Single-pass Membrane Protein Research Articles

Proteolytic control of the mitochondrial calcium uniporter complex

The mitochondrial calcium uniporter is a Ca2+-activated Ca2+ channel complex mediating mitochondrial Ca2+ uptake, a process crucial for Ca2+ signaling, bioenergetics, and cell death. The uniporter is composed of the pore-forming MCU protein, the gatekeeping MICU1 and MICU2 subunits, and EMRE, a single-pass membrane protein that links MCU and MICU1 together. As a bridging subunit required for channel function, EMRE could paradoxically inhibit uniporter complex formation if expressed in excess. Here, we show that mitochondrial mAAA proteases AFG3L2 and SPG7 rapidly degrade unassembled EMRE using the energy of ATP hydrolysis. Once EMRE is incorporated into the complex, its turnover is inhibited >15-fold. Protease-resistant EMRE mutants produce uniporter subcomplexes that induce constitutive Ca2+ leakage into mitochondria, a condition linked to debilitating neuromuscular disorders in humans. The results highlight the dynamic nature of uniporter subunit assembly, which must be tightly regulated to ensure proper mitochondrial responses to intracellular Ca2+ signals.

Role of the Transmembrane Domain of Mucin 1 in Nuclear Localization

Mucin 1 (MUC1) is a highly glycosylated, single pass membrane protein that provides the mucous surfaces of epithelial cells and protects against pathogens. It consists of two parts, MUC1‐N, which includes the extracellular domain, and MUC1‐C, which includes the cytosolic and transmembrane domains. MUC1 overexpression has been observed in 80 to 90% of human solid tissue cancers. Studies have shown that this overexpression may lead to malignant unregulated gene expression when MUC1‐C enters the nucleus. The transport of MUC1‐C, from the plasma membrane to the nucleus, appears to be the result of MUC1‐C homodimerization. Our lab has utilized ToxR Assays to elucidate the role of the transmembrane domain in MUC1‐C dimerization. Using this assay we have seen that an Ala1180Leu mutation decreases the propensity of MUC1‐C to dimerize, implying it may be of importance to the dimer interface. Because this mutation is shown to decrease the ability of MUC1‐C to form dimers, the hypothesis is that it will also decrease the transport of MUC1‐C to the nucleus. Currently, we are working with mammalian (HEK) cells to determine if the Ala1180Leu mutation also affects nuclear localization of MUC1. Understanding of the mechanisms of MUC1‐C dimerization and nuclear localization is important in designing effective therapeutics.Support or Funding InformationSaint Joseph's University Department of Biology Saint Joseph's University Summer Scholars Program Edwin Li, Ph.D.

Proteome-Wide Modeling of Transmembrane Alpha-Helical Homodimers by TMDOCK

A novel energy-based method, TMDOCK, has been developed for ab initio modeling of 3D structures of parallel homodimers in membranes. Instead of exploring the entire conformational space, the method employs a fast template-driven global energy optimization procedure that combines the knowledge of geometrically feasible packing arrangements and local energy minimization with a novel force field. Ranking and selection of optimal dimerization modes is based on the calculated free energy of α-helix association (ΔGassoc) which includes van der Waals, hydrogen bonding and dipole-dipole interactions between helices, side chain conformational entropy, and solvation energy in the anisotropic lipid environment. The fast version of the TMDOCK method is available through the web server (http://membranome.org/tm_server.php).TMDOCK successfully reproduced 26 experimental dimeric structures formed by transmembrane (TM) α-helices of 21 single-pass membrane proteins (including 4 mutants) with Cα atom r.m.s.d. from 1.0 to 3.3 A. Additional testing demonstrated consistency of calculated dimer structures with published TOXCAT, mutagenesis, or other experimental data for more than 80 bitopic proteins. Subsequent application of the method to 6041 bitopic proteins from six proteomes included in the Membranome database (membranome.org) shows that ∼70% of the proteins tend to form stable dimers with ΔGassoc<-3 kcal/mol. Results of modeling indicate three different types of dimerization behavior of TM α-helices. A unique dimerization mode is usually observed for TM domains carrying a single GxxxG-like motif, interhelical disulfide bridges, or polar residues in the middle of a helix. Proteins with several GxxxG-like motifs usually have a few dimerization modes with similar energies. Proteins with Phe-rich sequences often show multiple helix association modes. The preferred modes of dimerization are frequently evolutionarily conserved (e.g. in cadherin, HLA histocompatibility antigen, receptor tyrosine kinase, and cytochrome P450 families), but they can also change even after a single mutation.

