Inhibitory Mechanism of an Allosteric Antibody Targeting the Glucagon Receptor

Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.     

Voltage sensitivities and deactivation kinetics of histamine H(3) and H(4) receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by inhibitory autoreceptors. Likewise, receptor deactivation rates upon agonist removal have been implicated in autoreceptor function. Using G protein-coupled potassium (GIRK) channel activation in Xenopus oocytes as readout of receptor activity, we have investigated the voltage sensitivities and signaling kinetics of the hH(3)(445) and hH(3)(365) isoforms of the human histamine H(3) receptor, which functions as an inhibitory auto- and heteroreceptor in the nervous system. We have also investigated both the human and the mouse homologues of the related histamine H(4) receptor, which is expressed mainly on hematopoietic cells. We found that the hH(3)(445) receptor is the most sensitive to voltage, whereas the hH(3)(365) and H(4) receptors are less affected. We further observed a marked difference in response deactivation kinetics between the hH(3)(445) and hH(3)(365) isoforms, with the hH(3)(365) isoform being five to six-fold slower than the hH(3)(445) receptor. Finally, using synthetic agonists, we found evidence for agonist-specific voltage sensitivity at the hH(4) receptor. The differences in voltage sensitivities and deactivation kinetics between the hH(3)(445), hH(3)(365), and H(4) receptors might be relevant to their respective physiological roles.     

Role of the asialoglycoprotein receptor in binding and entry of hepatitis C virus structural proteins in cultured human hepatocytes

We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.     

Loop 5-directed compounds inhibit chimeric kinesin-5 motors: implications for conserved allosteric mechanisms

The human Eg5 (HsEg5) protein is unique in its sensitivity to allosteric agents even among phylogenetic kin. For example, S-trityl-l-cysteine (STC) and monastrol are HsEg5 inhibitors that bind to a surface pocket created by the L5 loop, but neither compound inhibits the Drosophila Kinesin-5 homologue (Klp61F). Herein we ask whether or not drug sensitivity can be designed into Klp61F. Two chimeric Klp61F motor domains were engineered, bacterially expressed, and purified to test this idea. We report that effector binding can elicit a robust allosteric response comparable with HsEg5 in both motor domain chimeras. Furthermore, isothermal titration calorimetry confirms that the Klp61F chimeras have de novo binding affinities for both STC and monastrol. These data show that the mechanism of intramolecular communication between the three ligand binding sites is conserved in the Kinesin-5 family, and reconstitution of a drug binding cassette within the L5 pocket is sufficient to restore allosteric inhibition. However, the two compounds were not equivalent in their allosteric inhibition. This surprising disparity in the response between the chimeras to monastrol and STC suggests that there is more than one allosteric communication network for these effectors.     

Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity

Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway.     

Functional Expression of GFP-linked Human Heart Sodium Channel (hH1) and Subcellular Localization of the a Subunit in HEK293 Cells and Dog Cardiac Myocytes

Recent evidence suggests that biosynthesis of the human heart Na+ channel (hH1) protein is rapidly modulated by sympathetic interventions. However, data regarding the intracellular processing of hH1 in vivo are lacking. In this study we sought to establish a model that would allow us to study the subcellular localization of hH1 protein. Such a model could eventually help us to better understand the trafficking of hH1 in vivo and its potential role in cardiac conduction. We labeled the C-terminus of hH1 with the green fluorescent protein (GFP) and compared the expression of this construct (hH1-GFP) and hH1 in transfected HEK293 cells. Fusion of GFP to hH1 did not alter its electrophysiological properties. Confocal microscopy revealed that hH1-GFP was highly expressed in intracellular membrane structures. Immuno-electronmicrographs showed that transfection of hH1-GFP and hH1 induced proliferation of three types of endoplasmic reticulum (ER) membranes to accommodate the heterologously expressed proteins. Labeling with specific markers for the ER and the Golgi apparatus indicated that the intracellular channels are almost exclusively retained within the ER. Immunocytochemical labeling of the Na+ channel in dog cardiomyocytes showed strong fluorescence in the perinuclear region of the cells, a result consistent with our findings in HEK293 cells. We propose that the ER may serve as a reservoir for the cardiac Na+ channels and that the transport from the ER to the Golgi apparatus is among the rate-limiting steps for sarcolemmal expression of Na+ channels.     

