Role of the conserved NPxxY motif of the 5-HT2A receptor in determining selective interaction with isoforms of ADP-ribosylation factor (ARF)

In this study we have shown that N376 to D mutation in the conserved NPxxY motif within the carboxy terminal tail domain (CT) of the 5-HT2A receptor alters the binding preference of GST-fusion protein constructs of the CT domain from ARF1 to an alternative isoform, ARF6. These findings were corroborated by experiments investigating co-immunoprecipitation of the wild type (WT) and N376D mutant of the 5-HT2A receptor with ARF1 or 6 or dominant negative ARF1/6 constructs co-expressed in COS7 cells. In functional assays of 5-HT-induced phospholipase D (PLD) activation responses of the WT receptor were inhibited by a dominant negative mutant of ARF1 but not ARF6, whereas responses of the N376D mutant were strongly inhibited by negative mutant ARF6. No equivalent effect of the ARF mutants was seen on phospholipase C activation. In experiments assaying 5-HT-induced increases in [35S]GTPgammaS binding to ARF 1/6 immunoprecipitates as a measure of ARF activation, increased ARF6 activation was seen only with the mutant receptor. When cellular PLD responses of other NPxxY- or a DPxxY-containing GPCRs were measured in the presence of dominant negative ARF1/6 constructs, the majority, but not all, fitted the pattern exemplified by the 5-HT2A receptor and its N376D mutant. These data suggest that the presence of the N or a D in this highly conserved motif is an important, but not exclusive, determinant of which ARF isoform interacts with the GPCR.     

Functional characterization of Glu298Asp mutant human endothelial nitric oxide synthase purified from a yeast expression system.

The Glu298Asp polymorphism of human endothelial nitric oxide synthase (eNOS) has been reported to be associated with several cardiovascular diseases, including hypertension and myocardial infarction. Therefore, we investigated the effect of the Glu298Asp (E298D) mutation on the function of purified recombinant eNOS expressed in the yeast Pichia pastoris. Wild type (WT) and mutant exhibited comparable affinities for L-arginine (K(m) values 4.4+/-0.6 and 5.2+/-0.8 microM, respectively) and V(max) values (142+/-36 and 159+/-29 nmol of L-citrulline/mg min, respectively). The E298D mutation affected neither electron transfer through the reductase domain (measured as cytochrome c reduction) nor reductive O(2) activation (measured either as NADPH oxidation or as H(2)O(2) formation in the absence of L-arginine and tetrahydrobiopterin (BH4)). The mutant was activated by BH4 with an EC(50) of 0.24+/-0.04 microM, a value comparable to that obtained with WT eNOS (0.22+/-0.02 microM). Activation of the enzyme by Ca(2+) was not affected (EC(50)=0.50+/-0.04 and 0.49+/-0.02 microM for WT and E298D eNOS, respectively). Calmodulin (CaM) affinity, studied by radioligand binding using 125I-labeled CaM, revealed virtually identical K(D) (3.2+/-0.5 and 4.0+/-0.3nM) and B(max) (1.4+/-0.2 and 1.2+/-0.3 pmol/pmol subunit) values for WT and E298D eNOS, respectively. Furthermore, E298D eNOS did not differ from the WT enzyme with respect to heme and flavin content or the ability to form SDS-resistant dimers. To summarize, we obtained no evidence for altered enzyme function of the eNOS mutant that could explain endothelial dysfunction associated with the E298D polymorphism.     

