C-terminal Cytoplasmic Domain Research Articles

Structural modeling and characterization of the Mycobacterium tuberculosis MmpL3 C-terminal domain.

The Mycobacterium tuberculosis (Mtb) cell envelope provides a protective barrier against the immune response and antibiotics. The mycobacterial membrane protein large (MmpL) family of proteins export cell envelope lipids and siderophores; therefore, these proteins are important for the basic biology and pathogenicity of Mtb. In particular, MmpL3 is essential and a known drug target. Despite interest in MmpL3, the structural data in the field are incomplete. Utilizing homology modeling, AlphaFold, and biophysical techniques, we characterized the cytoplasmic C-terminal domain (CTD) of MmpL3 to better understand its structure and function. Our in silico models of the MmpL11TB and MmpL3TB CTD reveal notable features including a long unstructured linker that connects the globular domain to the last transmembrane (TM) in each transporter, charged lysine and arginine residues facing the membrane, and a C-terminal alpha helix. Our predicted overall structure enables a better understanding of these transporters.

A C-terminal motif containing a PKC phosphorylation site regulates γ-Protocadherin-mediated dendrite arborization in the cerebral cortex in vivo.

The Pcdhg gene cluster encodes 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules that critically regulate multiple aspects of neural development, including neuronal survival, dendritic and axonal arborization, and synapse formation and maturation. Each γ-Pcdh isoform has unique protein domains-a homophilically interacting extracellular domain and a juxtamembrane cytoplasmic domain-as well as a C-terminal cytoplasmic domain shared by all isoforms. The extent to which isoform-specific versus shared domains regulate distinct γ-Pcdh functions remains incompletely understood. Our previous in vitro studies identified protein kinase C (PKC)phosphorylation of a serine residue within a shared C-terminal motif as a mechanism through which γ-Pcdh promotion of dendrite arborization via myristoylated alanine-rich C-kinase substrate (MARCKS) is abrogated. Here, we used CRISPR/Cas9 genome editing to generate two new mouse lines expressing only non-phosphorylatable γ-Pcdhs, due either to a serine-to-alanine mutation (PcdhgS/A) or to a 15-amino acid C-terminal deletion resulting from insertion of an early stop codon (PcdhgCTD). Both lines are viable and fertile, and the density and maturation of dendritic spines remain unchanged in both PcdhgS/A and PcdhgCTD cortex. Dendrite arborization of cortical pyramidal neurons, however, is significantly increased in both lines, as are levels of active MARCKS. Intriguingly, despite having significantly reduced levels of γ-Pcdh proteins, the PcdhgCTD mutation yields the strongest phenotype, with even heterozygous mutants exhibiting increased arborization. The present study confirms that phosphorylation of a shared C-terminal motif is a key γ-Pcdh negative regulation point and contributes to a converging understanding of γ-Pcdh family function in which distinct roles are played by both individual isoforms and discrete protein domains.

Significance of Zn2+ in RyR1 for Structural Integrity and Ligand Binding: Insight from Molecular Dynamics.

Ryanodine receptor type 1 (RyR1) is a Ca2+-release channel central to skeletal muscle excitation-contraction (EC) coupling. RyR1's cryo-EM structures reveal a zinc-finger motif positioned within the cytoplasmic C-terminal domain (CTD). Yet, owing to limitations in cryo-EM resolution, RyR1 structures lack precision in detailing the metal coordination structure, prompting the need for an accurate model. In this study, we employed molecular dynamics (MD) simulations and the density functional theory (DFT) method to refine the binding characteristics of Zn2+ in the zinc-finger site of the RyR1 channel. Our findings also highlight substantial conformational changes in simulations conducted in the absence of Zn2+. Notably, we observed a loss of contact at the interface between protein domains proximal to the zinc-finger site, indicating a crucial role of Zn2+ in maintaining structural integrity and interdomain interactions within RyR1. Furthermore, this study provides valuable insights into the modulation of ATP, Ca2+, and caffeine binding, shedding light on the intricate relationship between Zn2+ coordination and the dynamic behavior of RyR1. Our integrative approach combining MD simulations and DFT calculations enhances our understanding of the molecular mechanisms governing ligand binding in RyR1.