TMDOCK: An Energy-Based Method for Modeling α-Helical Dimers in Membranes

TMDOCK is a novel computational method for the modeling of parallel homodimers formed by transmembrane (TM) α-helices. Three-dimensional (3D) models of dimers are generated by threading a target amino acid sequence through several structural templates, followed by local energy minimization. This is the first method that identifies helix dimerization modes and ranks them based on the calculated free energy of α-helix association. Free energy components include van der Waals, hydrogen bonding, and dipole interactions; side-chain conformational entropy; and solvation energy in the anisotropic lipid environment. TMDOCK reproduced 26 experimental dimeric structures formed by TM α-helices of 21 single-pass membrane proteins (including 4 mutants) with Cα atom rmsd from 1.0 to 3.3Å. Assessment of dimerization heterogeneity of these TM domains demonstrated that 7 of them have a unique dimer structure, 12 have at least 2 alternative conformations, and 2 have a large number of different association modes. All unique experimental structures of proteins from the first group and eight structures from the second group were reproduced in computations as top-ranked models. A fast version of the method is available through the web server (http://membranome.org/tm_server.php).

Membranome: a database for proteome-wide analysis of single-pass membrane proteins.

The Membranome database was developed to assist analysis and computational modeling of single-pass (bitopic) transmembrane (TM) proteins and their complexes by providing structural information about these proteins on a genomic scale. The database currently collects data on >6000 bitopic proteins from Homo sapiens, Arabidopsis thaliana, Dictyostelium discoideum, Saccharomyces cerevisiae, Escherichia coli and Methanocaldococcus jannaschii. It presents the following data: (i) hierarchical classification of bitopic proteins into 15 functional classes, 689 structural superfamilies and 1404 families; (ii) 446 complexes of bitopic proteins with known three-dimensional (3D) structures classified into 129 families; (iii) computationally generated three-dimensional models of TM α-helices positioned in membranes; (iv) amino acid sequences, domain architecture, functional annotation and available experimental structures of bitopic proteins; (v) TM topology and intracellular localization, (vi) physical interactions between proteins from the database along with links to other resources. The database is freely accessible at http://membranome.org. There is a variety of options for browsing, sorting, searching and retrieval of the content, including downloadable coordinate files of TM domains with calculated membrane boundaries.

Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.

Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine.

The degradation requirements for topologically distinct quality control substrates in the yeast endoplasmic reticulum

In eukaryotic cells, nearly one third of all newly synthesized proteins are translocated into the endoplasmic reticulum (ER). Inside the ER, proteins are post‐translationally modified and folded by molecular chaperones into their native states. Mutations, transcriptional/translational errors, aberrant post‐translational modifications, or misassembly can result in ER‐associated degradation (ERAD), a quality control pathway which recognizes and degrades misfolded proteins to prevent toxic gain‐of‐function phenotypes. Given the vast array of conformers that proteins adopt, it is not surprising that many diseases result from protein degradation by the ERAD pathway.The rate‐limiting step during ERAD is substrate retrotranslocation from the ER into the cytoplasm, which occurs prior to proteasome‐mediated degradation. Therefore, the retrotranslocation step can tune degradation efficiency and potentially be targeted to mitigate ERAD‐related diseases. Retrotranslocation is driven by the AAA‐ATPase Cdc48p in yeast (p97 in mammals), which extracts integral membrane proteins either directly from the ER membrane or through an as‐yet‐elusive retrotranslocation channel. Although integral membrane proteins have diverse membrane spanning domains and orientation, little is known about how the placement of a misfolded domain in the ER lumen versus the cytoplasm influences ERAD efficiency. We predict that extraction of a large ER lumenal domain will slow retrotranslocation kinetics.To test this hypothesis, a pair of model ERAD substrates were designed to contain the same truncated nucleotide‐binding domain from a well‐characterized ERAD substrate, Ste6p*, but on opposite sides of the ER membrane. We previously demonstrated that a soluble version of this domain, NBD2*, when expressed in the cytoplasm is degraded by the proteasome, and therefore can be used as a transposable degron (Guerriero et al., JBC, 2013). Our first model substrate, Chimera A*, is a dual pass membrane protein fused to NBD2* positioned in the cytoplasm. By contrast, Chimera N*, is a single pass membrane protein which deposits NBD2* in the ER lumen. Initial characterization confirms the proper topology of these substrates and proteasome‐dependent degradation. Moreover, the substrates rely on ER resident E3 ligases, Hrd1p and Doa10p, for efficient degradation. We are currently determining the relative dependence on Cdc48p for both degradation and retrotranslocation in order to better understand the energetic barriers to ERAD (Supported by NIH grants DK101584 to C.J.G. and GM075061 to J.L.B.).