Coulombic and hydrophobic interactions in the first intracellular loop are vital for bradykinin B2 receptor ligand binding and consequent signal transduction

The role of the first intracellular loop (IC1) in the function of the rat bradykinin B2 receptor (BKB2R) was probed. On the basis of the bovine rhodopsin X-ray structure, the BKB2R IC1 consists of six residues: (60)HKTNCT. Exchange of this sequence with the bradykinin B1 receptor IC1 (PRRQLN) resulted in a chimera which bound bradykinin and signaled as wild-type (WT) BKB2R. In contrast, a chimera containing the IC1 of rat angiotensin II type Ia receptor (AT1aR) (YMKLKT) did not bind BK nor signal in response to BK at a concentration as high as 5 microM. ELISA illustrated that this receptor was still processed and inserted into the plasma membrane. Employing portions of the IC1, we observed that (60)HKT of BKB2R could be exchanged as a group with either the BKB1R (PRR) or AT1aR (YMK) with no change in receptor binding or signaling activities. When only the YM of AT1aR replaced the HK of BKB2R, leaving the N-terminal portion of IC1 without a positively charged residue, binding and signaling were reduced by more than 70%. When only N63 was replaced with the corresponding leucine of AT1aR, binding and signaling were ablated. In fact, replacement of the entire IC1 with the AT1aR except for N63 resulted in binding and signaling as WT BKB2R. However, N63 could be replaced by glutamine (in BKB1R) or aspartate and continued to function as WT BKB2R. NMR data indicated that the BKB2R IC1 extends beyond the bovine rhodopsin prototype to include HKTNCTVAEI. When E68 was exchanged with a serine (in AT1aR), ligand binding decreased by 60% and PI turnover decreased by 69%. Molecular modeling points to a strict requirement for a hydrophilic residue at position 63 (N) at the middle of the IC1 and a Coulombic charge interaction between the positive charges (H60 and K61) at the N-terminus and a negative charge (E68) at the C-terminus of the IC1.     

Expression of Na+,K+-ATPase in Pichia pastoris: analysis of wild type and D369N mutant proteins by Fe2+-catalyzed oxidative cleavage and molecular modeling

Na+,K+-ATPase (pig alpha1,beta1) has been expressed in the methylotrophic yeast Pichia pastoris. A protease-deficient strain was used, recombinant clones were screened for multicopy genomic integrants, and protein expression, and time and temperature of methanol induction were optimized. A 3-liter culture provides 300-500 mg of membrane protein with ouabain binding capacity of 30-50 pmol mg-1. Turnover numbers of recombinant and renal Na+,K+-ATPase are similar, as are specific chymotryptic cleavages. Wild type (WT) and a D369N mutant have been analyzed by Fe2+- and ATP-Fe2+-catalyzed oxidative cleavage, described for renal Na+,K+-ATPase. Cleavage of the D369N mutant provides strong evidence for two Fe2+ sites: site 1 composed of residues in P and A cytoplasmic domains, and site 2 near trans-membrane segments M3/M1. The D369N mutation suppresses cleavages at site 1, which appears to be a normal Mg2+ site in E2 conformations. The results suggest a possible role of the charge of Asp369 on the E1 <--> E2 conformational equilibrium. 5'-Adenylyl-beta,gamma-imidodi-phosphate(AMP-PNP)-Fe2+-catalyzed cleavage of the D369N mutant produces fragments in P (712VNDS) and N (near 440VAGDA) domains, described for WT, but only at high AMP-PNP-Fe2+ concentrations, and a new fragment in the P domain (near 367CSDKTGT) resulting from cleavage. Thus, the mutation distorts the active site. A molecular dynamic simulation of ATP-Mg2+ binding to WT and D351N structures of Ca2+-ATPase (analogous to Asp369 of Na+,K+-ATPase) supplies possible explanations for the new cleavage and for a high ATP affinity, which was observed previously for the mutant. The Asn351 structure with bound ATP-Mg2+ may resemble the transition state of the WT poised for phosphorylation.     