Connexin Gap Junction Channels and Hemichannels: Insights from High-Resolution Structures.

Connexins (Cxs) are a family of integral membrane proteins, which function as both hexameric hemichannels (HCs) and dodecameric gap junction channels (GJCs), behaving as conduits for the electrical and molecular communication between cells and between cells and the extracellular environment, respectively. Their proper functioning is crucial for many processes, including development, physiology, and response to disease and trauma. Abnormal GJC and HC communication can lead to numerous pathological states including inflammation, skin diseases, deafness, nervous system disorders, and cardiac arrhythmias. Over the last 15 years, high-resolution X-ray and electron cryomicroscopy (cryoEM) structures for seven Cx isoforms have revealed conservation in the four-helix transmembrane (TM) bundle of each subunit; an αβ fold in the disulfide-bonded extracellular loops and inter-subunit hydrogen bonding across the extracellular gap that mediates end-to-end docking to form a tight seal between hexamers in the GJC. Tissue injury is associated with cellular Ca2+ overload. Surprisingly, the binding of 12 Ca2+ ions in the Cx26 GJC results in a novel electrostatic gating mechanism that blocks cation permeation. In contrast, acidic pH during tissue injury elicits association of the N-terminal (NT) domains that sterically blocks the pore in a "ball-and-chain" fashion. The NT domains under physiologic conditions display multiple conformational states, stabilized by protein-protein and protein-lipid interactions, which may relate to gating mechanisms. The cryoEM maps also revealed putative lipid densities within the pore, intercalated among transmembrane α-helices and between protomers, the functions of which are unknown. For the future, time-resolved cryoEM of isolated Cx channels as well as cryotomography of GJCs and HCs in cells and tissues will yield a deeper insight into the mechanisms for channel regulation. The cytoplasmic loop (CL) and C-terminal (CT) domains are divergent in sequence and length, are likely involved in channel regulation, but are not visualized in the high-resolution X-ray and cryoEM maps presumably due to conformational flexibility. We expect that the integrated use of synergistic physicochemical, spectroscopic, biophysical, and computational methods will reveal conformational dynamics relevant to functional states. We anticipate that such a wealth of results under different pathologic conditions will accelerate drug discovery related to Cx channel modulation.

Mtc6/Ehg2 is a novel endoplasmic reticulum-resident glycoprotein essential for high-pressure tolerance.

Hydrostatic pressure is a common mechanical stressor that modulates metabolism and reduces cell viability. Eukaryotic cells have genetic programs to cope with hydrostatic pressure stress and maintain intracellular homeostasis. However, the mechanism underlying hydrostatic pressure tolerance remains largely unknown. We have recently demonstrated that maintenance of telomere capping protein 6 (Mtc6) plays a protective role in the survival of the budding yeast Saccharomyces cerevisiae under hydrostatic pressure stress by supporting the integrity of nutrient permeases. The current study demonstrates that Mtc6 acts as an endoplasmic reticulum (ER) membrane protein. Mtc6 comprises two transmembrane domains, a C-terminal cytoplasmic domain and a luminal region with 12 Asn (N)-linked glycans attached to it. Serial mutational analyses showed that the cytoplasmic C-terminal amino acid residues GVPS Mtc6 activity. Multiple N-linked glycans in the luminal region are involved in the structural conformation of Mtc6. Moreover, deletion of MTC6 led to increased degradation of the leucine permease Bap2 under hydrostatic pressure, suggesting that Mtc6 facilitates the proper folding of nutrient permeases in the ER under stress conditions. We propose a novel model of molecular function in which the glycosylated luminal domain and cytoplasmic GVPS sequences of Mtc6 cooperatively support the nutrient permease activity.