The calmodulin-like proteins AtCML4 and AtCML5 are single-pass membrane proteins targeted to the endomembrane system by an N-terminal signal anchor sequence.

Calmodulins (CaMs) are important mediators of Ca(2+) signals that are found ubiquitously in all eukaryotic organisms. Plants contain a unique family of calmodulin-like proteins (CMLs) that exhibit greater sequence variance compared to canonical CaMs. The Arabidopsis thaliana proteins AtCML4 and AtCML5 are members of CML subfamily VII and possess a CaM domain comprising the characteristic double pair of EF-hands, but they are distinguished from other members of this subfamily and from canonical CaMs by an N-terminal extension of their amino acid sequence. Transient expression of yellow fluorescent protein-tagged AtCML4 and AtCML5 under a 35S-promoter in Nicotiana benthamiana leaf cells revealed a spherical fluorescence pattern. This pattern was confirmed by transient expression in Arabidopsis protoplasts under the native promoter. Co-localization analyses with various endomembrane marker proteins suggest that AtCML4 and AtCML5 are localized to vesicular structures in the interphase between Golgi and the endosomal system. Further studies revealed AtCML5 to be a single-pass membrane protein that is targeted into the endomembrane system by an N-terminal signal anchor sequence. Self-assembly green fluorescent protein and protease protection assays support a topology with the CaM domain exposed to the cytosolic surface and not the lumen of the vesicles, indicating that AtCML5 could sense Ca(2+) signals in the cytosol. Phylogenetic analysis suggests that AtCML4 and AtCML5 are closely related paralogues originating from a duplication event within the Brassicaceae family. CML4/5-like proteins seem to be universally present in eudicots but are absent in some monocots. Together these results show that CML4/5-like proteins represent a flowering plant-specific subfamily of CMLs with a potential function in vesicle transport within the plant endomembrane system.

A Cytomegalovirus Peptide-Specific Antibody Alters Natural Killer Cell Homeostasis and Is Shared in Several Autoimmune Diseases.

Human cytomegalovirus (hCMV), a ubiquitous beta-herpesvirus, has been associated with several autoimmune diseases. However, the direct role of hCMV in inducing autoimmune disorders remains unclear. Here we report the identification of an autoantibody that recognizes a group of peptides with a conserved motif matching the Pp150 protein of hCMV (anti-Pp150) and is shared among patients with various autoimmune diseases. Anti-Pp150 also recognizes the single-pass membrane protein CIP2A and induces the death of CD56(bright) NK cells, a natural killer cell subset whose expansion is correlated with autoimmune disease. Consistent with this finding, the percentage of circulating CD56(bright) NK cells is reduced in patients with several autoimmune diseases and negatively correlates with anti-Pp150 concentration. CD56(bright) NK cell death occurs via both antibody- and complement-dependent cytotoxicity. Our findings reveal that a shared hCMV-induced autoantibody is involved in the decrease of CD56(bright) NK cells and may thus contribute to the onset of autoimmune disorders.

Development of a Novel FRET Assay to Characterize the Oligomerization State of Self-Associating Transmembrane Helices