Asparagine 285 plays a key role in transition state stabilization in rabbit muscle creatine kinase

To explore the possibility that asparagine 285 plays a key role in transition state stabilization in phosphagen kinase catalysis, the N285Q, N285D, and N285A site-directed mutants of recombinant rabbit muscle creatine kinase (rmCK) were prepared and characterized. Kinetic analysis of phosphocreatine formation showed that the catalytic efficiency of each N285 mutant was reduced by approximately four orders of magnitude, with the major cause of activity loss being a reduction in k(cat) in comparison to the recombinant native CK. The data for N285Q still fit a random-order, rapid-equilibrium mechanism, with either MgATP or creatine binding first with affinities very nearly equal to those for native CK. However, the affinity for the binding of the second substrate is reduced approximately 10-fold, suggesting that addition of a single methylene group at position 285 disrupts the symphony of substrate binding. The data for the N285A mutant only fit an ordered binding mechanism, with MgATP binding first. Isosteric replacement to form the N285D mutant has almost no effect on the K(M) values for either creatine or MgATP, thus the decrease in activity is due almost entirely to a 5000-fold reduction in k(cat). Using the quenching of the intrinsic CK tryptophan fluorescence by added MgADP (Borders et al. 2002), it was found that, unlike native CK, none of the mutants have the ability to form a quaternary TSAC. We use these data to propose that asparagine 285 indeed plays a key role in transition state stabilization in the reaction catalyzed by creatine kinase and other phosphagen kinases.     

Direct assessment of CXCR4 mutant conformations reveals complex link between receptor structure and G(alpha)(i) activation

Ligand binding to G protein-coupled receptors (GPCRs) is thought to induce changes in receptor conformation that translate into activation of downstream effectors. The link between receptor conformation and activity is still insufficiently understood, as current models of GPCR activation fail to take an increasing amount of experimental data into account. To elucidate structure-function relationships in GPCR activation, we used bioluminescence resonance energy transfer to directly assess the conformation of mutants of the chemokine receptor CXCR4. We analyzed substitutions in the arginine cage DRY motif and in the conserved asparagine N(3.35)119, which are pivotal molecular switches for receptor conformation and activation. G(alpha)(i) activation of the mutants was either similar to wild-type CXCR4 (D133N, Y135A, and N119D) or resulted in loss of activity (R134A and N119K). Mutant N119S was constitutively active but further activated by agonist. Bioluminescence resonance energy transfer analysis suggested no simple correlation between conformational changes in response to ligand binding and activation of G(alpha)(i) by the mutants. Different conformations of active receptors were detected (for wild-type CXCR4, D133N, and N119S), suggesting that different receptor conformations are able to trigger G(alpha)(i) activity. Several conformations were also found for inactive mutants. These data provide biophysical evidence for different receptor conformations being active with respect to a single readout. They support models of GPCR structure-activity relationships that take this conformational flexibility of active receptors into account.     

Evidence of the importance of the first intracellular loop of prokineticin receptor 2 in receptor function

Prokineticin receptors (PROKR) are G protein-coupled receptors (GPCR) that regulate diverse biological processes, including olfactory bulb neurogenesis and GnRH neuronal migration. Mutations in PROKR2 have been described in patients with varying degrees of GnRH deficiency and are located in diverse functional domains of the receptor. Our goal was to determine whether variants in the first intracellular loop (ICL1) of PROKR2 (R80C, R85C, and R85H) identified in patients with hypogonadotropic hypogonadism interfere with receptor function and to elucidate the mechanisms of these effects. Because of structural homology among GPCR, clarification of the role of ICL1 in PROKR2 activity may contribute to a better understanding of this domain across other GPCR. The effects of the ICL1 PROKR2 mutations on activation of signal transduction pathways, ligand binding, and receptor expression were evaluated. Our results indicated that the R85C and R85H PROKR2 mutations interfere only modestly with receptor function, whereas the R80C PROKR2 mutation leads to a marked reduction in receptor activity. Cotransfection of wild-type (WT) and R80C PROKR2 showed that the R80C mutant could exert a dominant negative effect on WT PROKR2 in vitro by interfering with WT receptor expression. In summary, we have shown the importance of Arg80 in ICL1 for PROKR2 expression and demonstrate that R80C PROKR2 exerts a dominant negative effect on WT PROKR2.     