Structural determinants of slow inactivation in human cardiac and skeletal muscle sodium channels.

Skeletal and heart muscle excitability is based upon the pool of available sodium channels as determined by both fast and slow inactivation. Slow inactivation in hH1 sodium channels significantly differs from slow inactivation in hSkM1. The beta(1)-subunit modulates fast inactivation in human skeletal sodium channels (hSkM1) but has little effect on fast inactivation in human cardiac sodium channels (hH1). The role of the beta(1)-subunit in sodium channel slow inactivation is still unknown. We used the macropatch technique on Xenopus oocytes to study hSkM1 and hH1 slow inactivation with and without beta(1)-subunit coexpression. Our results indicate that the beta(1)-subunit is partly responsible for differences in steady-state slow inactivation between hSkM1 and hH1 channels. We also studied a sodium channel chimera, in which P-loops from each domain in hSkM1 sodium channels were replaced with corresponding regions from hH1. Our results show that these chimeras exhibit hH1-like properties of steady-state slow inactivation. These data suggest that P-loops are structural determinants of sodium channel slow inactivation, and that the beta(1)-subunit modulates slow inactivation in hSkM1 but not hH1. Changes in slow inactivation time constants in sodium channels coexpressed with the beta(1)-subunit indicate possible interactions among the beta(1)-subunit, P-loops, and the slow inactivation gate in sodium channels.     

Proteolytic activation of pro-macrophage-stimulating protein by hepsin

Macrophage-stimulating protein (MSP) is a plasminogen-related growth factor and ligand for the receptor tyrosine kinase RON. The MSP/RON system promotes wound healing and invasive tumor growth and suppresses proinflammatory immune response. MSP binding to RON requires proteolytic conversion of the inactive single-chain form (pro-MSP) into the disulfide-linked α/β heterodimer. The pro-MSP cleavage sequence (Ser-Lys-Leu-Arg(483)↓Val(484)) closely matches the substrate recognition sequences of hepsin, a type II transmembrane serine protease, that is overexpressed in several cancers. Here, we show that recombinant hepsin cleaves pro-MSP at the consensus site Arg(483)-Val(484) with superior efficiency compared with the known activators MT-SP1 and hepatocyte growth factor activator (HGFA). At least 50% of pro-MSP was processed within 1 hour at a hepsin concentration of 2.4 nmol/L and at a molar enzyme to substrate ratio of 1:500. An uncleavable single-chain variant of MSP weakly bound to a RON-Fc fusion protein, whereas hepsin-cleaved MSP bound with a K(D) of 10.3 nmol/L, suggesting that the high-affinity binding site in MSP β-chain was properly formed. LNCaP prostate cancer cells overexpressing hepsin on the cell surface efficiently activated pro-MSP, which was blocked by a specific anti-hepsin antibody. Incubation of pro-MSP with hepsin led to robust RON-mediated phosphorylation of mitogen-activated protein kinase, ribosomal S6 protein, and Akt in human A2780 ovarian carcinoma cells stably expressing RON protein. In macrophages, pro-MSP with hepsin induced chemotaxis and attenuated lipopolysaccharide-dependent production of nitric oxide. These findings suggest that the MSP/RON signaling pathway may be regulated by hepsin in tissue homeostasis and in disease pathologies, such as in cancer and immune disorders.     

Molecular action of lidocaine on the voltage sensors of sodium channels

Block of sodium ionic current by lidocaine is associated with alteration of the gating charge-voltage (Q-V) relationship characterized by a 38% reduction in maximal gating charge (Q(max)) and by the appearance of additional gating charge at negative test potentials. We investigated the molecular basis of the lidocaine-induced reduction in cardiac Na channel-gating charge by sequentially neutralizing basic residues in each of the voltage sensors (S4 segments) in the four domains of the human heart Na channel (hH1a). By determining the relative reduction in the Q(max) of each mutant channel modified by lidocaine we identified those S4 segments that contributed to a reduction in gating charge. No interaction of lidocaine was found with the voltage sensors in domains I or II. The largest inhibition of charge movement was found for the S4 of domain III consistent with lidocaine completely inhibiting its movement. Protection experiments with intracellular MTSET (a charged sulfhydryl reagent) in a Na channel with the fourth outermost arginine in the S4 of domain III mutated to a cysteine demonstrated that lidocaine stabilized the S4 in domain III in a depolarized configuration. Lidocaine also partially inhibited movement of the S4 in domain IV, but lidocaine's most dramatic effect was to alter the voltage-dependent charge movement of the S4 in domain IV such that it accounted for the appearance of additional gating charge at potentials near -100 mV. These findings suggest that lidocaine's actions on Na channel gating charge result from allosteric coupling of the binding site(s) of lidocaine to the voltage sensors formed by the S4 segments in domains III and IV.     

