Type II Membrane Protein Research Articles

Abstract A24: Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) induces apoptosis via ectodomain shedding mediated by a disintegrin and metalloproteinase 9

Abstract Objectives: Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a type-II membrane protein that can induce apoptosis in immune cells including peripheral lymphocytes and natural killer cells after converted to a secreted type by proteolytic processing referred to as “ectodomain shedding.” So far, over 150 scientific reports have been published concerning the biological functions and clinical significance of RCAS1. Because RCAS1 not only helps tumor cells to evade immune surveillance but also induces cancer stromal tissue remodeling, RCAS1 is believed to exaggerate aggressive characteristics of human malignancies. Clinically, RCAS1 expression correlates with several pathological variables including tumor size, stage, invasion depth, and lymph node metastasis and is a negative predictor of overall survival in 15 different kinds of cancer derived from brain, oral cavity, lung, pleural mesothelium, esophagus, stomach, bile duct, gallbladder, pancreas, colon, gastrointestinal mesenchyme, kidney, prostate, uterine cervix, and endometrium. RCAS1 is secreted significantly higher in serum and pleural effusion in cancer patients than non-cancer individuals and its level is changed in response to treatment. We here studied regarding ectodomain shedding that affects the biological activity of membrane proteins such as growth factors, growth factor receptors, cell-adhesion molecules, extracellular matrix proteins by altering their localization and mode of action. The objective of this study is to investigate (1) a key protease involved in RCAS1 shedding and (2) an association between tumor protease expression and serum RCAS1 concentration in uterine cancer patients. Materials and Methods: (1) The key protease involved in RCAS1 shedding was investigated using two cell lines: a human cervical adenocarcinoma cell line SiSo and a breast adenocarcinoma cell line MCF-7. Both SiSo and MCF-7 express RCAS1, but RCAS1 secretion is undetectable in MCF-7. The protease expression was evaluated by microarray analysis and immnocytochemistry. The expression and secretion of RCAS1 were measured by using flow cytometry and ELISA after knockdown or introduction of protease expression. The induction of apoptosis in an erythroleukemia cell line K562, which express a putative RCAS1 receptor, was investigated. K562 cells were co-cultured with SiSo, MCF-7, and protease cDNA transfected MCF-7 to follow RCAS1 secretion in apoptosis initiation. (2) The association between tumor protease expression and serum RCAS1 concentration was studied in 50 cases of uterine cancer patients. The tumor protease expression was evaluated by immunohistochemistry and serum RCAS1 concentration was measured by ELISA. Results: (1) Microarray analysis revealed that SiSo expressed MMP-28, ADAM9, and ADAM21 at significantly higher levels than did MCF-7. The association of RCAS1 and ADAM9 was detected by immunocytochemistry in SiSo. ADAM9 cDNA transfection in MCF-7 induced RCAS1 secretion and reduced its expression, on the other hand, ADAM9 siRNA transfection in SiSo suppressed RCAS1 secretion and augmented its expression. Growth inhibition and apoptosis were induced by ADAM9 cDNA tansfected MCF-7 but not parent cell. Therefore, it was revealed that ADAM9 is a key protease for RCAS1 shedding. (2) There was a significant correlation between tumor ADAM9 expression and serum RCAS1 concentration in uterine cancer. Conclusions: Further exploration regarding the regulatory mechanisms involved in the conversion of membrane-anchored RCAS1 into its soluble form by ADAM9 should aid the development of novel therapeutic strategies against human malignancies by targeting RCAS1. Citation Format: Kenzo Sonoda. Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) induces apoptosis via ectodomain shedding mediated by a disintegrin and metalloproteinase 9. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A24.