A cell surface inactive mutant of the human lutropin receptor (hLHR) attenuates signaling of wild-type or constitutively active receptors via heterodimerization

The D405N and Y546F mutations of the human lutropin receptor (hLHR) have previously been shown to partially attenuate hCG-stimulated cAMP synthesis despite normal cell surface expression and hCG binding affinity (Min, L. and Ascoli, M. Mol. Endocrinol. 14:1797–1810, 2000). We now show that these mutations each stabilize a resting state of the hLHR. A combined mutant D405N,Y546F is similarly expressed at the cell surface and exhibits normal ligand-binding, but is profoundly signaling impaired. Introduction of hLHR(wt) into cells stably expressing the signaling inactive D405N,Y546F resulted in the attenuation of hCG-stimulated cAMP production by hLHR(wt) even if excess Gs is co-expressed. Similarly, co-expression of D405N,Y546F with hLHR constitutively active mutants (CAMs) attenuated their constitutive activity. Quantitative bioluminescence resonance energy transfer (BRET) analyses demonstrated that D405N,Y546F formed heterodimers with both wt and CAM hLHR. In contrast hLHR(D405N,Y546F) did not heterodimerize with the melanocortin 3 receptor (MC3R) and agonist-stimulated cAMP production through the MC3R was not attenuated when these two receptors were co-expressed. Taken altogether, our data demonstrate that a signaling inactive hLHR mutant (that is trafficked normally to the plasma membrane) attenuates the signaling of the cell surface localized wt or the constitutively active hLHR due to receptor heterodimerization. Our studies, therefore, suggest a novel ramification of GPCR signaling resulting from receptor dimerization.     

Replicative fitness of protease inhibitor-resistant mutants of human immunodeficiency virus type 1

The relative replicative fitness of human immunodeficiency virus type 1 (HIV-1) mutants selected by different protease inhibitors (PIs) in vivo was determined. Each mutant was compared to wild type (WT), NL4-3, in the absence of drugs by several methods, including clonal genotyping of cultures infected with two competing viral variants, kinetics of viral antigen production, and viral infectivity/virion particle ratios. A nelfinavir-selected protease D30N substitution substantially decreased replicative capacity relative to WT, while a saquinavir-selected L90M substitution moderately decreased fitness. The D30N mutant virus was also outcompeted by the L90M mutant in the absence of drugs. A major natural polymorphism of the HIV-1 protease, L63P, compensated well for the impairment of fitness caused by L90M but only slightly improved the fitness of D30N. Multiply substituted indinavir-selected mutants M46I/L63P/V82T/I84V and L10R/M46I/L63P/V82T/I84V were just as fit as WT. These results indicate that the mutations which are usually initially selected by nelfinavir and saquinavir, D30N and L90M, respectively, impair fitness. However, additional mutations may improve the replicative capacity of these and other drug-resistant mutants. Hypotheses based on the greater fitness impairment of the nelfinavir-selected D30N mutant are suggested to explain observations that prolonged responses to delayed salvage regimens, including alternate PIs, may be relatively common after nelfinavir failure.     

Interacting residues in an activated state of a G protein-coupled receptor

Ste2p, the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha-factor of Saccharomyces cerevisiae, was used as a model GPCR to investigate the role of specific residues in the resting and activated states of the receptor. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified Asn(205) as a potential interacting partner with the Tyr(266) residue. An N205H/Y266H double mutant showed pH-dependent functional activity, whereas the N205H receptor was non-functional and the Y266H receptor was partially active indicating that the histidine 205 and 266 residues interact in an activated state of the receptor. The introduction of N205K or Y266D mutations into the P258L/S259L constitutively active receptor suppressed the constitutive activity; in contrast, the N205K/Y266D/P258L/S259L quadruple mutant was fully constitutively active, again indicating an interaction between residues at the 205 and 206 positions in the receptor-active state. To further test this interaction, we introduced the N205C/Y266C, F204C/Y266C, and N205C/A265C double mutations into wild-type and P258L/S259L constitutively active receptors. After trypsin digestion, we found that a disulfide-cross-linked product, with the molecular weight expected for a receptor fragment with a cross-link between N205C and Y266C, formed only in the N205C/Y266C constitutively activated receptor. This study represents the first experimental demonstration of an interaction between specific residues in an active state, but not the resting state, of Ste2p. The information gained from this study should contribute to an understanding of the conformational differences between resting and active states in GPCRs.     

