Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. While 70% of CF chromosomes carry a deletion of the phenylalanine residue 508 (deltaF508) of CFTR, roughly 5% of all CF chromosomes carry a premature stop mutation. We reported that the aminoglycoside antibiotics G-418 and gentamicin can suppress two premature stop mutations [a stop codon in place of glycine residue 542 (G542X) and arginine residue 553 (R553X)] when expressed from a CFTR cDNA in HeLa cells. Suppression resulted in the synthesis of full-length CFTR protein and the appearance of a cAMP-activated anion conductance characteristic of CFTR function. However, it was unclear whether this approach could restore CFTR function in cells expressing mutant forms of CFTR from the nuclear genome. We now report that G-418 and gentamicin are also capable of restoring CFTR expression in a CF bronchial epithelial cell line carrying the CFTR W1282X premature stop mutation (a stop codon in place of tryptophan residue 1282). This conclusion is based on the reappearance of cAMP-activated chloride currents, the restoration of CFTR protein at the apical plasma membrane, and an increase in the abundance of CFTR mRNA levels from the W1282X allele.

Ligand-gated ion channels are a target for inhaled anesthetics and alcohols in the central nervous system. The inhibitory strychnine-sensitive glycine and gamma-aminobutyric acid type A receptors are positively modulated by anesthetics and alcohols, and site-directed mutagenesis techniques have identified amino acid residues important for the action of volatile anesthetics and alcohols in these receptors. A key question is whether these amino acids are part of an alcohol/anesthetic-binding site. In the present study, we used an alkanethiol anesthetic to covalently label its binding site by mutating selected amino acids to cysteine. We demonstrated that the anesthetic propanethiol, or alternatively, propyl methanethiosulfonate, covalently binds to cysteine residues introduced into a specific second transmembrane site in glycine receptor and gamma-aminobutyric acid type A receptor subunits and irreversibly enhances receptor function. Moreover, upon permanent occupation of the site by propyl disulfide, the usual ability of octanol, enflurane, and isoflurane to potentiate the function of the ion channels was lost. This approach provides strong evidence that the actions of anesthetics in these receptors are due to binding at a single site.

De novo antimicrobial peptides with the sequences: (KLAKKLA)n, (KLAKLAK)n (where n = 1,2,3), (KALKALK)3, (KLGKKLG)n, and (KAAKKAA)n (where n = 2,3), were prepared as the C-terminus amides. These peptides were designed to be perfectly amphipathic in helical conformations. Peptide antibacterial activity was tested against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Peptide cytotoxicity was tested against human erythrocytes and 3T3 mouse fibroblasts. The 3T3 cell testing was a much more sensitive test of cytotoxicity. The peptides were much less lytic toward human erythrocytes than 3T3 cells. Peptide secondary structure in aqueous solution, sodium dodecylsulfate micelles, and phospholipid vesicles was estimated using circular dichroism spectroscopy. The leucine/alanine-containing 21-mers were bacteriostatic at 3-8 microM and cytotoxic to 3T3 cells at about 10 microM concentrations. The leucine/alanine- or leucine/glycine-containing 14-mers and the leucine/glycine 21-mer were bacteriostatic at 6-22 microM but had much lower cytotoxicity toward 3T3 cells and higher selectivities than the natural antimicrobial peptides magainin 2 amide and cecropin B amide. The 7-mer peptides are devoid of biological activity and of secondary structure in membrane mimetic environments. The 14-mer peptides and the glycine-containing 21-mer show modest levels of helicity in model membranes. The leucine/alanine-containing 21-mer peptides have substantial helicity in model membranes. The propensity to alpha-helical conformation of the peptides in amphipathic media is proportional to their 3T3 cell cytotoxicity.

Corelease of GABA and glycine by mixed neurons is a prevalent mode of inhibitory transmission in the vertebrate hindbrain. However, little is known of the functional organization of mixed inhibitory networks. Golgi cells, the main inhibitory interneurons of the cerebellar granular layer, have been shown to contain GABA and glycine. We show here that, in the vestibulocerebellum, Golgi cells contact both granule cells and unipolar brush cells, which are excitatory relay interneurons for vestibular afferences. Whereas IPSCs in granule cells are mediated by GABA(A) receptors only, Golgi cell inhibition of unipolar brush cells is dominated by glycinergic currents. We further demonstrate that a single Golgi cell can perform pure GABAergic inhibition of granule cells and pure glycinergic inhibition of unipolar brush cells. This specialization results from the differential expression of GABA(A) and glycine receptors by target cells and not from a segregation of GABA and glycine in presynaptic terminals. Thus, postsynaptic selection of coreleased fast transmitters is used in the CNS to increase the diversity of individual neuronal outputs and achieve target-specific signaling in mixed inhibitory networks.

