Nature of protein family signatures: insights from singular value analysis of position-specific scoring matrices.

Position-specific scoring matrices (PSSMs) are useful for detecting weak homology in protein sequence analysis, and they are thought to contain some essential signatures of the protein families. In order to elucidate what kind of ingredients constitute such family-specific signatures, we apply singular value decomposition to a set of PSSMs and examine the properties of dominant right and left singular vectors. The first right singular vectors were correlated with various amino acid indices including relative mutability, amino acid composition in protein interior, hydropathy, or turn propensity, depending on proteins. A significant correlation between the first left singular vector and a measure of site conservation was observed. It is shown that the contribution of the first singular component to the PSSMs act to disfavor potentially but falsely functionally important residues at conserved sites. The second right singular vectors were highly correlated with hydrophobicity scales, and the corresponding left singular vectors with contact numbers of protein structures. It is suggested that sequence alignment with a PSSM is essentially equivalent to threading supplemented with functional information. In addition, singular vectors may be useful for analyzing and annotating the characteristics of conserved sites in protein families.

The human enhancer blocker CTC-binding factor interacts with the transcription factor Kaiso.

CTC-binding factor (CTCF) is a DNA-binding protein of vertebrates that plays essential roles in regulating genome activity through its capacity to act as an enhancer blocker. We performed a yeast two-hybrid screen to identify protein partners of CTCF that could regulate its activity. Using full-length CTCF as bait we recovered Kaiso, a POZ-zinc finger transcription factor, as a specific binding partner. The interaction occurs through a C-terminal region of CTCF and the POZ domain of Kaiso. CTCF and Kaiso are co-expressed in many tissues, and CTCF was specifically co-immunoprecipitated by several Kaiso monoclonal antibodies from nuclear lysates. Kaiso is a bimodal transcription factor that recognizes methylated CpG dinucleotides or a conserved unmethylated sequence (TNGCAGGA, the Kaiso binding site). We identified one consensus unmethylated Kaiso binding site in close proximity to the CTCF binding site in the human 5' beta-globin insulator. We found, in an insulation assay, that the presence of this Kaiso binding site reduced the enhancer-blocking activity of CTCF. These data suggest that the Kaiso-CTCF interaction negatively regulates CTCF insulator activity.

Hypoosmotic cell swelling as a novel mechanism for modulation of cloned HCN2 channels.

This work demonstrates cell swelling as a new regulatory mechanism for the cloned hyperpolarization-activated, cyclic nucleotide-gated channel 2 (HCN2). HCN2 channels were coexpressed with aquaporin1 in Xenopus laevis oocytes and currents were monitored using a two-electrode voltage-clamp. HCN2 channels were activated by hyperpolarization to -100 mV and the currents were measured before and during hypoosmotic cell swelling. Cell swelling increased HCN2 currents by 30% without changing the kinetics of the currents. Injection of 50 nl intracellular solution resulted in a current increase of 20%, indicating that an increase in cell volume also under isoosmotic conditions may lead to activation of HCN2. In the absence of aquaporin1 only negligible changes in oocyte cell volume occur during exposure to hypoosmotic media and no significant change in HCN2 channel activity was observed during perfusion with hypoosmotic media. This indicates that cell swelling and not a change in ionic strength of the media, caused the observed swelling-induced increase in current. The increase in HCN2 current induced by cell swelling could be abolished by cytochalasin D treatment, indicating that an intact F-actin cytoskeleton is a prerequisite for the swelling-induced current.

Oxidized human neuroglobin acts as a heterotrimeric Galpha protein guanine nucleotide dissociation inhibitor.

Neuroglobin (Ngb) is a newly discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. It has been reported that Ngb expression levels increase in response to oxygen deprivation and that it protects neurons from hypoxia in vitro and in vivo. However, the mechanism of this neuroprotection remains unclear. In the present study, we tried to clarify the neuroprotective role of Ngb under oxidative stress in vitro. By surface plasmon resonance, we found that ferric Ngb, which is generated spontaneously as a result of the rapid autoxidation, binds exclusively to the GDP-bound form of the alpha subunit of heterotrimeric G protein (Galphai). In GDP dissociation assays or guanosine 5'-O-(3-thio)triphosphate binding assays, ferric Ngb behaved as a guanine nucleotide dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP. The interaction of GDP-bound Galphai with ferric Ngb will liberate Gbetagamma, leading to protection against neuronal death. In contrast, ferrous ligand-bound Ngb under normoxia did not have GDI activities. Taken together, we propose that human Ngb may be a novel oxidative stress-responsive sensor for signal transduction in the brain.

The synaptic complex of RecA protein participates in hybridization and inverse strand exchange reactions.

RecA protein catalyzes strand exchange between homologous single-stranded and double-stranded DNAs. In the presence of ATPgammaS, the post-strand exchange synaptic complex is a stable end product that can be studied. Here we ask whether such complexes can hybridize to or exchange with DNA, 2'-OMe RNA, PNA, or LNA oligonucleotides. Using a gel mobility shift assay, we show that the displaced strand of a 45 bp synaptic complex can hybridize to complementary oligonucleotides with different backbones to form a four-stranded (double D-loop) joint that survives removal of the RecA protein. This hybridization reaction, which confirms the single-stranded character of the displaced strand in a synaptic complex, might initiate recombination-dependent DNA replication if it occurs in vivo. We also show that either strand of the heteroduplex in a 30 bp synaptic complex can be replaced with a homologous DNA oligonucleotide in a strand exchange reaction that is mediated by the RecA filament. Consistent with the important role that deoxyribose plays in strand exchange, oligonucleotides with non-DNA backbones did not participate in this reaction. The hybridization and strand exchange reactions reported here demonstrate that short synaptic complexes are dynamic structures even in the presence of ATPgammaS.