Using genetic reporter assays, significant self-interaction has been discovered in over 50 transmembrane (TM) helices from single-pass membrane proteins. To understand the biological functions of these TM interactions, which include signal transfer across the membrane in cancer-related pathways, it is necessary to construct accurate structural models of the TM oligomer. However in most cases this is not possible because the oligomerization state (stoichiometry) is unknown. It is often assumed that the helices form a dimer, though biologically relevant TM oligomers include dimers, trimers, and pentamers, as exemplified by GpA, HLA II Ii, and phospholamban, respectively.To characterize the stoichiometry of interacting single-pass membrane proteins, we are establishing a novel method based on Forster resonance energy transfer between identical fluorophores (homo-FRET). In order to allow the rapid bacterial expression and the purification of a wide range of fluorophore-coupled TM helices, we have developed TM fusion proteins with green fluorescent protein (GFP) and affinity tags. The measured homoFRET (via steady-state anisotropy) is proportional to both the affinity of the helices and the oligomerization state. Using only a fluorescence microplate reader, we demonstrate significant homo-FRET-coupling for GFP fusion proteins. Theoretical studies have shown that gradual photobleaching should yield a pattern of polarization that depends on the oligomerization state. High-profile experimental studies have confirmed this in-vivo using advanced microscopy. We show for the first time that the “fingerprint”-like curves after laser photobleaching can be achieved in-vitro with relatively simple equipment, allowing us to differentiate between oligomerization states. This demonstrates the feasibility of a rapid homo-FRET assay to determine the stoichiometry of transmembrane proteins in liposomes.

EPR and Electron Microscopy Study of the Influenza a M2 Transmembrane Domain Assembly and Drug Response

The M2 proton channel is essential to the influenza a virus life cycle. This single-pass helical membrane protein, which assembles in a tetramer, is a target for anti-influenza pharmacological development. The study of M2 is critical to our understanding of the mechanisms by which drugs inhibit its function and is also important in the more general context of protein-drug interactions.We report on the structural response of the M2 trans-membrane domain (M2TMD) C-terminal to amantadine drug binding at pH 5.5, i.e. at conditions close to those in endosomes where the M2 channel is active. We applied EPR spectroscopy and electron microscopy to the M2TMD core peptide extended with N- and C-terminus flanking residues, (M2TMD21-49). For purposes of this study, the peptide was labeled at position 46 using either nitroxide spin-label or nanogold particles and reconstituted into 85:15 mol% DOPC:POPS membranes, either with or without the addition of amantadine. To determine the oligomerization state, we analyzed the concentration dependence of double electron-electron resonance (DEER) data from spin-labeled samples prepared using peptide-to-lipid molar ratios (P/L) ranging from 1:160 to 1:18,800. We found that amantadine enhances M2TMD21-49 self-association. Moreover, binding of amantadine shortens inter-spin distances between protomers by 5-8 A. These large drug-induced structural changes are associated with M2TMD C-terminal pore closure and M2-channel inactivation. Additionally, continuous-wave EPR spectra analysis revealed increased ordering in the spin-label motional dynamics, pointing to local restructuring caused by compacting conformational changes. The electron microscopy images from samples with P/L of 1:10,000 clearly show the presence of monomers, dimers and tetramers of M2TMD21-49 in the presence and absence of amantadine, thereby supporting the model based on an intermediate strongly-bound dimeric state that emerged from our DEER study of the M2-channel assembly pathway.

Determining the CQC-Mediated Interactions in the Mucin 1 Homodimer

Mucin 1 (MUC1) is a highly glycosylated single pass membrane protein that serves as a selective barrier against pathogens and engages in signal transduction influencing cellular growth and differentiation. Overexpression of mucins have been linked to 80-90% of all human solid tissue cancers. Constitutive homodimerization, as a result of overexpression, leads to the formation of complexes with growth factor receptors and targeting to the nucleus, where MUC1 interacts with effector proteins regulating gene expression.Recent studies have shown that the MUC1 cytosolic membrane proximal CQC motif promotes dimerization under oxidizing conditions, suggesting that the motif may act as a redox switch in response to changes in oxidant levels. Preliminary studies have shown that the transmembrane domain (TMD) of MUC1 lacking the CQC motif form weak dimers, confirming their contribution to dimerization. Also, our results show that TMDs with the CQC motif dimerize strongly when the motif is in an oxidizing environment, further enforcing the role of the motif acts as a redox switch.Using the ToxR dimerization assay, the effects of single amino acid substitutions in TMDs with the CQC juxtamembrane area are analyzed. With these substitutions, the dimer interface can be elucidated. Our goal is to determine (1) whether certain amino acids in the TMD play a key role in forming MUC1 homodimers, and (2) whether the CQC motif induces a structural change when it functions as a redox switch. These conclusions will contribute to our understanding of MUC1 dimerization and provide insights for the development of novel therapeutic strategies.