Previous studies have shown large differences in taste responses to several sweeteners between mice of the C57BL/6ByJ (B6) and 129P3/J (129) inbred strains. The goal of this study was to compare behavioral responses of B6 and 129 mice to a wider variety of sweeteners. Seventeen sweeteners were tested using two-bottle preference tests with water. Three main patterns of strain differences were evident. First, sucrose, maltose, saccharin, acesulfame-K, sucralose and SC-45647 were preferred by both strains, but the B6 mice had lower preference thresholds and higher solution intakes. Second, the amino acids D-phenylalanine, D-tryptophan, L-proline and glycine were highly preferred by B6 mice, but not by 129 mice. Third, glycyrrhizic acid, neohesperidin dihydrochalcone, thaumatin and cyclamate did not evoke strong preferences in either strain. Aspartame was neutral to all 129 and some B6 mice, but other B6 mice strongly preferred it. Thus, compared with the 129 mice the B6 mice had higher preferences for sugars, sweet tasting amino acids and several but not all non-caloric sweeteners. Glycyrrhizic acid, neohesperidin, thaumatin and cyclamate are not palatable to B6 or 129 mice.

We have previously identified, in three British families having an index child with frequent infections, a point mutation (GGC->>GAC) in codon 54 of exon 1 of the gene for the human lectin mannose binding protein (MBP). This was associated with low serum levels of this complement activating protein and would be anticipated to impair opsonization of mannose rich microorganisms. We now report a second point mutation (GGA->>GAA) in Gambians from West Africa, involving codon 57 of exon 1. By substituting carboxylic acids for axial glycines in the translated proteins both mutations would be expected to disrupt the secondary structure of the collagenous triple helix of the 96 kDa MBP subunits. In the Gambians the codon 57 mutation was studied by PCR, sequence analysis and restriction analysis and found to be remarkably common (frequency of the mutant gene 0.29 in adults and 0.23 in newborns) whereas the codon 54 mutation was very rare (frequency 0.003). However, the codon 54 mutation was frequent in both a British Caucasian and a Hong Kong Chinese population (frequency of the mutant gene 0.17 and 0.11 respectively). It was predicted that both homozygous and heterozygous individuals would have profoundly reduced serum levels of the protein and this was confirmed by immunoassay as was the reduced capacity of such sera to activate complement through the MBP initiated classical pathway. Our data indicate that the two mutations have arisen independently since the divergence of African and non African populations and both have attained high frequencies.

The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.

flbA encodes an Aspergillus nidulans RGS (regulator of G protein signaling) domain protein that is required for control of mycelial proliferation and activation of asexual sporulation. We identified a dominant mutation in a second gene, fadA, that resulted in a very similar phenotype to flbA loss-of-function mutants. Analysis of fadA showed that it encodes the alpha-subunit of a heterotrimeric G protein, and the dominant phenotype resulted from conversion of glycine 42 to arginine (fadA(G42R)). This mutation is predicted to result in a loss of intrinsic GTPase activity leading to constitutive signaling, indicating that activation of this pathway leads to proliferation and blocks sporulation. By contrast, a fadA deletion and a fadA dominant-interfering mutation (fadA(G203R)) resulted in reduced growth without impairing sporulation. In fact, the fadA(G203R) mutant was a hyperactive asexual sporulator and produced elaborate sporulation structures, called conidiophores, under environmental conditions that blocked wild-type sporulation. Both the fadA(G203R) and the fadA deletion mutations suppressed the flbA mutant phenotype as predicted if the primary role of FlbA in sporulation is in blocking activation of FadA signaling. Because overexpression of flbA could not suppress the fadA(G42R) mutant phenotype, we propose that FlbA's role in modulating the FadA proliferation signal is dependent upon the intrinsic GTPase activity of wild-type FadA.