Constraining enzyme conformational change by an antibody leads to hyperbolic inhibition

Although it has been known for many years that antibodies display properties characteristic of allosteric effectors, the molecular mechanisms responsible for these effects remain poorly understood. Here, we describe a single-domain antibody fragment (nanobody) that modulates protein function by constraining conformational change in the enzyme dihydrofolate reductase (DHFR). Nanobody 216 (Nb216) behaves as a potent allosteric inhibitor of DHFR, giving rise to mixed hyperbolic inhibition kinetics. The crystal structure of Nb216 in complex with DHFR reveals that the nanobody binds adjacent to the active site. Half of the epitope consists of residues from the flexible Met20 loop. This loop, which ordinarily oscillates between occluded and closed conformations during catalysis, assumes the occluded conformation in the Nb216-bound state. Using stopped flow, we show that Nb216 inhibits DHFR by stabilising the occluded Met20 loop conformation. Surprisingly, kinetic data indicate that the Met20 loop retains sufficient conformational flexibility in the Nb216-bound state to allow slow substrate turnover to occur.     

Characterization of pyridoxal 5'-phosphate affinity labeling of band 3 protein. Evidence for allosterically interacting transport inhibitory subdomains

Pyridoxal 5'-phosphate (PLP) is a substrate of band 3, the erythrocyte anion transport protein. It competitively inhibits anion transport and labels two exofacial chymotryptic domains (the 17-kDa (CH17) and the 35-kDa (CH35) integral fragments). Two mol of PLP are bound/mol of each fragment at saturation. PLP labeling of both domains is competitive with chloride at constant ionic strength. Addition of DNDS (4,4'-dinitrostilbene-2,2'-disulfonate), protects PLP labeling of CH35 but exposes new, nonoverlapping sites on CH17.4,4'-Diisothiocyanostilbene-2,2'-disulfonate reduces PLP labeling to both domains with time, while NAP-taurine (N(-4-azido-2-nitrophenyl)2-aminosulfonate) has no effect on either domain. At low chloride (balance citrate) and high DNDS, we can strongly suppress CH35 labeling and selectively titrate CH17 with PLP. Correlation of fractional transport inhibition with fractional PLP covalent coverage of CH17, quantitatively follows the 1:2 correlation line indicating that full coverage of CH17 sites (which constitute half of the total PLP-labeling sites on band 3) exactly inhibits one-half of transport. PLP labeling of CH35 sites accounts for the other half of inhibition. The inhibition-labeling correlation plots are nonlinear in the absence of DNDS, indicating the presence of allosteric interactions between the domains. We conclude that CH17 and CH35 compose nonoverlapping, functionally equivalent, allosterically linked transport inhibitory subdomains on band 3.     

The b1 Subunit but not the b2 Subunit Colocalizes with the Human Heart Na+ Channel (hH1) already within the Endoplasmic Reticulum

Voltage-dependent Na+ channels are heteromultimers consisting of a pore-forming a subunit and accessory b subunits. In order to provide more insight into the trafficking and assembly of the cardiac Na+ channel complex, we investigated the subcellular localization of the Na+ channel beta1 and beta2 subunits, both in the absence and presence of the human heart Na+ channel (hH1). We fused spectrally distinct variants of the green fluorescent protein (GFP) to hH1 and to the beta1 and beta2 subunit, and expressed the optically labeled b subunits separately or in combination with hH1 in HEK293 cells. In contrast to the predominant localization of hH1 channels within the endoplasmic reticulum (ER), both beta subunits were clearly targeted to the plasma membrane when expressing their cDNAs alone. Upon coexpression of the a subunit, the beta1 subunit was efficiently retained within the ER and found to be colocalized with hH1. In contrast to this, hH1 and the beta2 subunit were not colocalized, i.e., they were detected mainly within the ER and the plasma membrane, respectively. These results indicate that hH1 and the b2 subunit are transported separately to the plasma membrane whereas the hH1/beta1 complex occurs already within the ER, which possibly facilitates trafficking of the channel complex to the plasma membrane.     

