The multifunctionality of regulatory peptides and the rule of "what--where--when" as a principle of their ordered action]

Consistent study of stages and history of discovery of regulatory peptides (RP) as a new class of multifunctional endogenic regulators puts forward some actual problems. A question what new notions on the manner of control of physiological functions have been introduced by RP investigation is considered. Two historical tendencies in the RP studies are estimated. A conception on peptides as multifunctional and colocated factors of neurohumoral regulation of organism was opposed to the idea of "single" substance--a regulator of "unique" function. In this connection a conception of peptide-regulatory continuum is considered and the author postulated the principle "what--where--when" as the main rule of RP well-regulated action. The statement on tissue (regional) specificity of the biogenesis processes of different RP as molecular basis of their regulatory function is grounded.     

Development and application of an expert system for analysis of the functional continuum of regulatory peptides

An expert computer system was developed to analyze the effects of regulatory peptides (RPs) and some other biologically active compounds. The database includes information on their structures and physiological and inductive effects taking into account the method of administration, doses, and types of organisms. The developed method of vector representation of RP effects and the software modules provide for the evaluation of the role of single RPs and their combinations in the regulatory system of organism. The expert system was used for solving the following tasks: (1) an analysis of the structural and functional organization of the RP continuum and a search for effective RP combinations regulating the levels of anxiety, depression, cognitive processes, etc; (2) design of a network of complicated cross inductive connections of major members of RP families; and (3) an analysis of the peculiar features of functioning of numerous internal reward factors.     

Development and application of an expert system for analysis of the functional continuum of regulatory peptides

An expert computer system was developed to analyze the effects of regulatory peptides (RPs) and some other biologically active compounds. The database includes information on their structures and physiological and inductive effects taking in account the method of administration, doses, and types of organisms. The developed method of vector representation of RP effects and the software modules provide for the estimation of the role of single RPs and their combinations in the regulatory system of organism. The expert system was used for solving the following tasks: (1) an analysis of the structural and functional organization of the RP continuum and a search for effective RP combinations regulating the levels of anxiety, depression, cognitive processes, etc; (2) design of a network of complicated cross inductive connections of major members of RP families; and (3) an analysis of the peculiar features of functioning of numerous internal reward factors. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.     

High pH reversed‐phase chromatography as a superior fractionation scheme compared to off‐gel isoelectric focusing for complex proteome analysis

MS/MS is the technology of choice for analyzing complex protein mixtures. However, due to the intrinsic complexity and dynamic range present in higher eukaryotic proteomes, prefractionation is an important step to maximize the number of proteins identified. Off‐gel IEF (OG‐IEF) and high pH RP (Hp‐RP) column chromatography have both been successfully utilized as a first‐dimension peptide separation technique in shotgun proteomic experiments. Here, a direct comparison of the two methodologies was performed on ex vivo peripheral blood mononuclear cell lysate. In 12‐fraction replicate analysis, Hp‐RP resulted in more peptides and proteins identified than OG‐IEF fractionation. Distributions of peptide pIs and hydropathy did not reveal any appreciable bias in either technique. Resolution, defined here as the ability to limit a specific peptide to one particular fraction, was significantly better for Hp‐RP. This leads to a more uniform distribution of total and unique peptides for Hp‐RP across all fractions collected. These results suggest that fractionation by Hp‐RP over OG‐IEF is the better choice for typical complex proteome analysis.     

Search for evolutionary ancient, relict, regulatory peptides

Among numerous regulatory peptides (RP) it is possible to presumably indicate the relict, evolutionary ancient RP. They combine three features: formation from non-specialized peptides-precursors, a comparatively high resistance to action of proteases in the organism media, and maximal simplicity of their structure. The examples of them are glyprolines—a recently identified RP family, as well as tuftsin. Several other praline-containing RP in terminal sites also seem to belong to the evolutionary ancient RP. The proposed approach to studies on the RP evolution is additional to those used traditionally in this problem.     

A way of functional classification of regulatory peptides. Parameters of divergent and convergent evolution of regulatory peptides

A mathematical method has been presented for systematization of functions of regulatory peptides (RP) and evaluation of directions of evolutionary development of RP systems. For this purpose, traditional methods of vector algebra and multi-dimensional space were used. Effects of various peptide regulators on anxiety, depression, and memory are considered by the example of the three-dimensional space. A way of the functional classification of peptides has been proposed.     