Cross-linking of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins on T cells can trigger cell activation. We and others have shown an association between GPI-anchored proteins and the protein tyrosine kinases (PTKs) p56lck and p59fyn, suggesting a pathway for signaling through GPI-anchored proteins. Studies of decay-accelerating factor (DAF) or CD59 in either the C32 cell line or the HeLa cell line transfected with PTK cDNA demonstrated that the GPI-anchored proteins associated noncovalently with p56lck and p59fyn but not with p60src. Nonmyristylated versions of p56lck and p59fyn also failed to associate with the GPI-anchored proteins. Mutational analysis of the PTK demonstrated that the association with the GPI-anchored proteins mapped to the unique amino-terminal domains of the PTK. A chimeric PTK consisting of the 10 amino-terminal residues of p56lck or p59fyn replacing the corresponding amino acids in p60src was sufficient for association with DAF, but the converse constructs containing the first 10 amino acids of p60src plus the remainder of p56lck or p59fyn did not associate with DAF. Mutation of cysteine to serine at positions 3 and 6 in p59fyn or positions 3 and 5 in p56lck abolished the association of these kinases with DAF. Mutation of serine to cysteine at positions 3 and 6 in p60src conferred on p60src the ability to associate with DAF. Direct labeling with [3H]palmitate demonstrated palmitylation of this amino-terminal cysteine motif in p56lck. Thus, palmitylation of the amino-terminal cysteine residue(s) together with myristylation of the amino-terminal glycine residue defines important motifs for the association of PTKs with GPI-anchored proteins.

Attempts to investigate the cellular localization of keratin mRNAs by in situ hybridization with specific [35S]-labelled cDNA probes to mouse epithelia have been seriously impeded by the uncontrollable detachment of frozen tissue sections on conventionally coated glass slides (i.e. those coated with egg white, gelatin, collagen). Similarly, a variety of other coating and attachment devices have proved to be unsatisfactory or impracticable for large scale investigations. These difficulties were completely overcome and in situ hybridization was possible after a short immersion of the glass slides in a 2% solution of 3-aminopropyltriethoxysilane in acetone. This treatment provides the glass surface with aminoalkyl groups which are apparently able to react covalently with aldehyde or ketone functions of frozen tissue sections. The resulting firm adhesion of the sections enabled us to investigate the influence of different fixation and prehybridization procedures on the quality of the in situ hybridization. It was found that especially harsh prehybridization, involving hydrochloric acid, heat and proteinase K treatment, drastically reduces the morphological integrity of the sections, thus rendering a reliable assignment of the label difficult. In contrast, a mild prehybridization, consisting mainly of a rehydration of the sections in phosphate-buffered saline and equilibration in 0.1 M glycine, leaves the morphology intact and leads to a highly efficient and specific in situ hybridization.

Strychnine-sensitive glycine receptors (GlyRs) are known to mediate synaptic inhibition in spinal cord, brainstem and other regions of the CNS. During the past 5 years, considerable progress has been made in delineating structural determinants of ligand binding and channel activation in recombinant GlyRs. Furthermore, immunohistochemical and gene inactivation studies have disclosed distinct distributions and functions of differentially expressed GlyR subtypes in retina, hippocampus and the dorsal horn of the spinal cord. Accordingly, GlyRs regulate not only the excitability of motor and sensory neurones, but are also essential for the processing of photoreceptor signals, neuronal development and inflammatory pain sensitization. Hence, these receptors constitute promising targets for the development of clinically useful compounds.

We have observed a common sequence motif in membrane proteins, which we call a glycine zipper. Glycine zipper motifs are strongly overrepresented and conserved in membrane protein sequences, and mutations in glycine zipper motifs are deleterious to function in many cases. The glycine zipper has a significant structural impact, engendering a strong driving force for right-handed packing against a neighboring helix. Thus, the presence of a glycine zipper motif leads directly to testable structural hypotheses, particularly for a subclass of glycine zipper proteins that form channels. For example, we suggest that the membrane pores formed by the amyloid-beta peptide in vitro are constructed by glycine zipper packing and find that mutations in the glycine zipper motif block channel formation. Our findings highlight an important structural motif in a wide variety of normal and pathological processes.

