Functional characterization of G-protein-coupled receptors: a bioinformatics approach.

Complex molecular and cellular mechanisms regulate G protein-coupled receptors (GPCRs). It is suggested that proteins intrinsically disordered regions (IDRs) are to play a role in GPCR's intra and extracellular regions plasticity, due to their potential for post-translational modification and interaction with other proteins. These regions are defined as lacking a stable three-dimensional (3D) structure. They are rich in hydrophilic and charged, amino acids and are capable to assume different conformations which allow them to interact with multiple partners. In this study we analyzed 75 GPCR involved in synaptic transmission using computational tools for sequence-based prediction of IDRs within a protein. We also evaluated putative ligand-binding motifs using receptor sequences. The disorder analysis indicated that neurotransmitter GPCRs have a significant amount of disorder in their N-terminus, third intracellular loop (3IL) and C-terminus. About 31%, 39% and 53% of human GPCR involved in synaptic transmission are disordered in these regions. Thirty-three percent of receptors show at least one predicted PEST motif, this being statistically greater than the estimate for the rest of human GPCRs. About 90% of the receptors had at least one putative site for dimerization in their 3IL or C-terminus. ELM instances sampled in these domains were 14-3-3, SH3, SH2 and PDZ motifs. In conclusion, the increased flexibility observed in GPCRs, added to the enrichment of linear motifs, PEST and heteromerization sites, may be critical for the nervous system's functional plasticity.

How adenylate cyclase choreographs the pas de deux of the receptors heteromerization dance.

Our work suggests that heteromer formation, mainly involves linear motifs (LMs) found in disordered regions of proteins. Local disorder imparts plasticity to LMs. Most molecular recognition of proteins occurs between short linear segments, known as LMs. Interaction of short continuous epitopes is not constrained by sequence and has the advantage of resulting in interactions with micromolar affinities which suit transient, reversible complexes such as receptor heteromers. Electrostatic interactions between epitopes of the G-protein coupled receptors (GPCR) involved, are the key step in driving heteromer formation forward. The first step in heteromerization, involves phosphorylating Ser/Thr in an epitope containing a casein kinase 1/2-consensus site. Our data suggest that dopaminergic neurotransmission, through cAMP-dependent protein kinase A (PKA) slows down heteromerization. The negative charge, acquired by the phosphorylation of a Ser/Thr in a PKA consensus site in the Arg-rich epitope, affects the activity of the receptors involved in heteromerization by causing allosteric conformational changes, due to the repulsive effect generated by the negatively charged phosphate. In addition to modulating heteromerization, it affects the stability of the heteromers' interactions and their binding affinity. So here we have an instance where phosphorylation is not just an on/off switch, instead by weakening the noncovalent bond, heteromerization acts like a rheostat that controls the stability of the heteromer through activation or inhibition of adenylate cyclase by the neurotransmitter Dopamine depending on which Dopamine receptor it docks at.

GPCR heteromers and their allosteric receptor-receptor interactions.

The concept of intramembrane receptor-receptor interactions and evidence for their existences were introduced in the beginning of the 1980's, suggesting the existence of receptor heterodimerization. The discovery of GPCR heteromers and the receptor mosaic (higher order oligomers, more than two) has been related to the parallel development and application of a variety of resonance energy transfer techniques such as bioluminescence (BRET), fluorescence (FRET) and sequential energy transfer (SRET). The assembly of interacting GPCRs, heterodimers and receptor mosaic leads to changes in the agonist recognition, signaling, and trafficking of participating receptors via allosteric mechanisms, sometimes involving the appearance of cooperativity. The receptor interface in the GPCR heteromers is beginning to be characterized and the key role of electrostatic epitope-epitope interactions for the formation of the receptor heteromers will be discussed. Furthermore, a "guide-and-clasp" manner of receptor-receptor interactions has been proposed where the "adhesive guides" may be the triplet homologies. These interactions probably represent a general molecular mechanism for receptor-receptor interactions. It is proposed that changes in GPCR function (moonlighting) may develop through the intracellular loops and C-terminii of the GPCR heteromers as a result of dynamic allosteric interactions between different types of G proteins and other receptor interacting proteins in these domains of the receptors. The evidence for the existence of receptor heteromers opens up a new field for a better understanding of neurophysiology and neuropathology. Furthermore, novel therapeutic approaches could be possible based on the use of heteromers as targets for drug development based on their unique pharmacology.