Dner inhibits neural progenitor proliferation and induces neuronal and glial differentiation in zebrafish

Delta/notch-like epidermal growth factor (EGF)-related receptor (DNER) is a single-pass transmembrane protein found to be a novel ligand in the Notch signaling pathway. Its function was previously characterized in the developing cerebellum and inner ear hair cells. In this study, we isolated a zebrafish homolog of DNER and showed that this gene is expressed in the developing nervous system. Overexpression of dner or the intracellular domain of dner was sufficient to inhibit the proliferation of neural progenitors and induce neuronal and glial differentiation. In contrast, the knockdown of endogenous Dner expression using antisense morpholino oligonucleotides increased the proliferation of neural progenitors and maintained neural cells in a progenitor status through inhibition of neuronal and glial differentiation. Through analysis of the antagonistic effect on the Delta ligand and the role of the potential downstream mediator Deltex1, we showed that Dner acts in Notch-dependent and Notch-independent manner. This is the first study to demonstrate a role for Dner in neural progenitors and neuronal differentiation and provides new insights into mediation of neuronal development and differentiation by the Notch signaling pathway.

Probing Cardiac Membrane Proteins with Fluorescence Resonance Energy Transfer

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study the structural basis of regulation of sarcoplasmic reticulum Ca2+ ATPase (SERCA) by a single-pass transmembrane protein, phospholamban (PLB). The most prominent feature of heart failure is calcium mishandling, which is largely due to impaired activity of sarcoplasmic reticulum calcium ATPase (SERCA), which actively transports Ca2+ from cytosol into the SR to relax the muscle. Unphosphorylated phospholamban inhibits SERCA by decreasing its apparent Ca2+ affinity. This inhibition can be relieved by either micromolar Ca2+ or by phosphorylation of PLB. The structural basis for relief of inhibition remains controversial. Cross-linking studies support the canonical dissociation model. However, recent EPR, NMR and FRET studies support the subunit model, in which subtle structural rearrangements are required to relieve the inhibition. The goal of this study is to elucidate the structural rearrangements during relief of inhibition. Previous EPR and NMR studies found that the cytoplasmic domain of PLB exists in a dynamic equilibrium between T and R states. We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to probe the complex directly by measuring the distances between SERCA (C674) and a series of positions on PLB in co-reconstituted vesicles. We found that the distances between SERCA and the cytoplasmic domain of PLB are best fitted with two Gaussian distance distributions, suggesting that the cytoplasmic domain of bound PLB exists in two states. Results will be reported on the effects of perturbations in this system which are known to relieve inhibition, including PLB phosphorylation, PLB mutation, and calcium binding to SERCA. We conclude that the function of the SERCA-PLB complex depends primarily on the structural equilibrium between two principal structural states of the complex.

E-cadherin–integrin crosstalk in cancer invasion and metastasis

E-cadherin is a single-pass transmembrane protein that mediates homophilic cell-cell interactions. Tumour progression is often associated with the loss of E-cadherin function and the transition to a more motile and invasive phenotype. This requires the coordinated regulation of both E-cadherin-mediated cell-cell adhesions and integrin-mediated adhesions that contact the surrounding extracellular matrix (ECM). Regulation of both types of adhesion is dynamic as cells respond to external cues from the tumour microenvironment that regulate polarity, directional migration and invasion. Here, we review the mechanisms by which tumour cells control the cross-regulation between dynamic E-cadherin-mediated cell-cell adhesions and integrin-mediated cell-matrix contacts, which govern the invasive and metastatic potential of tumours. In particular, we will discuss the role of the adhesion-linked kinases Src, focal adhesion kinase (FAK) and integrin-linked kinase (ILK), and the Rho family of GTPases.

EPR Detects Changes in the Transmembrane Region of the SERCA-Phospholamban Complex upon Ser16 Phosphorylation

We have used site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the effects of Ser16 phosphorylation on phospholamban's interaction with the sarcoplasmic reticulum Ca-ATPase (SERCA). Muscle contraction is signaled by the release of Ca from the sarcoplasmic reticulum (SR), while SERCA mediates muscle relaxation by actively pumping Ca back into the SR lumen. Cardiac SERCA is regulated by phospholamban (PLB), a single-pass transmembrane protein that inhibits the Ca pump unless phosphorylated at Ser16.We have shown that Ser16 phosphorylation does not dissociate PLB from SERCA to relieve inhibition, but instead induces a structural change in PLB's transmembrane (TM) helix that restores SERCA Ca sensitivity. To characterize this change, we have attached spin labels along the TM helix and performed EPR accessibility measurements to determine the effects of Ser16 phosphorylation on TM helix topology within the regulatory complex. Our results show that upon phosphorylation, the TM helix undergoes a vertical shift that could break inhibitory interactions between SERCA and PLB. Complementary ongoing studies employ these spin-labeled PLB constructs to determine changes in (a) the orientation of PLB and (b) distances to probes on SERCA, with the goal of obtaining a detailed structural model for the regulatory transitions within the SERCA-PLB complex.