The standard view of alpha helix formation in water, based on helix propensities determined by the host-guest method, is that differences in helix propensity among the amino acids are small, except for proline, and that the average value of the helix propagation parameter s is near 1. A contradictory view of alpha helix formation in water is emerging from substitution experiments with short, unique-sequence peptides that contain only naturally occurring amino acids. Short peptides that contain only alanine and lysine, or alanine and glutamate, form surprisingly stable monomeric helices in water and substitution of a single alanine residue by another amino acid in these or related peptides produces a wide range of changes in helix content, depending on which amino acid is substituted for alanine. We show here that the ratio of the helix propensities of alanine to glycine is large, about 100, in substitution experiments with a 17-residue reference peptide containing alanine and lysine. The helix propensity is identified with s, the helix propagation parameter of the statistical mechanics model for alpha helix formation, and the results are interpreted by the Lifson-Roig theory. Single alanine----glycine substitutions have been made at a series of positions in individual peptides. The helix-destabilizing effect of an Ala----Gly substitution depends strongly on its position in the helix, as predicted by the Lifson-Roig theory if the ratio of s values for Ala:Gly is large.

The postsynaptic glycine receptor of rat spinal cord is a glycosylated membrane protein that, after affinity purification, contains membrane-spanning subunits of Mr 48,000 and 58,000 and an associated peripheral polypeptide of Mr 93,000. Here, the quaternary structure of the transmembrane core of the receptor was investigated by chemically crosslinking its subunits. Upon treatment with crosslinking reagents of different side-chain specificities and lengths, a consistent set of adducts up to Mr 260,000 was detected after separation by NaDodSO4/PAGE. The observed pattern of adducts was similar irrespective of whether purified receptor protein or synaptosomal membranes were crosslinked. Compositional analysis revealed that the crosslinked adducts contained the Mr 48,000 and 58,000 subunits in varying ratios but not the peripheral Mr 93,000 polypeptide. Thus adducts of intermediate molecular weight represent dimers, trimers, and tetramers of the transmembrane subunits, whereas the major adduct of Mr 260,000 corresponds to a pentameric assembly of subunits forming the ion channel of the glycine receptor. This subunit arrangement is similar to that reported for the nicotinic acetylcholine receptor of fish electric organ and skeletal muscle. Hence, we suggest that the different ligand-gated ion channels of excitable membranes share a similar quaternary structure.

To characterize the impact of gut microbiota on host metabolism, we investigated the multicompartmental metabolic profiles of a conventional mouse strain (C3H/HeJ) (n=5) and its germ-free (GF) equivalent (n=5). We confirm that the microbiome strongly impacts on the metabolism of bile acids through the enterohepatic cycle and gut metabolism (higher levels of phosphocholine and glycine in GF liver and marked higher levels of bile acids in three gut compartments). Furthermore we demonstrate that (1) well-defined metabolic differences exist in all examined compartments between the metabotypes of GF and conventional mice: bacterial co-metabolic products such as hippurate (urine) and 5-aminovalerate (colon epithelium) were found at reduced concentrations, whereas raffinose was only detected in GF colonic profiles. (2) The microbiome also influences kidney homeostasis with elevated levels of key cell volume regulators (betaine, choline, myo-inositol and so on) observed in GF kidneys. (3) Gut microbiota modulate metabotype expression at both local (gut) and global (biofluids, kidney, liver) system levels and hence influence the responses to a variety of dietary modulation and drug exposures relevant to personalized health-care investigations.

The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was completely assigned in a sequential manner by two-dimensional NMR techniques. A large number of stereospecific assignments for beta-methylene protons, as well as ranges for the phi, psi, and chi 1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some NOE cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the alpha-methylene protons of glycine using information on phi torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 phi, 24 psi, and 25 chi 1 torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 +/- 0.04 A for the backbone atoms and 0.52 +/- 0.06 A for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel beta-sheet composed of residues 5-9 (beta 1), 24-28 (beta 2), and 33-36 (beta 3) in which strand beta 3 is hydrogen bonded to the other two strands.(ABSTRACT TRUNCATED AT 400 WORDS)

The specificity of the glycerol facilitator (glpF) of Escherichia coli was studied with an osmotic method. This transport system allowed the entry of polyols (glycerol and erythritol), pentitols, and hexitols. The analogous sugars were not transported. However, urea, glycine, and DL-glyceraldehyde could use this pathway to enter the cell. The glpF protein allowed the rapid efflux of preequilibrated xylitol. Glycerol surprisingly did not inhibit the uptake of xylitol, and xylitol only slightly reduced the uptake of glycerol. The observation and the insensitivity of the xylitol transport to low temperature suggest that the facilitator behaves as a membrane channel.