An update on adenosine A2A-dopamine D2 receptor interactions: implications for the function of G protein-coupled receptors.

Adenosine A(2A)-dopamine D(2) receptor interactions play a very important role in striatal function. A(2A)-D(2) receptor interactions provide an example of the capabilities of information processing by just two different G protein-coupled receptors. Thus, there is evidence for the coexistence of two reciprocal antagonistic interactions between A(2A) and D(2) receptors in the same neurons, the GABAergic enkephalinergic neurons. An antagonistic A(2A)-D(2) intramembrane receptor interaction, which depends on A(2A)-D(2) receptor heteromerization and G(q/11)-PLC signaling, modulates neuronal excitability and neurotransmitter release. On the other hand, an antagonistic A(2A)-D(2) receptor interaction at the adenylyl-cyclase level, which depends on G(s/olf)- and G(i/o)-type V adenylyl-cyclase signaling, modulates protein phosphorylation and gene expression. Finally, under conditions of upregulation of an activator of G protein signaling (AGS3), such as during chronic treatment with addictive drugs, a synergistic A(2A)-D(2) receptor interaction can also be demonstrated. AGS3 facilitates a synergistic interaction between G(s/olf) - and G(i/o)-coupled receptors on the activation of types II/IV adenylyl cyclase, leading to a paradoxical increase in protein phosphorylation and gene expression upon co-activation of A(2A) and D(2) receptors. The analysis of A(2)-D(2) receptor interactions will have implications for the pathophysiology and treatment of basal ganglia disorders and drug addiction.

On the key role played by altered protein conformation in Parkinson's disease.

On the basis of the previously proposed hierarchic organisation of the central nervous system (CNS) and of its syntropic behaviour, a view of neurodegenerative diseases focusing on the assemblage of abnormal multimeric proteins (pathologic protein mosaics (PMs)) is proposed. Thus, the main focus of the present paper is on Parkinson's disease (PD) as a neurodegenerative disease, which has as crucial feature protein conformational alterations and formation of pathological PMs. Two interconnected cellular dysfunctions are discussed as main pathogenic factors of PD syndromes, namely mitochondrial deficits (i.e. energy failure, especially critical for Substantia Nigra DA neurons) and conformational protein alterations (due to genetic or environmental causes). Conformational protein alterations can trigger pathological phenomena via the loss and/or the gain of new functions. In particular, altered proteins can lead to the formation of abnormal PMs, which can, inter alia, cause distortion of cellular structures, toxic functions and/or formation of improper membrane ion channels. In view of the fact that disordered proteins can easily acquire unwanted conformation, the "disorder index" (DI) for proteins involved in PD has been evaluated. It has been found that both alpha-synuclein and tau-protein have high DI. This datum is in agreement with the observation that these two proteins synergistically promote polymerisation of each other into amyloid fibrils, favouring the formation of Lewy bodies.

Tag-mass: specific molecular imaging of transcriptome and proteome by mass spectrometry based on photocleavable tag.

MALDI tissue imaging of tissues has become a promising technique for tracking biomarkers while determining their location and structural characterization. We have now developed specific targeting probes (oligonucleotides, antibodies), named Tag-Mass. This approach is based on probes modified with a photocleavable linker coupled with a tag cleaved and detected using mass spectrometry. Tag-Mass development is the key for a rapid, sensitive, and accurate approach to correlate levels of expression of different mRNA or proteins in diseases.