FliH and FliI help FlhA bring strict order to flagellar protein export in Salmonella

The flagellar type III secretion system (fT3SS) switches substrate specificity from rod-hook-type to filament-type upon hook completion, terminating hook assembly and initiating filament assembly. The C-terminal cytoplasmic domain of FlhA (FlhAC) forms a homo-nonameric ring and is directly involved in substrate recognition, allowing the fT3SS to coordinate flagellar protein export with assembly. The highly conserved GYXLI motif (residues 368–372) of FlhAC induces dynamic domain motions of FlhAC required for efficient and robust flagellar protein export by the fT3SS, but it remains unknown whether this motif is also important for ordered protein export by the fT3SS. Here we analyzed two GYXLI mutants, flhA(GAAAA) and flhA(GGGGG), and provide evidence suggesting that the GYXLI motif in FlhAC requires the flagellar ATPase complex not only to efficiently remodel the FlhAC ring structure for the substrate specificity switching but also to correct substrate recognition errors that occur during flagellar assembly.

CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23.

CRISPR-Cas provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using an N-terminal transmembrane α-helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.

Multiple roles for the cytoplasmic C-terminal domains of the yeast cell surface receptors Rgt2 and Snf3 in glucose sensing and signaling

The plasma membrane proteins Rgt2 and Snf3 are glucose sensing receptors (GSRs) that generate an intracellular signal for the induction of gene expression in response to high and low extracellular glucose concentrations, respectively. The GSRs consist of a 12-transmembrane glucose recognition domain and a cytoplasmic C-terminal signaling tail. The GSR tails are dissimilar in length and sequence, but their distinct roles in glucose signal transduction are poorly understood. Here, we show that swapping the tails between Rgt2 and Snf3 does not alter the signaling activity of the GSRs, so long as their tails are phosphorylated in a Yck-dependent manner. Attachment of the GSR tails to Hxt1 converts the transporter into a glucose receptor; however, the tails attached to Hxt1 are not phosphorylated by the Ycks, resulting in only partial signaling. Moreover, in response to non-fermentable carbon substrates, Rgt2 and Hxt1-RT (RT, Rgt2-tail) are efficiently endocytosed, whereas Snf3 and Hxt1-ST (ST, Snf3-tail) are endocytosis-impaired. Thus, the tails are important regulatory domains required for the endocytosis of the Rgt2 and Snf3 glucose sensing receptors triggered by different cellular stimuli. Taken together, these results suggest multiple roles for the tail domains in GSR-mediated glucose sensing and signaling.

CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism.

CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating among closely related homologs has been a long-standing mystery, in part because few CLC channel structures are available. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl--permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct conformations involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl--permeation pathway. This peptide is highly conserved among species variants of CLC-2 but is not present in other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a "ball-and-chain" gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl--permeation pathway.

CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism

CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating among closely related homologs has been a long-standing mystery, in part because few CLC channel structures are available. Here, we report cryoEM structures of human CLC-2 at 2.46 – 2.76 Å, in the presence and absence of the selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl–-permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct conformations involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl–-permeation pathway. This peptide is highly conserved among species variants of CLC-2 but is not present in other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a “ball-and-chain” gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl–-permeation pathway.

Lysosomal membrane proteins LAMP1 and LIMP2 are segregated in the Golgi apparatus independently of their clathrin adaptor binding motif.

To reach the lysosome, lysosomal membrane proteins (LMPs) are translocated in the endoplasmic reticulum after synthesis and then transported to the Golgi apparatus. The existence of a direct transport from the Golgi apparatus to the endosomes but also of an indirect route through the plasma membrane has been described. Clathrin adaptor binding motifs contained in the cytosolic tail of LMPs have been described as key players in their intracellular trafficking. Here we used the RUSH assay to synchronize the biosynthetic transport of multiple LMPs. After exiting the Golgi apparatus, RUSH-synchronized LAMP1 was addressed to the cell surface both after overexpression or at endogenous level. Its YXXΦ motif was not involved in the transport from the Golgi apparatus to the plasma membrane but in its endocytosis. LAMP1 and LIMP2 were sorted from each other after reaching the Golgi apparatus. LIMP2 was incorporated in punctate structures for export from the Golgi apparatus from which LAMP1 is excluded. LIMP2-containing post-Golgi transport intermediates did not rely neither on its adaptor binding signal nor on its C-terminal cytoplasmic domain.