The Impact of Nephrectomy and Renal Transplantation on Serum Levels of Soluble Klotho Protein

BackgroundKlotho, a single-pass transmembrane protein primarily expressed in the kidneys, parathyroid glands, and choroid plexus of the brain, has a short cytoplasmic tail and a long extracellular domain, which can be cleaved and released as a soluble form. However, information regarding the origins and kinetics of soluble serum Klotho remains poorly understood. We evaluated serial changes in serum Klotho levels among living donors before and after retroperitoneoscopic nephrectomy as well as in their renal transplant recipients. MethodsThe levels of soluble Klotho in serum obtained from 10 living donors and their renal transplant recipients were determined using a sandwich enzyme-linked immunosorbent assay system. ResultsSerum soluble Klotho was detectable in all subjects. The baseline serum Klotho concentrations in the living donors ranged from 726.4 to 1417.1 pg/mL (median, 909.8 pg/mL; interquartile ranges [IR], 754.8–1132.4), whereas that in the concomitant renal transplant recipients ranged from 397.5 to 1047.2 pg/mL (median, 613.0 pg/mL; IR, 445.9–750.8; P = .003). The levels of soluble serum Klotho measured 5 days after retroperitoneoscopic nephrectomy (median, 619.0 pg/mL; IR, 544.6–688.5; P = .001) were significantly lower than the baseline values. Among the renal transplant recipients, no significant changes in serum Klotho levels were observed during the observation period. ConclusionOur data regarding soluble serum Klotho levels obtained from living donors support the idea that the kidneys are a major source of soluble serum Klotho in human subjects without a deterioration of renal function. In recipients, concomitant acute kidney injuries and immunosuppressive protocols might modulate the release of soluble Klotho from the grafts into the circulation.

Neto2 Interacts with the Scaffolding Protein GRIP and Regulates Synaptic Abundance of Kainate Receptors

Kainate receptors (KARs) are a class of ionotropic glutamate receptors that are expressed throughout the central nervous system. The function and subcellular localization of KARs are tightly regulated by accessory proteins. We have previously identified the single-pass transmembrane proteins, Neto1 and Neto2, to be associated with native KARs. In the hippocampus, Neto1, but not Neto2, controls the abundance and modulates the kinetics of postsynaptic KARs. Here we evaluated whether Neto2 regulates synaptic KAR levels in the cerebellum where Neto1 expression is limited to the deep cerebellar nuclei. In the cerebellum, where Neto2 is present abundantly, we found a ∼40% decrease in GluK2-KARs at the postsynaptic density (PSD) of Neto2-null mice. No change, however, was observed in total level of GluK2-KARs, thereby suggesting a critical role of Neto2 on the synaptic localization of cerebellar KARs. The presence of a putative class II PDZ binding motif on Neto2 led us to also investigate whether it interacts with PDZ domain-containing proteins previously implicated in regulating synaptic abundance of KARs. We identified a PDZ-dependent interaction between Neto2 and the scaffolding protein GRIP. Furthermore, coexpression of Neto2 significantly increased the amount of GRIP associated with GluK2, suggesting that Neto2 may promote and/or stabilize GluK2:GRIP interactions. Our results demonstrate that Neto2, like Neto1, is an important auxiliary protein for modulating the synaptic levels of KARs. Moreover, we propose that the interactions of Neto1/2 with various scaffolding proteins is a critical mechanism by which KARs are stabilized at diverse synapses.