Identification of active site residues in E. coli ketopantoate reductase by mutagenesis and chemical rescue

Ketopantoate reductase (EC 1.1.1.169) catalyzes the NADPH-dependent reduction of alpha-ketopantoate to D-(-)-pantoate in the biosynthesis of pantothenate. The pH dependence of V and V/K for the E. coli enzyme suggests the involvement of a general acid/base in the catalytic mechanism. To identify residues involved in catalysis and substrate binding, we mutated the following six strictly conserved residues to Ala: Lys72, Lys176, Glu210, Glu240, Asp248, and Glu256. Of these, the K176A and E256A mutant enzymes showed 233- and 42-fold decreases in V(max), and 336- and 63-fold increases in the K(m) value of ketopantoate, respectively, while the other mutants exhibited WT kinetic properties. The V(max) for the K176A and E256A mutant enzymes was markedly increased, up to 25% and 75% of the wild-type level, by exogenously added primary amines and formate, respectively. The rescue efficiencies for the K176A and E256A mutant enzymes were dependent on the molecular volume of rescue agents, as anticipated for a finite active site volume. The protonated form of the amine is responsible for recovery of activity, suggesting that Lys176 functions as a general acid in catalysis of ketopantoate reduction. The rescue efficiencies for the K176A mutant by primary amines were independent of the pK(a) value of the rescue agents (Bronsted coefficient, alpha = -0.004 +/-0.008). Insensitivity to acid strength suggests that the chemical reaction is not rate-limiting, consistent with (a) the catalytic efficiency of the wild-type enzyme (k(cat)/K(m) = 2x10(6) M(-1) s(-1) and (b) the small primary deuterium kinetic isotope effects, (D)V = 1.3 and (D)V/K = 1.5, observed for the wild-type enzyme. Larger primary deuterium isotope effects on V and V/K were observed for the K176A mutant ((D)V = 3.0, (D)V/K = 3.7) but decreased nearly to WT values as the concentration of ethylamine was increased. The nearly WT activity of the E256A mutant in the presence of formate argues for an important role for this residue in substrate binding. The double mutant (K176A/E256A) has no detectable ketopantoate reductase activity. These results indicate that Lys176 and Glu256 of the E. coli ketopantoate reductase are active site residues, and we propose specific roles for each in binding ketopantoate and catalysis.     

Functional role of a conserved motif in TM6 of the rat mu opioid receptor: constitutively active and inactive receptors result from substitutions of Thr6.34(279) with Lys and Asp.

Mutations within the "X1BBX2X3B" motif or its variants in the junction of the third intracellular (i3) loop and the sixth transmembrane domain (TM6) have been shown to lead to constitutive activation of several G protein-coupled receptors (GPCRs). In this study, T6.34(279) at the X3 locus of the rat mu opioid receptor was mutated to Lys and Asp, and the mutants were examined for binding and signaling properties. The T6.34(279)K mutant was poorly expressed, and pretreatment with naloxone greatly enhanced its expression. This construct exhibited properties identified previously with constitutive activation: (1) compared with the wild type, it produced much higher agonist-independent [35S]GTPgammaS binding, which was abolished by pertussis toxin treatment; (2) it displayed an enhanced affinity for the agonist DAMGO similar to that of the high-affinity state of the wild type, which was not altered by GTPgammaS, while having unchanged affinity for the antagonist diprenorphine. The T6.34(279)K mutant displayed a higher intracellular receptor pool than the wild type. Naloxone inhibited the basal [35S]GTPgammaS binding of the T6.34(279)K mutant, demonstrating inverse agonist activity at this mutant receptor. In contrast, the T6.34(279)D substitution did not increase basal [35S]GTPgammaS binding, greatly reduced agonist-promoted [35S]GTPgammaS binding, and markedly decreased affinity for DAMGO. Thus, the T6.34(279)D mutant adopts conformations corresponding to inactive states of the receptor. The results were interpreted in the structural context of a model for the mu opioid receptor that incorporates the information from the crystal structure of rhodopsin. The interaction of T6.34(279) with R3.50(165) in the mu opioid receptor is considered to stabilize the inactive conformations. The T6.34(279)K substitution would then disrupt this interaction and support agonist-free activation, while T6.34(279)D mutation should strengthen this interaction which keeps the receptor in inactive states. T6.34(279) may, in addition, interact with the neighboring R6.35(280) to help constrain the receptor in inactive states, and T6.34(279)K and T6.34(279)D mutations would affect this interaction by disrupting or strengthening it, respectively. To the best of our knowledge, the results presented here represent the first structurally rationalized demonstration that mutations of this locus can lead to dramatically different properties of a GPCR.     