NMR Structural Study of Syndecan-4 Transmembrane Domain with Cytoplasmic Region

Syndecan-4 (SDC4) consists of transmembrane heparan sulfate proteoglycan (HSPG) belonging to the syndecan family. It is present in most cell types of Mammalia. Its structure contains a heparan-sulfate-modified extracellular domain, a single transmembrane domain, and a short C-terminal cytoplasmic domain. Regarding the overall cellular function of SDC4, other cells or ligands can bind to its ecto-domain. In addition, 4,5-bisphosphate phosphatidylinositol (PIP2) or protein kinase Cα can bind to its cyto-domain to activate downstream signaling pathways. To understand the signal transduction mechanism of syndecan, it is important to know the interactions between their actual structure and function in vivo. Therefore, it is important to identify the structure of SDC4 to understand the ligand binding behavior of SDC4. In this study, expression and purification were performed to reveal structures of the short ecto-domain, the transmembrane domain, and the cytoplasmic domain of Syd4-eTC (SDC4). Solution-state NMR spectroscopy and solid-state NMR spectroscopy were used to study the structure of Syd4-eTC in membrane environments and to demonstrate the interaction between Syd4-eTC and PIP2.

Anionic Phospholipids Stimulate the Proton Pumping Activity of the Plant Plasma Membrane P-Type H+-ATPase.

The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H+-ATPase Arabidopsis thaliana H+-ATPase isoform 2 (AHA2) upon purification and reconstitution into liposomes of defined lipid compositions. We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. This activation was independent of the cytoplasmic C-terminal regulatory domain of the pump. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partially conserved in functionally different P-type ATPases from different organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H+-ATPase activity.

HIV-1 virological synapse formation enhances infection spread by dysregulating Aurora Kinase B.

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity.

The Src-Family Kinases SRC and BLK Contribute to the CLDN6-Adhesion Signaling.

Cell adhesion molecules, including integrins, cadherins, and claudins (CLDNs), are known to activate Src-family kinases (SFKs) that organize a variety of physiological and pathological processes; however, the underlying molecular basis remains unclear. Here, we identify the SFK members that are coupled with the CLDN6-adhesion signaling. Among SFK subtypes, BLK, FGR, HCK, and SRC were highly expressed in F9 cells and concentrated with CLDN6 along cell borders during epithelial differentiation. Immunoprecipitation assay showed that BLK and SRC, but not FGR or HCK, form a complex with CLDN6 via the C-terminal cytoplasmic domain. We also demonstrated, by pull-down assay, that recombinant BLK and SRC proteins directly bind to the C-terminal cytoplasmic domain of CLDN6 (CLDN6C). Unexpectedly, both recombinant SFK proteins recognized the CLDN6C peptide in a phosphotyrosine-independent manner. Furthermore, by comparing phenotypes of F9:Cldn6:Blk-/- and F9:Cldn6:Src-/- cells with those of wild-type F9 and F9:Cldn6 cells, we revealed that BLK and SRC are essential for CLDN6-triggered cellular events, namely epithelial differentiation and the expression of retinoid acid receptor target genes. These results indicate that selective SFK members appear to participate in the CLDN-adhesion signaling.

The cytoplasmic tail of the mechanosensitive channel Pkd2 regulates its internalization and clustering in eisosomes.