Genetic Interactions Between UNC-17/VAChT and a Novel Transmembrane Protein in Caenorhabditis elegans

The unc-17 gene encodes the vesicular acetylcholine transporter (VAChT) in Caenorhabditis elegans. unc-17 reduction-of-function mutants are small, slow growing, and uncoordinated. Several independent unc-17 alleles are associated with a glycine-to-arginine substitution (G347R), which introduces a positive charge in the ninth transmembrane domain (TMD) of UNC-17. To identify proteins that interact with UNC-17/VAChT, we screened for mutations that suppress the uncoordinated phenotype of UNC-17(G347R) mutants. We identified several dominant allele-specific suppressors, including mutations in the sup-1 locus. The sup-1 gene encodes a single-pass transmembrane protein that is expressed in a subset of neurons and in body muscles. Two independent suppressor alleles of sup-1 are associated with a glycine-to-glutamic acid substitution (G84E), resulting in a negative charge in the SUP-1 TMD. A sup-1 null mutant has no obvious deficits in cholinergic neurotransmission and does not suppress unc-17 mutant phenotypes. Bimolecular fluorescence complementation (BiFC) analysis demonstrated close association of SUP-1 and UNC-17 in synapse-rich regions of the cholinergic nervous system, including the nerve ring and dorsal nerve cords. These observations suggest that UNC-17 and SUP-1 are in close proximity at synapses. We propose that electrostatic interactions between the UNC-17(G347R) and SUP-1(G84E) TMDs alter the conformation of the mutant UNC-17 protein, thereby restoring UNC-17 function; this is similar to the interaction between UNC-17/VAChT and synaptobrevin.

Characteristics of urinary and serum soluble Klotho protein in patients with different degrees of chronic kidney disease

BackgroundKlotho is a single-pass transmembrane protein, which appears to be implicated in aging. The purpose of the present study was to characterize the relationship between the soluble Klotho level and renal function in patients with various degrees of chronic kidney disease (CKD).MethodsThe levels of soluble Klotho in the serum and urine obtained from one hundred thirty-one CKD patients were determined by a sandwich enzyme-linked immunosorbent assay system.ResultsThe amount of urinary excreted Klotho during the 24 hr period ranged from 1.6 to 5178 ng/day (median 427 ng/day; interquartile range [IR] 56.8-1293.1), and the serum Klotho concentration ranged from 163.9 to 2123.7 pg/ml (median 759.7 pg/ml; IR 579.5-1069.1). The estimated glomerular filtration rate (eGFR) was significantly correlated with the log-transformed values of the amount of 24 hr urinary excreted Klotho (r = 0.407, p < 0.01) and the serum Klotho levels (r = 0.232, p < 0.01). However, a stepwise multiple regression analysis identified eGFR to be a variable independently associated only with the log-transformed value of the amount of 24-hr urinary excreted Klotho but not with the log-transformed serum Klotho concentration. Despite the strong correlation between random urine protein-to-creatinine ratio and the 24 hr urinary protein excretion (r = 0.834, p < 0.01), a moderate linear association was observed between the log-transformed value of the amount of 24 hr urinary excreted Klotho and that of the urinary Klotho-to-creatinine ratio (Klotho/Cr) in random urine specimens (r = 0.726, p < 0.01).ConclusionsThe amount of urinary Klotho, rather than the serum Klotho levels, should be linked to the magnitude of the functioning nephrons in CKD patients. The use of random urine Klotho/Cr as a surrogate for the amount of 24-hr urinary excreted Klotho needs to be evaluated more carefully.

CNTNAP2 is significantly associated with schizophrenia and major depression in the Han Chinese population

CNTNAP2, located on 7q35–36.1, encodes a single-pass transmembrane protein mediating cell–cell interactions in the nervous system. CNTNAP2 has been suggested to play an important role in mental diseases such as autism and language disorder. However, we still do not know whether it also confers risk to major psychiatric disorders such as schizophrenia, major depression and bipolar disorder. We analysed single nucleotide polymorphisms (SNPs) previously reported to be associated with autism or language impairment in 1135 schizophrenia patients, 1135 unrelated major depression patients, 1135 unrelated bipolar disorder patients and 1135 unrelated normal controls recruited from the Han Chinese population. We found that the genotypes of rs17236239 were significantly associated with schizophrenia and the alleles of rs2710102 and rs2710117 were significantly associated with major depression. According to the location of significant signals, our study indicated that exon 13–15 of CNTNAP2 may play important roles in both schizophrenia and major depression in the Han Chinese population.