Crystallographic studies on acyl ureas, a new class of glycogen phosphorylase inhibitors, as potential antidiabetic drugs

Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular target to control hyperglycemia in type 2 diabetics. This series is exemplified by 6-{2,6-Dichloro- 4-[3-(2-chloro-benzoyl)-ureido]-phenoxy}-hexanoic acid, which inhibits human liver glycogen phosphorylase a with an IC(50) of 2.0 microM. Here we analyze four crystal structures of acyl urea derivatives in complex with rabbit muscle glycogen phosphorylase b to elucidate the mechanism of inhibition of these inhibitors. The structures were determined and refined to 2.26 Angstroms resolution and demonstrate that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Acyl ureas induce conformational changes in the vicinity of the allosteric site. Our findings suggest that acyl ureas inhibit glycogen phosphorylase by direct inhibition of AMP binding and by indirect inhibition of substrate binding through stabilization of the T' state.     

Modes of caldesmon binding to actin: sites of caldesmon contact and modulation of interactions by phosphorylation

Smooth muscle caldesmon binds actin and inhibits actomyosin ATPase activity. Phosphorylation of caldesmon by extracellular signal-regulated kinase (ERK) reverses this inhibitory effect and weakens actin binding. To better understand this function, we have examined the phosphorylation-dependent contact sites of caldesmon on actin by low dose electron microscopy and three-dimensional reconstruction of actin filaments decorated with a C-terminal fragment, hH32K, of human caldesmon containing the principal actin-binding domains. Helical reconstruction of negatively stained filaments demonstrated that hH32K is located on the inner portion of actin subdomain 1, traversing its upper surface toward the C-terminal segment of actin, and forms a bridge to the neighboring actin monomer of the adjacent long pitch helical strand by connecting to its subdomain 3. Such lateral binding was supported by cross-linking experiments using a mutant isoform, which was capable of cross-linking actin subunits. Upon ERK phosphorylation, however, the mutant no longer cross-linked actin to polymers. Three-dimensional reconstruction of ERK-phosphorylated hH32K indeed indicated loss of the interstrand connectivity. These results, together with fluorescence quenching data, are consistent with a phosphorylation-dependent conformational change that moves the C-terminal end segment of caldesmon near the phosphorylation site but not the upstream region around Cys(595), away from F-actin, thus neutralizing its inhibitory effect on actomyosin interactions. The binding pattern of hH32K suggests a mechanism by which unphosphorylated, but not ERK-phosphorylated, caldesmon could stabilize actin filaments and resist F-actin severing or depolymerization in both smooth muscle and nonmuscle cells.     

Proline- and arginine-rich peptides constitute a novel class of allosteric inhibitors of proteasome activity

Substrate-specific inhibition of the proteasome has been unachievable despite great interest in proteasome inhibitors as drugs. Recent studies demonstrated that PR39, a natural proline- and arginine-rich antibacterial peptide, stimulates angiogenesis and inhibits inflammatory responses by specifically blocking degradation of IkappaBalpha and HIF-1alpha by the proteasome. However, molecular events involved in the PR39-proteasome interaction have not been elucidated. Here we show that PR39 is a noncompetitive and reversible inhibitor of the proteasome function. This effect is achieved by a unique allosteric mechanism allowing for specific inhibition of degradation of selected proteins without affecting total proteasome-dependent proteolysis. Atomic force microscopy (AFM) studies demonstrate that 20S and 26S proteasomes treated with PR39 or its derivatives exhibit serious perturbations in their structure and their normal allosteric movements. These effects are universal for proteasomes from yeast to human. The shortest functional sequence derived from PR39 still showing the allosteric inhibitory effect consists of eleven NH(2)-terminal residues containing essential three NH(2)-terminal arginines. The noncompetitive and reversible in vitro action of PR39 and its truncated derivatives is matched by the ability of the peptides to induce angiogenesis in vivo. We postulate that PR39 changes conformational dynamics of the proteasomes by interactions with the noncatalytic subunit alpha7 in a way that prevents the enzyme from cleaving the substrates of unique structural constraints.     