Functional continuum of the regulatory peptides enhancing anxiety. Search for the optimal complexes as a basis for developing inhibitory therapeutic agents

Regulatory peptides (RP) take an active part in managing the majority of physiological processes. One of these functional applications is the monitoring of the anxiety level, of panic state. This work represents the presumptive analysis of literature data of 1960-2004 on the effects of regulatory peptides enhancing anxiety (RP-AT). This information database was used for researching the characteristics of organization and functionality of the system of anxiogenic RP. Taking into account the method of vector representation of RP effects, estimation of spectra of physiological effects that can go with each of RP-AT and their combination, was carried out. The most perspective RP complexes of anxiogenic profile for further experimental development of inhibitory therapeutic agents are proposed.     

The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release.

Substrates enter the proteasome core particle (CP) through a channel that opens upon association with the regulatory particle (RP). Using yeast mutants, we show that channel opening is mediated by the ATPase domain of Rpt2, one of six ATPases in the RP. To test whether degradation products exit through this channel, we analyzed their size distribution. Their median length from an open-channel CP mutant was 40% greater than that from the wild-type. Thus, channel opening may enhance the yield of peptides long enough to function in antigen presentation. These experiments demonstrate that gating of the RP channel controls both substrate entry and product release, and is specifically regulated by an ATPase in the base of the RP.     

Anticipated and unexpected physiological effects of oligopeptides (glyprolines, ACTH (4-10) analogs, taftsin, and thyroliberin)

A short review of investigations resulting in determination of a new family of regulatory peptides (glyprolines) participating in modulation of hemostasis and protection of mucous membranes, obtaining of the ACTH4-10 new analogues which are potent neuroprotectors and nootrops, and revealing of unexpected biological activities of some other peptides (behavioural effects of tuftsin, lymph flow stimulation by thyroliberin, etc.). Further investigations into the functional peptide continuum is discussed.     

Solution conformational preferences of immunogenic peptides derived from the principal neutralizing determinant of the HIV-1 envelope glycoprotein gp120

With standard one- and two-dimensional proton NMR techniques, a common structural motif has been identified in water solutions of short peptide sequences derived from the envelope glycoprotein gp120 of HIV-1. Three peptides of lengths 12, 24, and 40 residues (termed RP342, RP142, and RP70, respectively) were synthesized, each containing a central amino acid sequence common to many HIV-1 isolates. In addition, RP70 contained a disulfide bond between cysteine residues close to the ends of the molecule, forming a loop that is thought to constitute an important structural and immunological component of the intact glycoprotein. Peptides RP70 and RP142 showed evidence for the presence of a significant population of conformations containing a beta-turn in the conserved sequence Gly-Pro-Gly-Arg. Strong nuclear Overhauser effect (NOE) connectivities were observed between the amide protons of the arginine and the adjacent glycine. A weak NOE connectivity was observed between the C alpha H of the proline residue and the NH of the Arg [a d alpha N(i,i + 2) NOE connectivity], confirming the presence of a conformational preference for a turn conformation in this sequence. The remainder of the peptide showed evidence of conformational averaging: no NMR evidence for a uniquely folded structure was obtained for any of the peptides in water solution. Circular dichroism (CD) spectra indicated that no ordered helix was present in water solutions of RP70, although a CD spectrum that indicated the presence of approximately 30% helix could be induced by the addition of trifluoroethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional analysis of the proteasome regulatory particle

We have developed S. cerevisiae as a model system for mechanistic studies of the 26S proteasome. The subunits of the yeast 19S complex, or regulatory particle (RP), have been defined, and are closely related to those of mammalian proteasomes. The multiubiquitin chain binding subunit (S5a/Mcb1/Rpn10) was found, surprisingly, to be nonessential for the degradation of a variety of ubiquitin-protein conjugates in vivo. Biochemical studies of proteasomes from rpn10 mutants revealed the existence of two structural subassemblies within the RP, the lid and the base. The lid and the base are both composed of 8 subunits. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The base is sufficient to activate the 20S core particle for degradation of peptides, but the lid is required for ubiquitin-dependent degradation. The lid subunits share sequence motifs with components of the COP9/signalosome complex, suggesting that these functionally diverse particles have a common evolutionary ancestry. Analysis of equivalent point mutations in the six ATPases of the base indicate that they have well-differentiated functions. In particular, mutations in one ATPase gene, RPT2, result in an unexpected defect in peptide hydrolysis by the core particle. One interpretation of this result is that Rpt2 participates in gating of the channel through which substrates enter the core particle.     