Neutralization of the aspartic acid residue Asp-367, but not Asp-454, inhibits binding of Na+ to the glutamate-free form and cycling of the glutamate transporter EAAC1

Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na(+) ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, Asp-367 and Asp-454, in Na(+) cotransport. To test the effect of charge neutralization mutations in these positions on Na(+) binding to the glutamate-free transporter, we recorded the Na(+)-induced anion leak current to determine the K(m) of EAAC1 for Na(+). For EAAC1(WT), this K(m) was determined as 120 mm. When the negative charge of Asp-367 was neutralized by mutagenesis to asparagine, Na(+) activated the anion leak current with a K(m) of about 2 m, indicating dramatically impaired Na(+) binding to the mutant transporter. In contrast, the Na(+) affinity of EAAC1(D454N) was virtually unchanged compared with the wild type transporter (K(m) = 90 mm). The reduced occupancy of the Na(+) binding site of EAAC1(D367N) resulted in a dramatic reduction in glutamate affinity (K(m) = 3.6 mm, 140 mm [Na(+)]), which could be partially overcome by increasing extracellular [Na(+)]. In addition to impairing Na(+) binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which Asp-367, but not Asp-454, is involved in coordinating the bound Na(+) in the glutamate-free transporter form.     

Mechanisms regulating the cell surface residence time of the alpha 2A-adrenergic receptor

Despite considerable insights concerning the mechanisms regulating short-term agonist-mediated G protein-coupled receptor (GPCR) internalization, little is known about the mechanisms regulating GPCR surface residence over long periods of time. Herein, we experimentally evaluated mechanisms regulating the surface t(1/2) of various alpha(2A)-adrenergic receptor (alpha(2A)AR) structures. The Delta 3i alpha(2A)AR (lacking the third intracellular loop), D79N alpha(2A)AR (impaired G protein coupling), and CAM alpha(2A)AR (enhanced G protein coupling) all exhibited a cell surface alpha(2A)AR turnover in Chinese hamster ovary cells that was faster than that of the wild type (WT). Cell surface receptor turnover could be slowed with ligand occupancy of D79N alpha(2A)AR (agonist or antagonist) and CAM alpha(2A)AR (antagonist only) but not the Delta 3i- or WT alpha(2A)AR. This selective ligand-induced surface stabilization was paralleled by a dramatic ligand-dependent receptor density upregulation for D79N- and CAM alpha(2A)AR structures. Receptors which exhibited surface turnover and density that could be modulated by ligand (D79N and CAM) also demonstrated structural instability, measured by a loss of radioligand binding capacity in detergent solution over time without parallel changes in receptor protein content. In contrast, the shorter surface t(1/2) of the Delta 3i alpha(2A)AR, whose cell surface t(1/2) and steady state density were not altered by ligand occupancy, occurred in the context of a structurally stable receptor in detergent solution. These results demonstrate that changes in receptor structure which alter receptor-G protein coupling (either an increase or decrease) are paralleled by structural instability and ligand-induced surface stabilization. These studies also provide criteria for assessing the structural instability of the alpha(2A)AR that can likely be generalized to all GPCRs.     