Laminin-332 is a substrate for hepsin, a protease associated with prostate cancer progression

Hepsin, a cell surface protease, is widely reported to be overexpressed in more than 90% of human prostate tumors. Hepsin expression correlates with tumor progression, making it a significant marker and target for prostate cancer. Recently, it was reported that in a prostate cancer mouse model, hepsin up-regulation in tumor tissue promotes progression and metastasis. The underlying mechanisms, however, remain largely uncharacterized. Hepsin transgenic mice displayed reduced laminin-332 (Ln-332) expression in prostate tumors. This is an intriguing cue, since proteolytic processing of extracellular matrix macromolecules, such as Ln-332, is believed to be involved in cancer progression, and Ln-332 expression is lost during human prostate cancer progression. In this study, we provide the first direct evidence that hepsin cleaves Ln-332. Cleavage is specific, since it is both inhibited in a dose-dependent manner by a hepsin inhibitor (Kunitz domain-1) and does not occur when catalytically inactive hepsin is used. By Western blotting and mass spectrometry, we determined that hepsin cleaves the beta3 chain of Ln-332. N-terminal sequencing identified the cleavage site at beta3 Arg(245), in a sequence context (SQLR(245) LQGSCFC) conserved among species and in remarkable agreement with reported consensus target sequences for hepsin activity. In vitro cell migration assays showed that hepsin-cleaved Ln-332 enhanced motility of DU145 prostate cancer cells, which was inhibited by Kunitz domain-1. Further, hepsin-overexpressing LNCaP prostate cancer cells also exhibited increased migration on Ln-332. Direct cleavage of Ln-332 may be one mechanism by which hepsin promotes prostate tumor progression and metastasis, possibly by up-regulating prostate cancer cell motility.     

Allosteric modulation of the human P-glycoprotein involves conformational changes mimicking catalytic transition intermediates

The drug transport function of human P-glycoprotein (Pgp, ABCB1) can be inhibited by a number of pharmacological agents collectively referred to as modulators or reversing agents. In this study, we demonstrate that certain thioxanthene-based Pgp modulators with an allosteric mode of action induce a distinct conformational change in the cytosolic domain of Pgp, which alters susceptibility to proteolytic digestion. Both cis and trans-isomers of the Pgp modulator flupentixol confer considerable protection of an 80 kDa Pgp fragment against trypsin digestion, that is recognized by a polyclonal antibody specific for the NH(2)-terminal half to Pgp. The protection by flupentixol is abolished in the Pgp F983A mutant that is impaired in modulation by flupentixols, indicating involvement of the allosteric site in generating the conformational change. A similar protection to an 80 kDa fragment is conferred by ATP, its nonhydrolyzable analog ATPgammaS, and by trapping of ADP-vanadate at the catalytic domain, but not by transport substrate vinblastine or by the competitive modulator cyclosporin A, suggesting different outcomes from modulator interaction at the allosteric site and at the substrate site. In summary, we demonstrate that allosteric interaction of flupentixols with Pgp generates conformational changes that mimic catalytic transition intermediates induced by nucleotide binding and hydrolysis, which may play a crucial role in allosteric inhibition of Pgp-mediated drug transport.     

Measurement of antibody to Jo-1 by ELISA and comparison to enzyme inhibitory activity