Abeta peptides as one of the crucial volume transmission signals in the trophic units and their interactions with homocysteine. Physiological implications and relevance for Alzheimer's disease.

Amyloid peptides (Abeta) can operate as volume transmission (VT) signals since they are continuously released from cells of the central nervous system and diffuse in the extra-cellular space of the brain. They have both regulatory and trophic functions on cellular networks. In agreement with Abeta regulatory actions on glial-neuronal networks, the present paper reports new findings demonstrating that intrastriatal injections of Abeta peptides reduce striatal tyrosine hydroxylase, increase striatal GFAP immunoreactivities and lower pain threshold in experimental rats. Furthermore, it has been demonstrated that exogenous homocysteine (Hcy) binds Abeta(1-40) favouring its beta-sheet conformation both in vitro and in vivo and hence the formation of beta-fibrils and development of neurotoxicity. Thus, the hypothesis is discussed that Abeta peptides represent crucial VT-signals in the brain and their action is altered by dysmetabolic signals such as high Hcy extra-cellular levels, known to be an important risk factor for Alzheimer's disease.

Matrix implanted laser desorption ionization (MILDI) combined with ion mobility-mass spectrometry for bio-surface analysis.

The implantation of low velocity massive gold cluster ions allows homogeneous incorporation of a metallic matrix into the near-surface region of rat brain tissues. Subsequent analysis by laser desorption ionization mass spectrometry yields spectra exhibiting molecular ion peaks in the mass range up to 35 kDa similar to those observed by matrix-assisted LDI. Matrix-implanted LDI when combined with ion-mobility preseparation promises to be a useful technique for molecular imaging of biotissues with a laser microprobe.

A study of peptide--peptide interaction by matrix-assisted laser desorption/ionization.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to study peptide-peptide interaction. The interaction was seen when 6-aza-2-thiothymine was used as a matrix (pH 5.4), but was disrupted with a more acidic matrix, alpha-cyano-4-hydroxycinnamic acid (pH 2.0). In the present study, we show that dynorphin, an opioid peptide, and five of its fragments that contain two adjacent basic residues (Arg6-Arg7), all interact noncovalently with peptides that contain two to five adjacent acidic residues (Asp or Glu). Two other nonrelated peptides containing two (Arg6-Arg7) or three (Arg1-Lys2-Arg3) adjacent basic amino acid residues were studied and exhibited the same behavior. However, peptides containing adjacent Lys or His did not form noncovalent complexes with acidic peptides. The noncovalent bonding was sufficiently stable that digestion with trypsin only cleaved Arg and Lys residues that were not involved in hydrogen bonding with the acidic residues. In an equimolar mixture of dynorphin, dynorphin fragments (containing the motif RR), and an acidic peptide (minigastrin), the acidic peptide preferentially complexed with dynorphin. If the concentration of minigastrin was increased 10 fold, noncovalent interaction was seen with dynorphin and all its fragments containing the motif RR. In the absence of dynorphin, minigastrin formed noncovalent complexes with all dynorphin fragments. These findings suggest that conformation, equilibrium, and concentration do play a role in the occurrence of peptide-peptide interaction. Observations from this study include: (1) ionic bonds were not disrupted by enzymatic digests, (2) conformation and concentration influenced complex formation, and (3) the complex did not form with fragments of dynorphin or unrelated peptides that did not contain the motifs RR or RKR, nor with a fragment of dynorphin where Arg7 was mutated to a phenylalanine residue. These findings strongly suggest that peptide-peptide interaction does occur, and can be studied by MALDI if near physiologic pH is maintained.

Glycosylphosphatidylinositol anchors of Plasmodium falciparum: molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis.