Mutations in the ubiquitin-binding domain of OPTN/optineurin interfere with autophagy-mediated degradation of misfolded proteins by a dominant-negative mechanism

OPTN (optineurin) is an autophagy receptor and mutations in the OPTN gene result in familial glaucoma (E50K) and amyotrophic lateral sclerosis (ALS) (E478G). However, the mechanisms through which mutant OPTN leads to human diseases remain to be characterized. Here, we demonstrated that OPTN colocalized with inclusion bodies (IBs) formed by mutant HTT/huntingtin protein (mHTT) in R6/2 transgenic mice and IBs formed by 81QNmHTT (nuclear form), 109QmHTT (cytoplasmic form) or the truncated form of TARDBP/TDP-43 (TARDBP^ND251) in Neuro2A cells. This colocalization required the ubiquitin (Ub)-binding domain (UbBD, amino acids 424 to 511) of OPTN. Overexpression of wild-type (WT) OPTN decreased IBs through K63-linked polyubiquitin-mediated autophagy. E50K or 210 to 410Δ (with amino acids 210 to 410 deleted) whose mutation or deletion was outside the UbBD decreased the IBs formed by 109QmHTT or TARDBP^ND251, as was the case with WT OPTN. In contrast, UbBD mutants, including E478G, D474N, UbBDΔ, 411 to 520Δ and 210 to 520Δ, increased accumulation of IBs. UbBD mutants (E478G, UbBDΔ) retained a substantial ability to interact with WT OPTN, and were found to colocalize with polyubiquitinated IBs, which might occur indirectly through their WT partner in a WT-mutant complex. They decreased autophagic flux evidenced by alteration in LC3 level and turnover and in the number of LC3-positive puncta under stresses like starvation or formation of IBs. UbBD mutants exhibited a weakened interaction with MYO6 (myosin VI) and TOM1 (target of myb1 homolog [chicken]), important for autophagosome maturation, in cells or sorted 109QmHtt IBs. Taken together, our data indicated that UbBD mutants acted as dominant-negative traps through the formation of WT-mutant hybrid complexes to compromise the maturation of autophagosomes, which in turn interfered with OPTN-mediated autophagy and clearance of IBs.     

Mutations in BIN1 Associated with Centronuclear Myopathy Disrupt Membrane Remodeling by Affecting Protein Density and Oligomerization

The regulation of membrane shapes is central to many cellular phenomena. Bin/Amphiphysin/Rvs (BAR) domain-containing proteins are key players for membrane remodeling during endocytosis, cell migration, and endosomal sorting. BIN1, which contains an N-BAR domain, is assumed to be essential for biogenesis of plasma membrane invaginations (T-tubules) in muscle tissues. Three mutations, K35N, D151N and R154Q, have been discovered so far in the BAR domain of BIN1 in patients with centronuclear myopathy (CNM), where impaired organization of T-tubules has been reported. However, molecular mechanisms behind this malfunction have remained elusive. None of the BIN1 disease mutants displayed a significantly compromised curvature sensing ability. However, two mutants showed impaired membrane tubulation both in vivo and in vitro, and displayed characteristically different behaviors. R154Q generated smaller membrane curvature compared to WT N-BAR. Quantification of protein density on membranes revealed a lower membrane-bound density for R154Q compared to WT and the other mutants, which appeared to be the primary reason for the observation of impaired deformation capacity. The D151N mutant was unable to tubulate liposomes under certain experimental conditions. At medium protein concentrations we found ‘budding’ structures on liposomes that we hypothesized to be intermediates during the tubulation process except for the D151N mutant. Chemical crosslinking assays suggested that the D151N mutation impaired protein oligomerization upon membrane binding. Although we found an insignificant difference between WT and K35N N-BAR in in vitro assays, depolymerizing actin in live cells allowed tubulation of plasma membranes through the K35N mutant. Our results provide insights into the membrane-involved pathophysiological mechanisms leading to human disease.