Association of the SEL1L protein transmembrane domain with HRD1 ubiquitin ligase regulates ERAD‐L

Misfolded proteins in the endoplasmic reticulum (ER) are transported to the cytoplasm for degradation by the ubiquitin-proteasome system, a process otherwise known as ER-associated degradation (ERAD). Mammalian HRD1, an integral membrane ubiquitin ligase that ubiquitinates ERAD substrates, forms a large assembly in the ER membrane including SEL1L, a single-pass membrane protein, and additional components. The mechanism by which these molecules export misfolded proteins through the ER membrane remains unclear. Unlike Hrd3p, the homologue in Saccharomyces cerevisiae, human SEL1L is an unstable protein, which is restored by the association with HRD1. Here we report that the inherently unstable nature of the human SEL1L protein lies in its transmembrane domain, and that association of HRD1 with the SEL1L transmembrane domain restored its stability. On the other hand, we found that the SEL1L luminal domain escaped degradation, and inhibited the degradation of misfolded α1 -antitrypsin variant null Hong Kong by retaining the misfolded cargo in the ER. Overexpression of HRD1 inhibited the degradation of unfolded secretory cargo, which was restored by the interaction of HRD1 with the SEL1L transmembrane domain. Hence, we propose that SEL1L critically regulates HRD1-mediated disposal of misfolded cargo through its short membrane spanning stretch.

Role of GxxxG Motifs in Transmembrane Domain Interactions.

Transmembrane (TM) helices of integral membrane proteins can facilitate strong and specific noncovalent protein-protein interactions. Mutagenesis and structural analyses have revealed numerous examples in which the interaction between TM helices of single-pass membrane proteins is dependent on a GxxxG or (small)xxx(small) motif. It is therefore tempting to use the presence of these simple motifs as an indicator of TM helix interactions. In this Current Topic review, we point out that these motifs are quite common, with more than 50% of single-pass TM domains containing a (small)xxx(small) motif. However, the actual interaction strength of motif-containing helices depends strongly on sequence context and membrane properties. In addition, recent studies have revealed several GxxxG-containing TM domains that interact via alternative interfaces involving hydrophobic, polar, aromatic, or even ionizable residues that do not form recognizable motifs. In multipass membrane proteins, GxxxG motifs can be important for protein folding, and not just oligomerization. Our current knowledge thus suggests that the presence of a GxxxG motif alone is a weak predictor of protein dimerization in the membrane.

Mechanism of influenza A M2 transmembrane domain assembly in lipid membranes

M2 from influenza A virus functions as an oligomeric proton channel essential for the viral cycle, hence it is a high-priority pharmacological target whose structure and functions require better understanding. We studied the mechanism of M2 transmembrane domain (M2TMD) assembly in lipid membranes by the powerful biophysical technique of double electron-electron resonance (DEER) spectroscopy. By varying the M2TMD-to-lipid molar ratio over a wide range from 1:18,800 to 1:160, we found that M2TMD exists as monomers, dimers, and tetramers whose relative populations shift to tetramers with the increase of peptide-to-lipid (P/L) molar ratio. Our results strongly support the tandem mechanism of M2 assembly that is monomers-to-dimer then dimers-to-tetramer, since tight dimers are abundant at small P/L’s, and thereafter they assemble as dimers of dimers in weaker tetramers. The stepwise mechanism found for a single-pass membrane protein oligomeric assembly should contribute to the knowledge of the association steps in membrane protein folding.

Abstract 40: Discovery and Characterization of the Novel Muscle-Specific Membrane Protein Smco1

Mutations in numerous membrane proteins cause debilitating myopathies. The discovery of novel muscle-specific, membrane proteins would likely provide insight into mechanisms of disease and potentially yield new therapeutic targets. Through bioinformatics screening for muscle-specific membrane proteins with unknown function, we identified C3orf43 or single-pass membrane protein with coiled-coil domains 1 (Smco1). Consistent with bioinformatics predictions, Smco1 is expressed exclusively in cardiac and skeletal muscle. We demonstrate with chromatin immunoprecipitation and luciferase promoter assays that Smco1 is a Mef2-regulated gene with robust expression occurring shortly after birth. Immunofluorescent analysis demonstrates Scmo1 localizes to the cardiomyocyte sarcolemma and intercalated disks. Talen-mediated disruption of Smco1 in mice results in stunted postnatal growth, cardiac hypoplasia and skeletal muscle myopathy as early as postnatal day 15. While studies are on going to determine the function of Smco1, our findings reveal an essential role of Smco1 in striated muscle structure and function. The identification of heart- and muscle-specific membrane proteins will likely illuminate the mechanisms of muscular membrane diseases.