Regulation of synaptic vesicles pools within motor nerve terminals during short-term facilitation and neuromodulation.

The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals were physiologically differentiated into distinctly separate functional groups. This was accomplished in glutamatergic nerve terminals by blocking the glutamate transporter with dl-threo-beta-benzyloxyaspartate (TBOA; 10 microM) during electrical stimulation with either 40 Hz of 10 pulses within a train or 20- or 50-Hz continuous stimulation. The 50-Hz continuous stimulation decreased the excitatory postsynaptic potential amplitude 60 min faster than for the 20-Hz continuous stimulation in the presence of TBOA (P < 0.05). There was no significant difference between the train stimulation and 20-Hz continuous stimulation in the run-down time in the presence of TBOA. After TBOA-induced synaptic depression, the excitatory postsynaptic potentials were rapidly (<1 min) revitalized by exposure to serotonin (5-HT, 1 microM) in every preparation tested (P < 0.05). At this glutamatergic nerve terminal, 5-HT promotes an increase probability of vesicular docking and fusion. Quantal recordings made directly at nerve terminals revealed smaller quantal sizes with TBOA exposure with a marked increase in quantal size as well as a continual appearance of smaller quanta upon 5-HT treatment after TBOA-induced depression. Thus 5-HT was able to recruit vesicles from the RP that were not rapidly depleted by acute TBOA treatment and electrical stimulation. The results support the notion that the RRP is selectively activated during rapid electrical stimulation sparing the RP; however, the RP can be recruited by the neuromodulator 5-HT. This suggests at least two separate kinetic and distinct regulatory paths for vesicle recycling within the presynaptic nerve terminal.     

Enhancing Proteomic Throughput in Capillary Electrophoresis–Mass Spectrometry by Sequential Sample Injection

In this study we demonstrate the potential of sequential injection of samples in capillary electrophoresis–mass spectrometry for rapid and sensitive proteome characterization of human lymphoblastic T‐cells (line CCRF–CEM). Proteins were extracted, enzymatically digested, and the resulting peptides fractionated by RP–HPLC. Twenty fractions were thereafter analyzed by CE–MS within a single MS analysis. The CE–MS method was designed so that every 10 min a new fraction was injected into the CE system. Without any rinsing or equilibration steps we were able to generate a continuous stream of peptides feeding the mass analyzer. In 250 min, the total analysis time of a single sequential injection experiment, we were able to identify roughly 28 000 peptide sequences counting for 4800 proteins. These numbers could be increased to 62 000 peptides and more than 6100 proteins identified, when performing three experiments analyzing a total of 60 fractions, all within 12.5 h. We found that the electrophoretic mobility of peptides can be used to trace back peptides and assign them to the fraction they originate from.     

Positive and negative regulations by FGF8 contribute to midbrain roof plate developmental plasticity

The roof plate (RP) of the midbrain shows an unusual plasticity, as it is duplicated or interrupted by experimental manipulations involving the mid/hindbrain organizer or FGF8. In previous experiments, we have found that FGF8 induces a local patterning center, the isthmic node, that is essential for the local development of a RP. Here, we show that the plasticity of the midbrain RP derives from two apparently antagonistic influences of FGF8. On the one hand, FGF8 widens beyond the neural folds the competence of the neuroepithelium to develop a RP by inducing the expression of LMX1B and WNT1. Ectopic overexpression of these two factors is sufficient to induce widely the expression of markers of the mature RP in the midbrain. On the other hand, FGF8 exerts a major destabilizing influence on RP maturation by controlling signaling by members of the TGFbeta superfamily belonging to the BMP, GDF and activin subgroups. We show in particular that FGF8 tightly modulates follistatin expression, thus progressively restraining the inhibitory influence of activin B on RP differentiation. These regulations, together with FGF8 triggered apoptosis, allow the formation of a RP progress zone at some distance from the FGF8 source. Posterior elongation of the RP is permitted when the source of FGF8 withdraws. Growth of the posterior midbrain neuroepithelium and convergent extension movements induced by FGF8 both contribute to increase the distance between the source of FGF8 and the maturing RP. Normally, the antagonistic regulatory interactions spread smoothly across the midbrain. Plasticity of midbrain RP differentiation probably results from an experimentally induced imbalance between regulatory pathways.     

Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses

Identifying deamidated peptides using low-resolution mass spectrometry is difficult because traditional database search programs cannot accurately detect modified peptides when the mass differences are only 0.984 Da. In this study, we utilized differential reversed-phase elution behavior of deamidated and corresponding unmodified peptide forms to significantly improve deamidation detection on a low-resolution LCQ ion trap instrument. We also improved the mass measurements of unmodified and deamidated peptide forms by averaging survey scans across each chromatogram peak. Tryptic digests of a series of normal (3-day old, 2-year old, 18-year old, 35-year old, and 70-year old) and cataractous (93-year old) human lens samples were used to produce large numbers of potentially deamidated peptides. The complex peptide mixtures were separated by strong cation exchange (SCX) chromatography followed by reversed-phase (RP) chromatography. Synthetic peptides were used to show that unmodified and deamidated peptides coeluted during the SCX separation and were completely resolved with the RP conditions used. Retention time shifts (RTS) and mass differences (DeltaM) of deamidated lens peptides and their corresponding unmodified forms were manually determined for the 70-year old lens sample. These values were used to assign correct or incorrect deamidation identifications from SEQUEST searches where deamidation was specified as a variable modification. Manual validation of SEQUEST identifications from synthetic peptides, 3-day old, and 70-year old samples had an overall 42% deamidation detection accuracy. Filtering SEQUEST identifications using RTS and DeltaM constraints resulted in >93% deamidation detection accuracy. An algorithm was developed to automate this method, and 72 Crystallin deamidation sites, 18 of which were not previously reported in human lens tissue, were detected.     

Graphite powder as an alternative or supplement to reversed-phase material for desalting and concentration of peptide mixtures prior to matrix-assisted laser desorption/ionization-mass spectrometry

The success attributed to identification and characterization of gel separated proteins by mass spectrometry (MS) is highly dependent on the percentage of an entire sequence covered by matching peptides derived from enzymatic digestion. Desalting and concentration of peptide mixtures on reversed-phase (RP) microcolumns prior to mass spectrometric analysis have resulted in increased signal-to-noise ratio and sensitivity, and consequently higher sequence coverage. A large proportion of peptides, however, remains undetected by MS presumably because they are lost during sample preparation on microcolumns, or are suppressed in the ionization process. We report here the use of graphite powder packed in constricted GELoader tips as an alternative to RP microcolumns for desalting and concentration of peptide mixtures prior to MS. Such columns are able to retain small and/or hydrophilic peptides that can be lost when using RP microcolumns. In addition, we show that samples contaminated with small biological polymers can readily be analyzed using graphite powder rather than RP microcolumns, since the polymer molecules bind strongly to graphite and are not eluted with the peptides.     

Rapid prototyping as a tool for manufacturing bioartificial livers

Rapid prototyping (RP) technologies are a set of manufacturing processes that can produce very complex structures directly from computer-aided design models without structure-specific tools or knowledge. These technologies might eventually enable the manufacture of human livers to create functional substitutes for treating liver failure or dysfunctionality. However, the approaches used currently face many challenges, such as the complex branched vascular and bile ductular systems and the variety of cell types, matrices and regulatory factors involved in liver development. Here, we discuss the challenges and provide evidence for the usefulness of RP in overcoming them.     

Immunoreactivity of human tissue mast cells: nonspecific binding of primary antibodies against regulatory peptides by ionic linkage

Tissue mast cells (TMC) are known to react with antibodies against various regulatory peptides (RP). The specificity of such reactions was investigated by various methods in this study. When normal immunohistochemical staining procedures were employed. TMC in the vermiform appendix and in a cutaneous mastocytoma reacted with antibodies against ACTH, Leu-enkephalin, Met-enkephalin, and peptide histidine isoleucine (PHI). Antibody specificity was tested by absorption controls, and staining specificity by varying the concentration of the primary antibodies and the pH and sodium chloride concentration of the buffer used for rinsing and diluting. In absorption controls, staining of the TMC by anti-PHI was diminished but staining by anti-ACTH, anti-Leu-enkephalin, and anti-Met-enkephalin remained unchanged. Unlike control reactions, immunostaining of TMC with antibodies against RP exhibited marked dependence on antibody concentration and the pH and sodium chloride concentration of the buffer. Alkalization of the buffer led to an obvious increase in the reaction with antibodies against RP, and lowering the pH to 6.0 usually resulted in abolition of the reaction. These results indicate that the immunostaining of TMC with antibodies against RP, including PHI, was nonspecific. It is postulated that the granules of TMC bind certain antibodies by a cation-exchange mechanism involving ionic interactions with positively charged groups in the F(ab')2 and/or Fc segments.