RNA Interference: Systemic RNAi SIDes with Endosomes

Systemic RNAi, the intercellular spreading of RNAi silencing, requires SID-1 and SID-3 to import silencing signals in Caenorhabditis elegans. How are these signals exported? SID-5, an endosome-associated protein, is a candidate for the job.

Biochemical and functional characterization of human phospholipid scramblase 4 (hPLSCR4)

Human phospholipid scramblase 4 (hPLSCR4), an isoform of the scramblase family, is a type II single-pass transmembrane protein whose function remains unknown. To understand its role, recombinant hPLSCR4 was obtained by cloning the ORF into a pET28 a(+) vector and overexpressed in Escherichia coli. Functional assay showed that Ca2+, Mg2+, and Zn2+ activate hPLSCR4 and mediate scrambling activity independent of the phospholipid head group. Far-UV-CD and fluorescence spectroscopy revealed that Ca2+ and Mg2+ binding induces conformation change in hPLSCR4, exposing hydrophobic patches of the protein, and Ca2+ has more affinity than Mg2+ and Zn2+. Stains-all studies further confirm that hPLSCR4 is a Ca2+-binding protein. Point mutation (Asp290→Ala) in hPLSCR4 decreased the Ca2+-binding affinity as well as Tb3+ luminescence, suggesting residues of the predicted Ca2+-binding motif are involved in Ca2+ binding. Functional reconstitution with (Asp290→Ala) mutant led to ~50% and ~40% decrease in scramblase activity in the presence of Ca2+ and Mg2+, respectively.

Protein disorder and short conserved motifs in disordered regions are enriched near the cytoplasmic side of single-pass transmembrane proteins.

Intracellular juxtamembrane regions of transmembrane proteins play pivotal roles in cell signalling, mediated by protein-protein interactions. Disordered protein regions, and short conserved motifs within them, are emerging as key determinants of many such interactions. Here, we investigated whether disorder and conserved motifs are enriched in the juxtamembrane area of human single-pass transmembrane proteins. Conserved motifs were defined as short disordered regions that were much more conserved than the adjacent disordered residues. Human single-pass proteins had higher mean disorder in their cytoplasmic segments than their extracellular parts. Some, but not all, of this effect reflected the shorter length of the cytoplasmic tail. A peak of cytoplasmic disorder was seen at around 30 residues from the membrane. We noted a significant increase in the incidence of conserved motifs within the disordered regions at the same location, even after correcting for the extent of disorder. We conclude that elevated disorder within the cytoplasmic tail of many transmembrane proteins is likely to be associated with enrichment for signalling interactions mediated by conserved short motifs.

Multistate Organization of Transmembrane Helical Protein Dimers Governed by the Host Membrane

Association of transmembrane (TM) helices taking place in the cell membrane has an important contribution to the biological function of bitopic proteins, among which receptor tyrosine kinases represent a typical example and a potent target for medical applications. Since this process depends on a complex interplay of different factors (primary structures of TM domains and juxtamembrane regions, composition and phase of the local membrane environment, etc.), it is still far from being fully understood. Here, we present a computational modeling framework, which we have applied to systematically analyze dimerization of 18 TM helical homo- and heterodimers of different bitopic proteins, including the family of epidermal growth factor receptors (ErbBs). For this purpose, we have developed a novel surface-based modeling approach, which not only is able to predict particular conformations of TM dimers in good agreement with experiment but also provides screening of their conformational heterogeneity. Using all-atom molecular dynamics simulations of several of the predicted dimers in different model membranes, we have elucidated a putative role of the environment in selection of particular conformations. Simulation results clearly show that each particular bilayer preferentially stabilizes one of possible dimer conformations, and that the energy gain depends on the interplay between structural properties of the protein and the membrane. Moreover, the character of protein-driven perturbations of the bilayer is reflected in the contribution of a particular membrane to the free energy gain. We have found that the approximated dimerization strength for ErbBs family can be related to their oncogenic ability.