BACKGROUND

Disturbances of N-methyl-D-aspartate (NMDA) receptor-mediated glutamatergic neurotransmission may play an important role in the pathophysiology of negative symptoms of schizophrenia. Glycine, a small nonessential amino acid, functions as an obligatory coagonist at NMDA receptors through its action at a strychnine-insensitive binding site on the NMDA receptor complex. Glycine-induced augmentation of NMDA receptor-mediated neurotransmission may thus offer a potentially safe and feasible approach for ameliorating persistent negative symptoms of schizophrenia.

METHODS

Twenty-two treatment-resistant schizophrenic patients participated in a double-blind, placebo-controlled, 6-week, crossover treatment trial with 0.8 g/kg per day of glycine added to their ongoing antipsychotic medication. Clinical assessments, including the Brief Psychiatric Rating Scale (BPRS), the Positive and Negative Syndrome Scale (PANSS), the Simpson-Angus Scale for Extrapyramidal Symptoms, and the Abnormal Involuntary Movement Scale, were performed biweekly throughout the study. Clinical laboratory values and amino acid serum levels were monitored.

RESULTS

Glycine treatment was well tolerated and induced increased glycine (P=.001) and serine (P=.001) serum levels. Glycine administration resulted in (1) a significant (P<.001) 30%+/-16% reduction in negative symptoms, as measured by the PANSS, and (2) a significant (P<.001) 30%+/-18% improvement in the BPRS total scores. The improvement in negative symptoms was unrelated to alterations in extrapyramidal effects or symptoms of depression. Low pretreatment glycine serum levels significantly predicted (r= 0.80) clinical response.

CONCLUSIONS

These findings support hypoglutamatergic hypotheses of schizophrenia and suggest a novel approach for the pharmacotherapy of negative symptoms associated with this illness.

Glycine and GABAA (gamma-aminobutyric acid A) receptors are inhibitory neurotransmitter-gated Cl- channels localized in postsynaptic membranes. In some cases, GABAA receptors are also found presynaptically, but they retain their inhibitory effect as their activation reduces excitatory transmitter release. Here we report evidence for presynaptic ionotropic glycine receptors, using pre- and postsynaptic recordings of a calyceal synapse in the medial nucleus of the trapezoid body (MNTB). Unlike the classical action of glycine, presynaptic glycine receptors triggered a weakly depolarizing Cl- current in the nerve terminal. The depolarization enhanced transmitter release by activating Ca2+ channels and increasing resting intraterminal Ca2+ concentrations. Repetitive activation of glycinergic synapses on MNTB neurons also enhanced glutamatergic synaptic currents, indicating that presynaptic glycine receptors are activated by glycine spillover. These results reveal a novel site of action of the transmitter glycine, and indicate that under certain conditions presynaptic Cl- channels may increase transmitter release.

The influence of external [H+] on whole-cell and single-channel currents activated by glutamate agonists was studied in rat hippocampal neurons. In the pH range between 6.6 and 8.0, changes in external [H+] had negligible influence on the amplitude and kinetics of the monovalent ion-carrying currents activated by the agonists quisqualate and kainate. The divalent ion-carrying N-methyl-D-aspartate (NMDA)-activated current, on the other hand, was strongly modulated by extracellular [H+]. Increased external [H+] suppressed, whereas decreased external [H+] enhanced, the NMDA-activated current. Changes in internal [H+] had little or no effect on the NMDA-activated current. Modulation of the NMDA-activated current resulted primarily from changes in the number of channel openings. Neither the unitary conductance nor the individual open dwell-times were significantly affected. These results suggest that the protonation site is on the external aspect of the channel and is far removed from the channel permeation pathway. Because interactions between H+, NMDA, and glycine in activating the current were predominantly noncompetitive, our results suggest that the modulatory effect of H+ was not associated with changes in receptor-agonist affinities. These results suggest that modulation of the NMDA-receptor channel by [H+] may be an intrinsic protective mechanism by which calcium influx into neurons is regulated, particularly in hypoxic/ischemic conditions.

beta-1,4-Endoglucanases (EGases, EC 3.2.1.4) degrade polysaccharides possessing beta-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes Globodera rostochiensis and Heterodera glycines. From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37-44% overall amino acid identity with EGases from the bacteria Erwinia chrysanthemi, Clostridium acetobutylicum, and Bacillus subtilis. One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3' end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation.