Polycystins are a family of conserved ion channels, mutations of which lead to one of the most common human genetic disorders, namely, autosomal dominant polycystic kidney disease. Schizosacchromyces pombe possesses an essential polycystin homologue, Pkd2, which directs Ca2+ influx on the cell surface in response to membrane tension, but its structure remains unsolved. Here, we analyzed the structure-function relationship of Pkd2 based on its AlphaFold-predicted structure. Pkd2 consists of three domains, the extracellular lipid-binding domain (LBD), nine-helix transmembrane domain (TMD) and C-terminal cytoplasmic domain (CCD). Our genetic and microscopy data revealed that LBD and TMD are essential for targeting Pkd2 to the plasma membrane from the endoplasmic reticulum. In comparison, CCD ensures the polarized distribution of Pkd2 by promoting its internalization and preventing its clustering in the eisosome, a caveolae-like membrane compartment. The domains of Pkd2 and their functions are conserved in other fission yeast species. We conclude that both extracellular and cytoplasmic domains of Pkd2 are crucial for its intracellular trafficking and function. We propose that mechanosensitive channels can be desensitized through either internalization or clustering in low-tension membrane compartments.

Epitope specific antibodies to N- and C cytoplasmic domains of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) differentiate native and post-translationally modified variant

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (or PfCRT) were shown to be causative of decreased sensitivity to diverse quinoline-based antimalarials. In this report we describe the identification of a post-translational variant of PfCRT using highly characterized antibodies raised against its N- and C-terminal cytoplasmic domains (e.g., 58 and 26 amino acids, respectively). Western blot analyses of P. falciparum protein extracts with anti N-PfCRT antiserum revealed two polypeptides with apparent molecular masses of 52 kDa and 42 kDa, relative to the calculated molecular mass of PfCRT of 48.7 kDa. The 52 kDa polypeptide was detectable with anti C-PfCRT antiserum, only after alkaline phosphatase treatment of P. falciparum extracts. Detailed epitope mapping of anti N- and C-PfCRT antisera revealed epitopes covering two previously identified phosphorylation sites, Ser411 and Thr416, whereby substitution of these residues with Asp amino acid, to mimic phosphorylated residues, dramatically inhibited anti C-PfCRT binding. Consistently, alkaline phosphatase treatment of P. falciparum extract unmasked the binding of anti C-PfCRT to the 52 kDa polypeptide, suggesting that the 52 kDa but not 42 kDa polypeptide is phosphorylated at its C-terminal Ser411 and Thr416. Interestingly, Pfcrt expressed in HEK-293F human kidney cells showed the same reactive polypeptides with anti N- and C-PfCRT antisera, consistent with PfCRT origin of the two polypeptides (e.g., 42 kDa and 52 kDa), but lacking PfCRT phosphorylation at its C-terminal. Immunohistochemical staining of late trophozoite-infected erythrocytes with anti N-or C-PfCRT antisera showed both polypeptides are localized to the parasite's digestive vacuole. Moreover, both polypeptides are detected in chloroquine-susceptible and -resistant strains of P. falciparum. This is the first report describing a post-translationally modified variant of PfCRT. The physiologic role of the 52 kDa phosphorylated PfCRT in P. falciparum remains to be determined.

Ca2+ -binding-region-dependent cell surface localization of NADPH oxidase Nox5.

Six gene splice variants of superoxide-generating NADPH oxidase 5 (Nox5) have been identified in humans, and they differ in the sequence of their N-terminal cytoplasmic domains, which comprise four EF-hand motifs. Here, we demonstrated that the Ca2+ -dependent association and dissociation between the N- and C-terminal cytoplasmic domains of the Nox5β variant are affected by the alanine substitution of the conserved Ile-113 or Leu-115 at the connecting loop between the third and fourth EF-hand motifs. These substitutions impair the cell surface localization of Nox5β. In addition, the Nox5ε/S variant, lacking all EF-hand motifs, does not localize to the plasma membrane. Thus, the Ca2+ -sensitive intramolecular interaction determines the Nox5 subcellular localization, that is, whether Nox5 variants generate superoxide in the extracellular or intracellular space.

A Unique Role for Protocadherin γC3 in Promoting Dendrite Arborization through an Axin1-Dependent Mechanism.