Induction of proinflammatory cytokine responses by glycosylphosphatidylinositols (GPIs) of intraerythrocytic Plasmodium falciparum is believed to contribute to malaria pathogenesis. In this study, we purified the GPIs of P. falciparum to homogeneity and determined their structures by biochemical degradations and mass spectrometry. The parasite GPIs differ from those of the host in that they contain palmitic (major) and myristic (minor) acids at C-2 of inositol, predominantly C18:0 and C18:1 at sn-1 and sn-2, respectively, and do not contain additional phosphoethanolamine substitution in their core glycan structures. The purified parasite GPIs can induce tumor necrosis factor alpha release from macrophages. We also report a new finding that adults who have resistance to clinical malaria contain high levels of persistent anti-GPI antibodies, whereas susceptible children lack or have low levels of short-lived antibody response. Individuals who were not exposed to the malaria parasite completely lack anti-GPI antibodies. Absence of a persistent anti-GPI antibody response correlated with malaria-specific anemia and fever, suggesting that anti-GPI antibodies provide protection against clinical malaria. The antibodies are mainly directed against the acylated phosphoinositol portion of GPIs. These results are likely to be valuable in studies aimed at the evaluation of chemically defined structures for toxicity versus immunogenicity with implications for the development of GPI-based therapies or vaccines.

Peptide sequence information derived by pronase digestion and ammonium sulfate in-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We present the use of Pronase digestion and in-source decay in the presence of ammonium sulfate as complementary techniques to confirm the amino acid sequence of a peptide. Pronase, a commercial preparation from Streptomyces griseus, is a combination of proteolytic enzymes. It produces carboxypeptidase and aminopeptidase ladders using a single Pronase digestion and represents an inexpensive, nonspecific, and fast supplement to traditional sequencing enzymes. However, N-terminal peptidase activity appears dependent on the terminal amino acid residue. We also introduce the use of saturated ammonium sulfate as an "on-slide" sample additive to promote in-source fragmentation of peptides. Use of saturated ammonium sulfate resulted in a simple way to increase peptide backbone fragmentation and essentially produced either a cn or yn ion series. Together these techniques provide useful supplements to existing methods for peptide sequence information.

Characterization of the "helix clamp" motif of HIV-1 reverse transcriptase using MALDI-TOF MS and surface plasmon resonance.

A helix-turn-helix motif in the crystal structure of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was proposed to be a conserved nucleic acid binding domain among several nucleotide polymerizing enzymes (Hermann, T.; Meier, T.; Götte, M.; Heumann, H. Nucleic Acids Res. 1994, 22, 4625-4633). The sequence of this domain is homologous to 259KLVGKL-(X)16KLLR284 of HIV-1 RT, which acts as a "helix clamp" grasping the template-primer (T-P) complex. We characterized the helix clamp motif using MALDI-TOF MS and surface plasmon resonance (BIAcore). Our studies showed that the "helix clamp" has a nucleic acid binding function that may not be sequence specific. This evidence suggests that ionic interactions between the helix clamp and oligonucleotide backbone are not solely responsible for binding. Secondary and tertiary structures of the protein may also play a significant role in nucleic acid binding. The association and dissociation constants, ka and kd, for the binding of single-stranded oligonucleotide to the helix clamp were determined to be 7.03 x 10(3) M(-1) s(-1) and 1.22 x 10(3) s(-1), respectively.

Elevated abeta42 in skeletal muscle of Alzheimer disease patients suggests peripheral alterations of AbetaPP metabolism.