STIM/Orai signalling complexes in vascular smooth muscle

Stromal interaction molecules (STIM1 and STIM2) are single pass transmembrane proteins located mainly in the endoplasmic reticulum (ER). STIM proteins contain an EF-hand in their N-termini that faces the lumen side of the ER allowing them to act as ER calcium (Ca(2+)) sensors. STIM1 has been recognized as central to the activation of the highly Ca(2+) selective store-operated Ca(2+) (SOC) entry current mediated by the Ca(2+) release-activated Ca(2+) (CRAC) channel; CRAC channels are formed by tetramers of the plasma membrane (PM) protein Orai1. Physiologically, the production of inositol 1,4,5-trisphosphate (IP(3)) upon stimulation of phospholipase C-coupled receptors and the subsequent emptying of IP(3)-sensitive ER Ca(2+) stores are sensed by STIM1 molecules which aggregate and move closer to the PM to interact physically with Orai1 channels and activate Ca(2+) entry. Orai1 has two homologous proteins encoded by separate genes, Orai2 and Orai3. Other modes of receptor-regulated Ca(2+) entry into cells are store-independent; for example, arachidonic acid activates a highly Ca(2+) selective store-independent channel formed by heteropentamers of Orai1 and Orai3 and regulated by the PM pool of STIM1. Here, I will discuss results pertaining to the roles of STIM and Orai proteins in smooth muscle Ca(2+) entry pathways and their role in vascular remodelling.

Constitutive Cleavage of the Single-Pass Transmembrane Protein Alcadeinα Prevents Aberrant Peripheral Retention of Kinesin-1

Various membrane proteins are shed by proteinases, constitutively and/or when stimulated by external signals. While the physiological significance of external signal-induced cleavages has been intensely investigated, relatively little is known about the function of constitutive cleavages. Alcadeinα (Alcα; also called Calsyntenin-1) is an evolutionarily conserved type I single-pass transmembrane protein that binds to kinesin-1 light chain (KLC) to activate kinesin-1's transport of Alcα-containing vesicles. We found that Alcα was constitutively and efficiently cleaved to liberate its ectodomain into the extracellular space, and that full-length Alcα protein was rarely detected on the cell surface. The secretion efficiency of the ectodomain was unaltered by a mutation that both abolished Alcα's KLC-binding activity and attenuated its peripheral transport, suggesting that Alcα's cleavage occurred, at least partly, en route to the cell surface. We further demonstrated that uncleavable mutant Alcα proteins readily accumulated on the cell surface and induced aberrant peripheral recruitment of KLC1 and kinesin heavy chain. Our observations suggest that Alcα is efficiently processed in part to minimize the inappropriate peripheral retention of kinesin-1. This role might exemplify the functional relevance of the constitutive cleavage of single-pass transmembrane proteins.

Klotho as a potential biomarker and therapy for acute kidney injury

Klotho is a single-pass transmembrane protein that is highly expressed in the kidney and is known to act as a coreceptor for fibroblast growth factor 23. The extracellular domain can be produced independently or shed from membrane-bound Klotho and functions as an endocrine substance with multiple functions including antioxidation, modulation of ion transport, suppression of fibrosis, and preservation of stem cells. Emerging evidence has revealed that Klotho deficiency is an early event in acute kidney injury (AKI), and a pathogenic factor that exacerbates acute kidney damage and contributes to long-term consequences. Restoration by exogenous supplementation or stimulation of endogenous Klotho might prevent and ameliorate injury, promote recovery, and suppress fibrosis to mitigate development of chronic kidney disease. Although data are still emerging, in this Perspectives article we discuss why this renal-derived protein is a highly promising candidate as both an early biomarker and therapeutic agent for AKI.