The establishment of a functional cerebral cortex depends on the proper execution of multiple developmental steps, culminating in dendritic and axonal outgrowth and the formation and maturation of synaptic connections. Dysregulation of these processes can result in improper neuronal connectivity, including that associated with various neurodevelopmental disorders. The γ-Protocadherins (γ-Pcdhs), a family of 22 distinct cell adhesion molecules that share a C-terminal cytoplasmic domain, are involved in multiple aspects of neurodevelopment including neuronal survival, dendrite arborization, and synapse development. The extent to which individual γ-Pcdh family members play unique versus common roles remains unclear. We demonstrated previously that the γ-Pcdh-C3 isoform (γC3), via its unique "variable" cytoplasmic domain (VCD), interacts in cultured cells with Axin1, a Wnt-pathway scaffold protein that regulates the differentiation and morphology of neurons. Here, we confirm that γC3 and Axin1 interact in the cortex in vivo and show that both male and female mice specifically lacking γC3 exhibit disrupted Axin1 localization to synaptic fractions, without obvious changes in dendritic spine density or morphology. However, both male and female γC3 knock-out mice exhibit severely decreased dendritic complexity of cortical pyramidal neurons that is not observed in mouse lines lacking several other γ-Pcdh isoforms. Combining knock-out with rescue constructs in cultured cortical neurons pooled from both male and female mice, we show that γC3 promotes dendritic arborization through an Axin1-dependent mechanism mediated through its VCD. Together, these data identify a novel mechanism through which γC3 uniquely regulates the formation of cortical circuitry.SIGNIFICANCE STATEMENT The complexity of a neuron's dendritic arbor is critical for its function. We showed previously that the γ-Protocadherin (γ-Pcdh) family of 22 cell adhesion molecules promotes arborization during development; it remained unclear whether individual family members played unique roles. Here, we show that one γ-Pcdh isoform, γC3, interacts in the brain with Axin1, a scaffolding protein known to influence dendrite development. A CRISPR/Cas9-generated mutant mouse line lacking γC3 (but not lines lacking other γ-Pcdhs) exhibits severely reduced dendritic complexity of cerebral cortex neurons. Using cultured γC3 knock-out neurons and a variety of rescue constructs, we confirm that the γC3 cytoplasmic domain promotes arborization through an Axin1-dependent mechanism. Thus, γ-Pcdh isoforms are not interchangeable, but rather can play unique neurodevelopmental roles.

Serine 408 phosphorylation is a molecular switch that regulates structure and function of the occludin α-helical bundle

Occludin is a tetramembrane-spanning tight junction protein. The long C-terminal cytoplasmic domain, which represents nearly half of occludin sequence, includes a distal bundle of three α-helices that mediates interactions with other tight junction components. A short unstructured region just proximal to the α-helical bundle is a phosphorylation hotspot within which S408 phosphorylation acts as molecular switch that modifies tight junction protein interactions and barrier function. Here, we used NMR to define the effects of S408 phosphorylation on intramolecular interactions between the unstructured region and the α-helical bundle. S408 pseudophosphorylation affected conformation at hinge sites between the three α-helices. Further studies using paramagnetic relaxation enhancement and microscale thermophoresis indicated that the unstructured region interacts with the α-helical bundle. These interactions between the unstructured domain are enhanced by S408 phosphorylation and allow the unstructured region to obstruct the binding site, thereby reducing affinity of the occludin tail for zonula occludens-1 (ZO-1). Conversely, S408 dephosphorylation attenuates intramolecular interactions, exposes the binding site, and increases the affinity of occludin binding to ZO-1. Consistent with an increase in binding to ZO-1, intravital imaging and fluorescence recovery after photobleaching (FRAP) analyses of transgenic mice demonstrated increased tight junction anchoring of enhanced green fluorescent protein (EGFP)-tagged nonphosphorylatable occludin relative to wild-type EGFP-occludin. Overall, these data define the mechanisms by which S408 phosphorylation modifies occludin tail conformation to regulate tight junction protein interactions and paracellular permeability.