Antibody to the Jo-1 antigen (histidyl-tRNA synthetase) is found almost exclusively in myositis patients, usually those with adult PM, but has been found in only 30% of that group by immunodiffusion or other techniques thus far reported. We have reexamined the prevalence of antibody to Jo-1 in sera from 130 patients and 82 controls by using the sensitive ELISA technique. The ELISA used affinity-purified, enzymatically active bovine Jo-1 antigen. A wide range of antibody level by ELISA was found among 24 immunodiffusion positive sera. Six myositis and two control sera had apparent specific antibody detectable only by ELISA. Overall, however, the antibody continued to show high myositis specificity with predominance in adult PM (35.8% in that group). Because the antibody inhibits enzymatic activity of the synthetase antigen, we also studied the quantitative inhibitory activity of these sera to compare with the antibody activity as determined by ELISA. Twenty-four immunodiffusion-positive sera, 29 immunodiffusion-negative sera, and 15 normal sera were tested at 1/50 dilution in the reaction mixture. There was background inhibition by all normal sera tested that averaged 30.5%. All but one immunodiffusion negative myositis sera (a high binder by ELISA) inhibited less than 50% of the average with normal serum. Twenty-three of 24 immunodiffusion positive sera inhibited greater than 80% of this normal average; the other inhibited 66%. The serum dilution giving 50% inhibition was highly correlated (R = 0.83) with the ELISA activity. Thus, inhibition of histidyl-tRNA synthetase activity is a relatively accurate measure of Jo-1 antibody. This method should be applicable to measuring antibody to other aminoacyl-tRNA synthetases.     

Linker histone subtypes differ in their effect on nucleosomal spacing in vivo

Linker histone H1 is located on the surface of the nucleosome where it interacts with the linker DNA region and stabilizes the 30-nm chromatin fiber. Vertebrates have several different, relatively conserved subtypes of H1; however, the functional reason for this is unclear. We have previously shown that H1 can be reconstituted in Xenopus oocytes, cells that lack somatic H1, by cytosolic mRNA injection and incorporated into in vivo assembled chromatin. Using this assay, we have expressed individual H1 subtypes in the oocytes to study their effect on chromatin structure using nucleosomal repeat length (NRL) as readout. We have compared chicken differentiation-specific histone H5, Xenopus differentiation-specific xH1(0) and the somatic variant xH1A as well as the ubiquitously expressed human somatic subtypes hH1.2, hH1.3, hH1.4 and hH1.5. This shows that all subtypes, except for human H1.5, result in a saturable increase in NRL. hH1.4 results in an increase of approximately 13-20 bp as does xH1(0) and xH1A. chH5 gives rise to the same or slightly longer increase compared to hH1.4. Interestingly, both hH1.2 and hH1.3 show a less extensive increase of only 4.5-7 bp in the NRL, thus yielding the shortest increase of the studied subtypes. We show for the first time in an in vivo system lacking H1 background that ubiquitously expressed and redundant H1 subtypes that coexist in most types of cells of higher eukaryotes differ in their effects on the nucleosomal spacing in vivo. This suggests that H1 subtypes have different roles in the organization and functioning of the chromatin fiber.     

Identification and characterization of the genes encoding the core histones and histone variants of Neurospora crassa

We have identified and characterized the complete complement of genes encoding the core histones of Neurospora crassa. In addition to the previously identified pair of genes that encode histones H3 and H4 (hH3 and hH4-1), we identified a second histone H4 gene (hH4-2), a divergently transcribed pair of genes that encode H2A and H2B (hH2A and hH2B), a homolog of the F/Z family of H2A variants (hH2Az), a homolog of the H3 variant CSE4 from Saccharomyces cerevisiae (hH3v), and a highly diverged H4 variant (hH4v) not described in other species. The hH4-1 and hH4-2 genes, which are 96% identical in their coding regions and encode identical proteins, were inactivated independently. Strains with inactivating mutations in either gene were phenotypically wild type, in terms of growth rates and fertility, but the double mutants were inviable. As expected, we were unable to isolate null alleles of hH2A, hH2B, or hH3. The genomic arrangement of the histone and histone variant genes was determined. hH2Az and the hH3-hH4-1 gene pair are on LG IIR, with hH2Az centromere-proximal to hH3-hH4-1 and hH3 centromere-proximal to hH4-1. hH3v and hH4-2 are on LG IIIR with hH3v centromere-proximal to hH4-2. hH4v is on LG IVR and the hH2A-hH2B pair is located immediately right of the LG VII centromere, with hH2A centromere-proximal to hH2B. Except for the centromere-distal gene in the pairs, all of the histone genes are transcribed toward the centromere. Phylogenetic analysis of the N. crassa histone genes places them in the Euascomycota lineage. In contrast to the general case in eukaryotes, histone genes in euascomycetes are few in number and contain introns. This may be a reflection of the evolution of the RIP (repeat-induced point mutation) and MIP (methylation induced premeiotically) processes that detect sizable duplications and silence associated genes.