed in prostate cancer, if it is alternatively spliced and whether any functional differences between the variants could be observed. EXPERIMENTAL DESIGN: The expression of NRG4 was determined using immunohistochemical staining of 40 cases of primary prostate cancer. Bioinformatic analysis and reverse transcription-PCR (RT-PCR) using NRG4 isotype-specific primers on a panel of normal and prostate cancer cell lines were used to identify alternatively spliced NRG4 variants. Expression of these variants was determined using isotype-specific antibodies. Transfection into Cos-7 cells of two of these green fluorescent protein-tagged variants allowed analysis of their subcellular location. Four of the variants were chemically synthesized and tested for their ability to activate the ErbB4 receptor. RESULTS: NRG4 was variably expressed in the cytoplasm in the majority of prostate cancer cases, and in a subset of cases in the membrane, high levels were associated with advanced disease stage. Four novel NRG4 splice variants (NRGA2, NRG4 B1-3) were characterized, where each seemed to have a different subcellular location and were also expressed in the cytoplasm of the prostate tumors. NRG4 B3 was also present in endothelial cells. In transfected cells, the A type variant (NRG4 A1) was localized to the membrane, whereas the B type variant (NRG4 B1), which lacks the predicted transmembrane region, had an intracellular localization. Only the variants with an intact epidermal growth factor-like domain activated ErbB4 signaling. CONCLUSION: NRG4 overexpression is associated with advanced-stage prostate cancer. The alternative splice variants may have different roles in cell signaling, some acting as classic receptor ligands and some with as-yet unknown functions.

Impact of Naja nigricollis Venom on the Production of Methaemoglobin.

Snakebite envenomation is an affliction currently estimated to be killing upwards of 100,000 people annually. Snakebite is associated with a diverse pathophysiology due to the magnitude of variation in venom composition that is observed worldwide. The haemolytic (i.e., lysis of red blood cells) actions of snake venoms are well documented, although the direct impact of venoms on haemoglobin is not fully understood. Here we report on the varied ability of a multitude of snake venoms to oxidise haemoglobin into methaemoglobin. Moreover, our results demonstrate that the venom of an elapid, the black necked spitting cobra, Naja nigricollis, oxidises oxyhaemoglobin (Fe2+) into methaemoglobin (Fe3+) in a time- and concentration-dependent manner that is unparalleled within the 47 viper and elapid venoms evaluated. The treatment of venom with a reducing agent, dithiothreitol (DTT) is observed to potentiate this effect at higher concentrations, and the use of denatured venom demonstrates that this effect is dependent upon the heat-sensitive proteinaceous elements of the venom. Together, our results suggest that Naja nigricollis venom appears to promote methaemoglobin production to a degree that is rare within the Elapidae family, and this activity appears to be independent of proteolytic activities of venom components on haemoglobin.

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS.

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.

A FERM-adjacent (FA) region defines a subset of the 4.1 superfamily and is a potential regulator of FERM domain function.

BACKGROUND: Proteins containing FERM domains comprise a diverse group of eukaryotic proteins that bind membrane proteins and lipids. In doing so, they organise membrane microstructure, and coordinate the disposition of signalling and cell adhesion complexes. In protein 4.1R, phosphorylation adjacent to the FERM domain regulates its activity, and membrane mechanical properties. RESULTS: A novel sequence domain has been detected in a subset of proteins that contain FERM domains. This subset includes the true 4.1 proteins, some tyrosine phosphatases, rho-GEF proteins and type II transmembrane proteins, as well as some uncharacterised FERM proteins. This FERM-adjacent region is always closely proximate to the C-terminal of the FERM domain. This sequence is likely to be folded with elements of alpha and beta structure. The FERM-adjacent region of 4.1R contains serine residues phosphorylated by PKC and PKA; these appear conserved in about half of all other FERM-adjacent regions. Phylogenetic analyses indicate that all proteins containing a FERM-adjacent region arose from a single ancestor after FERM domains had started to proliferate in genomes of animals, plants and mycetozoa. CONCLUSION: The FERM-adjacent region defines a subset of the FERM proteins in animals. The conservation of motifs in this region that are potential substrates for kinases together with the known regulatory phosphorylation of 4.1R in this region raises the possibility that the FERM-adjacent region is a regulatory adaptation in this subset of the FERM proteins.