Molecular Docking of Amino Acid Analogues of Rimantidine

M2 channel is a 97-residue single-pass membrane protein with its N-and C-termini directed toward the outside and inside of the virion, re-spectively; it is a homotetramer in its native state. The four TM helicesform a channel in which His37 is the pH sensor and Trp41, the gate. Theadamantane-based drugs, amantadine and rimantadine, which target theM2 channel, have been used as rst-choice antiviral drugs against commu-nity outbreaks of inuenza A viruses for many years, but resistance to theadamantanes has recently become widespread. To overcome this dierentanalogues of rimantidine have been synthesized. The aim of this study isto predict the biological activity of amino acid analogues of rimantidinewith a help of docking studies in order to synthesize only promising can-didates. Twenty analogues of rimantidine with natural amino acids wereused. Docking was performed with the M2 channel as it is very impor-tant in the replicative cycle of inuenza A virus. Crystal structure of thechannel was obtained from RCSB (PDB id: 2rlf). Docking was performedwith GOLD 5.2 using GoldScore tness function. The complexes of riman-tidine analogues with M2 channel were analyzed and their total energieswere calculated in Molegro Molecular Viewer. Total energy of the com-plex rimantidine/M2 channel is -29.437 kJ/mole. All complexes of aminoacid analogues of rimantidine with the channel have lower energies, butthe lowest are the energies of the complexes of Asn-Rim/M2 channel andHis-Rim/M2 channel with -72.475 and -76.440 kJ/mole, respectively. Fromthe energetically point of view complexes will be more stables. This meansthat all rimantidine analogues will block the M2 channel thus will aectthe viral replication cycle. Docking studies are an useful tool for predictionof biological eect of dierent type of compounds and could be applied inshortening the drug design process.

Elucidating the structural consequences of CQC‐mediated dimerization of MUC1

Mucin 1 (MUC1) is a highly glycosylated single pass membrane protein that serves as a selective barrier against pathogens and engages in signal transduction influencing cellular growth and differentiation. Overexpression of mucins have been linked to 80‐90% of all human solid tissue cancers. Constitutive homodimerization, as a result of overexpression, leads to the formation of complexes with growth factor receptors and targeting to the nucleus, where MUC1 interacts with effector proteins regulating gene expression.Recent studies have shown that the MUC1 cytosolic membrane proximal CQC motif promotes dimerization under oxidizing conditions, suggesting that the motif may act as a redox switch in response to changes of oxidant levels. Preliminary studies have shown that the transmembrane domain (TMD) of MUC1 lacking the CQC motif form weak dimers, confirming their contribution to dimerization. Also, results show that TMDs containing the CQC in an oxidizing environment exhibit increased dimerization, further enforcing the role of the motif acts as a redox switch.Using the ToxR dimerization assay, the effects of single amino acid substitutions in TMDs with the CQC motif and TMDs with the CQC‐&gt;AQA motif are being measured. Our goal is to determine (1) whether certain amino acids in the TMD play a key role in forming MUC1 homodimers, and (2) whether the CQC motif induces a structural change when it functions as a redox switch. These conclusions will contribute to our understanding of MUC1 dimerization and provide insights for the development of novel therapeutic strategies.

Biological Role of Anti-aging Protein Klotho

Klotho-deficient mice have accelerated aging phenotypes, whereas overexpression of Klotho in mice extends lifespan. Klotho is an anti-aging single-pass membrane protein predominantly produced in the kidney, with shedding of the amino-terminal extracellular domain into the systemic circulation. Circulating levels of soluble Klotho decrease with age, and the klotho gene is associated with increased risk of age-related diseases. The three forms of Klotho protein have distinct functions. Membrane Klotho forms a complex with fibroblast growth factor (FGF) receptors, functions as an obligatory co-receptor for FGF23, which is involved in aging and the development of chronic diseases via regulation of Pi and vitamin D metabolism. Secreted Klotho functions as a humoral factor with pleiotropic activities including regulation of oxidative stress, growth factor signaling, and ion homeostasis. Secreted Klotho is also involved in organ protection. The intracellular form of Klotho suppresses inflammation-mediated cellular senescence and mineral metabolism. Herein we provide a brief overview of the structure and function and recent research about Klotho.