Intracellular Trafficking of FXYD1 (Phospholemman) and FXYD7 Proteins in Xenopus Oocytes and Mammalian Cells

FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na(+)/K(+) ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na(+)/K(+) ATPase. On the other hand, the Na(+)/Ca(2+) exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na(+)/K(+) ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1β1 Na(+)/K(+) ATPase. The Na(+)/K(+) ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.

Analysis of Delta–Notch interaction by molecular modeling and molecular dynamic simulation studies

Notch is a single-pass transmembrane receptor protein. Delta (member of the DSL protein family), a Notch ligand, is also single-pass transmembrane protein that can interact with Notch to form the Delta–Notch complex. It has been demonstrated that of the 36 Epidermal Growth Factor (EGF) repeats of Notch, 11th and 12th are sufficient to mediate interactions with Delta. Crystal structure of mammalian Notch1 extracellular ligand binding domain shows the presence of 11th and 12th EGF-like repeats. Here a portion of the Drosophila Delta protein, known to interact with Notch extracellular domain, has been modeled using homology modeling. The structure of the Delta–Notch complex was subsequently modeled by protein-docking method using GRAMM. Molecular dynamic simulations of the modeled structures were performed. The probable structures for Delta–Notch complex have been proposed based on interaction energy parameter and planarity studies.

Analysis of molecular recognition features in membrane proteins

Motivations. During the past few years there has been a growing interest in the field of intrinsically disordered proteins-related research. Intrinsically disordered proteins (IDPs) possess no rigid 3D structure under physiological conditions, yet they are functionally active. A protein may be fully or partly disordered, containing long or short intrinsically disordered regions (IDRs). Molecular Recognition Features (MoRFs), known also as Molecular Recognition Elements (MoREs), are defined as short regions that undergo disorder-to-order transition upon binding to their partners. As their name suggests, they are considered to be implicated in molecular recognition, which serves as the initial step for protein-protein interactions. Membrane proteins comprise approximately 30% of fully sequenced proteomes and they are responsible for a wide variety of cellular functions, including cell signalling. The aim of the current study was to identify and analyse MoRFs in membrane proteins. Methods. A dataset of putative MoRFs was constructed from the Protein Data Bank, selecting membrane protein fragments between 10 and 70 residues, which interact with proteins longer than 100 residues. The assumption was made that such short aminoacid sequences would be less likely to form a rigid 3D structure prior to interaction. The initial dataset was further filtered for ambiguous information and a non-redundant dataset was created applying length-dependent thresholds. Subsequently, sequence, structural and functional analysis of the membrane MoRF non-redundant dataset was conducted. Results. Initialy, we sought to characterize our dataset and assess membrane MoRF associations with intrinsic disorder. Approximately half of the membrane MoRFs are short, between 10 and 20 residues. In addition, membrane MoRFs' aminoacid composition was found to differ not only from that of a typical IDR, as expected, but from globular MoRFs' aminoacid preferences as well. A structure-based criterion, evaluating per-residue surface and interface areas, supported the idea that membrane MoRFs are intrinsically disordered when isolated, and undergo a disorder-to-order transition upon binding. Missing density residues, often associated with disorder, were also assessed and were found to comprise, along with irregular residues, more than half of membrane MoRFs' secondary structure. Membrane MoRFs were categorized as alpha, beta, irregular and complex, depending on their secondary structure after the interaction with their partners. Further studies were focused on MoRF-containing proteins and revealed that the vast majority are eukaryotic single-spanning transmembrane proteins. Moreover, the position of MoRFs in relation to the protein's topology was determined. Finally, functional analyses of MoRF-containing proteins and MoRF-binding partners are currently under way. In conclusion, our goal is to provide insight into potential disorder-based protein-protein interactions involving membrane proteins. A comparison between membrane and globular MoRFs will allow us to determine if they are likewise implicated in molecular recognition procedures and whether a similar mechanism is involved. In the long term, the above information will facilitate identification, and possibly prediction, of membrane MoRFs.