The levels of amyloid-beta40 (Abeta40) and Abeta42 peptides were quantified in temporalis muscles and brain of neuropathologically diagnosed Alzheimer disease (AD) and of nondemented individuals. This was achieved by using a novel analytical approach consisting of a combination of fast-performance liquid chromatographic (FPLC) size exclusion chromatography developed under denaturing conditions and europium immunoassay on the 4.0- to 4.5-kd fractions. In the temporalis muscles of the AD and nondemented control groups, the average values for Abeta42 were 15.7 ng/g and 10.2 ng/g (P = 0.010), and for Abeta40 they were 37.8 ng/g and 29.8 ng/g (P = 0.067), respectively. Multiple regression analyses of the AD and control combined populations indicated that 1) muscle Abeta40 and muscle Abeta42 levels were correlated with each other (P < 0.001), 2) muscle Abeta40 levels were positively correlated with age (P = 0. 036), and 3) muscle Abeta42 levels were positively correlated with Braak stage (P = 0.042). Other forms of the Abeta peptide were discovered by mass spectrometry, revealing the presence of Abeta starting at residues 1, 6, 7, 9, 10, and 11 and ending at residues 40, 42, 44, 45, and 46. It is possible that in AD the skeletal muscle may contribute to the elevated plasma pool of Abeta and thus indirectly to the amyloid deposits of the brain parenchyma and cerebral blood vessels. The increased levels of Abeta in the temporalis muscles of AD patients suggest that alterations in AbetaPP and Abeta metabolism may be manifested in peripheral tissues.

Improving the sensitivity of the end-cap reflectron time-of-flight mass spectrometer.

A miniaturized time-of-flight (TOF) mass spectrometer utilizes an end-cap reflectron to achieve high kinetic energy focusing and improved mass resolution. However, the coaxial geometry gives rise to considerable losses in sensitivity resulting from reflected ion trajectories close to the center. These trajectories were modeled, using initial ion velocity distributions in the radial direction up to 300 m s(-1), and the portion of the active area of the detector that is utilized was evaluated experimentally using a variable diameter iris diaphragm. The sensitivity was improved by modification of the reflectron by tilting the end-cap electrode 4 degrees and redirecting the ions to a portion of the detector active area. Sensitivity was then measured as 3 fmol of the peptide substance P.

Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens.

The development of many autoimmune diseases has been etiologically linked to exposure to infectious agents. For example, a subset of patients with a history of Salmonella infection develop reactive arthritis. The persistence of bacterial antigen in arthritic tissue and the isolation of Salmonella or Yersinia reactive CD8+ T cells from the joints of patients with reactive arthritis support the etiological link between Gram-negative bacterial infection and autoimmune disease. Models proposed to account for the link between infection and autoimmunity include inflammation-induced presentation of cryptic self-epitopes, antigen persistence and molecular mimicry. Several studies support molecular mimicry as a mechanism for the involvement of class II epitopes in infectious disease-induced self-reactivity. Here, we have identified an immunodominant epitope derived from the S. typhimurium GroEL molecule. This epitope is presented by the mouse H2-T23-encoded class Ib molecule Qa-1 and was recognized by CD8+ cytotoxic T lymphocytes induced after natural infection. S. typhimurium-stimulated cytotoxic T lymphocytes recognizing the GroEL epitope cross-reacted with a peptide derived from mouse heat shock protein 60 and recognized stressed macrophages. Our results indicate involvement of MHC class Ib molecules in infection-induced autoimmune recognition and indicate a mechanism for the etiological link between Gram-negative bacterial infection and autoimmunity.

Phosphorylation of simian cytomegalovirus assembly protein precursor (pAPNG.5) and proteinase precursor (pAPNG1): multiple attachment sites identified, including two adjacent serines in a casein kinase II consensus sequence.