Archaea, one of three major evolutionary lineages of life, encode proteasomes highly related to those of eukaryotes. In contrast, archaeal ubiquitin-like proteins are less conserved and not known to function in protein conjugation. This has complicated our understanding of the origins of ubiquitination and its connection to proteasomes. Here we report two small archaeal modifier proteins, SAMP1 and SAMP2, with a beta-grasp fold and carboxy-terminal diglycine motif similar to ubiquitin, that form protein conjugates in the archaeon Haloferax volcanii. The levels of SAMP-conjugates were altered by nitrogen-limitation and proteasomal gene knockout and spanned various functions including components of the Urm1 pathway. LC-MS/MS-based collision-induced dissociation demonstrated isopeptide bonds between the C-terminal glycine of SAMP2 and the epsilon-amino group of lysines from a number of protein targets and Lys 58 of SAMP2 itself, revealing poly-SAMP chains. The widespread distribution and diversity of pathways modified by SAMPylation suggest that this type of protein conjugation is central to the archaeal lineage.

Osteopontin is an arginine-glycine-aspartate containing acidic glycoprotein postulated to mediate adhesion, migration, and biomineralization in diverse tissues. The mechanisms explaining this multifunctionality are not well understood, although it is known that one osteopontin receptor is the alpha v beta 3 integrin. In this work, we studied human smooth muscle cells varying in alpha v beta 3 levels to identify additional osteopontin receptors. We report that, in addition to alpha v beta 3, both alpha v beta 5 and alpha v beta 1 are osteopontin receptors. Moreover, the presence or absence of alpha v beta 3 on the cell surface altered the adhesive and migratory responses of smooth muscle cells to osteopontin. Adhesion of alpha v beta 3-deficient cell populations to osteopontin was only half that of cells containing alpha v beta 3, and migration toward an osteopontin gradient in the Boyden chamber was dependent on cell surface alpha v beta 3. Although alpha v beta 3-deficient smooth muscle cells were unable to migrate to osteopontin, they did migrate significantly in response to vitronectin and fibronectin. These findings represent the first description of alpha v beta 5 and alpha v beta 1 as osteopontin receptors and suggest that, while adhesion to osteopontin is supported by integrins containing beta 1, beta 3, and beta 5, migration in response to osteopontin appears to depend on alpha v beta 3. Thus, interaction with distinct receptors is one mechanism by which osteopontin may initiate multiple functions.

Determinacy is an agronomically important trait associated with the domestication in soybean (Glycine max). Most soybean cultivars are classifiable into indeterminate and determinate growth habit, whereas Glycine soja, the wild progenitor of soybean, is indeterminate. Indeterminate (Dt1/Dt1) and determinate (dt1/dt1) genotypes, when mated, produce progeny that segregate in a monogenic pattern. Here, we show evidence that Dt1 is a homolog (designated as GmTfl1) of Arabidopsis terminal flower 1 (TFL1), a regulatory gene encoding a signaling protein of shoot meristems. The transition from indeterminate to determinate phenotypes in soybean is associated with independent human selections of four distinct single-nucleotide substitutions in the GmTfl1 gene, each of which led to a single amino acid change. Genetic diversity of a minicore collection of Chinese soybean landraces assessed by simple sequence repeat (SSR) markers and allelic variation at the GmTfl1 locus suggest that human selection for determinacy took place at early stages of landrace radiation. The GmTfl1 allele introduced into a determinate-type (tfl1/tfl1) Arabidopsis mutants fully restored the wild-type (TFL1/TFL1) phenotype, but the Gmtfl1 allele in tfl1/tfl1 mutants did not result in apparent phenotypic change. These observations indicate that GmTfl1 complements the functions of TFL1 in Arabidopsis. However, the GmTfl1 homeolog, despite its more recent divergence from GmTfl1 than from Arabidopsis TFL1, appears to be sub- or neo-functionalized, as revealed by the differential expression of the two genes at multiple plant developmental stages and by allelic analysis at both loci.