The assembly protein precursor (pAP) of cytomegalovirus (CMV), and its homologs in other herpesviruses, functions at several key steps during the process of capsid formation. This protein, and the genetically related maturational proteinase, is distinguished from the other capsid proteins by posttranslational modifications, including phosphorylation. The objective of this study was to identify sites at which pAP is phosphorylated so that the functional significance of this modification and the enzyme(s) responsible for it can be determined. In the work reported here, we used peptide mapping, mass spectrometry, and site-directed mutagenesis to identify two sets of pAP phosphorylation sites. One is a casein kinase II (CKII) consensus sequence that contains two adjacent serines, both of which are phosphorylated. The other site(s) is in a different domain of the protein, is phosphorylated less frequently than the CKII site, does not require preceding CKII-site phosphorylation, and causes an electrophoretic mobility shift when phosphorylated. Transfection/expression assays for proteolytic activity showed no gross effect of CKII-site phosphorylation on the enzymatic activity of the proteinase or on the substrate behavior of pAP. Evidence is presented that both the CKII sites and the secondary sites are phosphorylated in virus-infected cells and plasmid-transfected cells, indicating that these modifications can be made by a cellular enzyme(s). Apparent compartmental differences in phosphorylation of the CKII-site (cytoplasmic) and secondary-site (nuclear) serines suggest the involvement of more that one enzyme in these modifications.

High levels of circulating Abeta42 are sequestered by plasma proteins in Alzheimer's disease.

A previously unrecognized large pool of Abeta was discovered in freshly drawn plasma of patients diagnosed with Alzheimer's disease (AD) and non-demented control subjects. This Abeta pool was revealed after acid denaturation and chromatographic separation of plasma proteins followed by Abeta quantitation in the 4.5 kDa fractions by europium immunoassay. The mean values of Abeta42 in the AD and control individuals amounted to 236 ng/ml and 38 ng/ml, respectively. These Abeta values are on the average far higher than previously measured. Surprisingly, the circulating Abeta42 is about 16 times more abundant than Abeta40 in the AD population. Addition of Abeta to freshly drawn plasma demonstrated the rapid disappearance of Abeta epitopes, as detected by immunochemical techniques, suggesting either proteolytic degradation or Abeta sequestration. Incubation of Abeta with purified plasma proteins and lipoproteins rapidly decreases detectable levels of free Abeta suggesting epitope masking as the likely mechanism. The free and protein-bound Abetab in the circulation may represent a potential source for deposition in the cerebrovasculature and brain parenchyma of AD.

Detection of non-covalent interaction of single and double stranded DNA with peptides by MALDI-TOF.

DNA-histone interaction facilitates packaging of huge amounts of DNA in the confined space of the nucleus. The importance of this interaction underscores the need for new analytical techniques to acquire a better understanding of nuclear dynamics. Electrospray-ionization mass spectrometry made it possible to investigate non-covalently-bound biopolymers. We are enlarging the scope of available analytical tools by studying non-covalent interaction between single and double stranded DNA and peptides with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The interaction is an ionic one, between the negatively charged sugar-phosphate backbone of single stranded DNA and positively charged side chains of Arg- and Lys-rich peptides as demonstrated by Vertes' group with the dipeptides Arg-Lys and His-His. We replicated Lecchi and Pannell's work, which showed that double stranded DNA could be seen by MALDI using 6-aza-2-thiothymine (ATT) as matrix. We tried various peptides and found that as was demonstrated in DNA-histone interaction, a certain ratio and arrangement of basic residues was needed in order to generate ionic binding between DNA and peptide. We tested various single and double stranded DNA with the peptide of choice, and found that other variables such as pH value of solution, ionic strength, and matrix system did play a role.

Natural ligand of mouse CD1d1: cellular glycosylphosphatidylinositol.

Mouse CD1d1, a member of the CD1 family of evolutionarily conserved major histocompatibility antigen-like molecules, controls the differentiation and function of a T lymphocyte subset, NK1+ natural T cells, proposed to regulate immune responses. The CD1d1 crystal structure revealed a large hydrophobic binding site occupied by a ligand of unknown chemical nature. Mass spectrometry and metabolic radiolabeling were used to identify cellular glycosylphosphatidylinositol as a major natural ligand of CD1d1. CD1d1 bound glycosylphosphatidylinositol through its phosphatidylinositol aspect with high affinity. Glycosylphosphatidylinositol or another glycolipid could be a candidate natural ligand for CD1d1-restricted T cells.