Inherent instability of plasminogen activator inhibitor type 2 mRNA is regulated by tristetraprolin.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that is subject to regulation at the post-transcriptional level. At least two mRNA instability elements reside within the PAI-2 transcript; one in the coding region and another within the 3'-untranslated region (UTR). For the latter, a functional AU-rich motif (ARE) has been identified that provides a binding site for a number of cellular proteins, including the mRNA stability protein, HuR. In this study, we used the yeast three-hybrid system to screen a human leukocyte cDNA library to identify other proteins that associate with the PAI-2 ARE. This screen identified tristetraprolin (TTP) as a PAI-2 mRNA ARE-binding protein. UV cross-linking and immunoprecipitation experiments showed that TTP expressed in HEK293 cells could associate with the PAI-2 ARE in vitro. Co-transfection of plasmids expressing TTP and PAI-2 in HEK293 cells resulted in an increase in the decay rate of PAI-2 mRNA and loss of PAI-2 protein in a process that was dependent upon the PAI-2 3'-UTR. The 29-nt PAI-2 AU-rich element alone was also capable of conferring TTP-dependent mRNA instability to a reporter transcript. The extent of PAI-2 mRNA stability was remarkably sensitive to TTP since TTP-dependent PAI-2 mRNA decay occurred at TTP levels that were below Western blot detection limits. This study identifies TTP as a functional PAI-2 ARE-binding protein that modulates the post-transcriptional regulation of the PAI-2 gene.

Using CLUSTAL for multiple sequence alignments.

We have tested CLUSTAL W in a wide variety of situations, and it is capable of handling some very difficult protein alignment problems. If the data set consists of enough closely related sequences so that the first alignments are accurate, then CLUSTAL W will usually find an alignment that is very close to ideal. Problems can still occur if the data set includes sequences of greatly different lengths or if some sequences include long regions that are impossible to align with the rest of the data set. Trying to balance the need for long insertions and deletions in some alignments with the need to avoid them in others is still a problem. The default values for our parameters were tested empirically using test cases of sets of globular proteins where some information as to the correct alignment was available. The parameter values may not be very appropriate with nonglobular proteins. We have argued that using one weight matrix and two gap penalties is too simplistic to be of general use in the most difficult cases. We have replaced these parameters with a large number of new parameters designed primarily to help encourage gaps in loop regions. Although these new parameters are largely heuristic in nature, they perform surprisingly well and are simple to implement. The underlying speed of the progressive alignment approach is not adversely affected. The disadvantage is that the parameter space is now huge; the number of possible combinations of parameters is more than can easily be examined by hand. We justify this by asking the user to treat CLUSTAL W as a data exploration tool rather than as a definitive analysis method. It is not sensible to automatically derive multiple alignments and to trust particular algorithms as being capable of always getting the correct answer. One must examine the alignments closely, especially in conjunction with the underlying phylogenetic tree (or estimate of it) and try varying some of the parameters. Outliers (sequences that have no close relatives) should be aligned carefully, as should fragments of sequences. The program will automatically delay the alignment of any sequences that are less than 40% identical to any others until all other sequences are aligned, but this can be set from a menu by the user. It may be useful to build up an alignment of closely related sequences first and to then add in the more distant relatives one at a time or in batches, using the profile alignments and weighting scheme described earlier and perhaps using a variety of parameter settings. We give one example using SH2 domains. SH2 domains are widespread in eukaryotic signalling proteins where they function in the recognition of phosphotyrosine-containing peptides. In the chapter by Bork and Gibson ([11], this volume), Blast and pattern/profile searches were used to extract the set of known SH2 domains and to search for new members. (Profiles used in database searches are conceptually very similar to the profiles used in CLUSTAL W: see the chapters [11] and [13] for profile search methods.) The profile searches detected SH2 domains in the JAK family of protein tyrosine kinases, which were thought not to contain SH2 domains. Although the JAK family SH2 domains are rather divergent, they have the necessary core structural residues as well as the critical positively charged residue that binds phosphotyrosine, leaving no doubt that they are bona fide SH2 domains. The five new JAK family SH2 domains were added sequentially to the existing alignment of 65 SH2 domains using the CLUSTAL W profile alignment option. Figure 6 shows part of the resulting alignment. Despite their divergent sequences, the new SH2 domains have been aligned nearly perfectly with the old set. No insertions were placed in the original SH2 domains. In this example, the profile alignment procedure has produced better results than a one-step full alignment of all 70 SH2 domains, and in considerably less time. (ABSTRACT TRUNCATED)

The structure of deoxy- and oxy-leghaemoglobin from lupin.

The leghaemoglobins have oxygen affinities 11 to 24 times higher than that of sperm whale myoglobin, due mainly to higher rates of association. To find out why, we have determined the structures of deoxy- and oxy-leghaemoglobin II of the lupin at 1.7 A resolution. Results confirm the general features found in previous X-ray analyses of this protein. The unique feature that has now emerged is the rotational freedom of the proximal histidine. In deoxy-leghaemoglobin the imidazole oscillates between two alternative orientations, eclipsing either the lines N1-N3 or N2-N4 of the porphyrin; in oxy-leghaemoglobin it is fixed in a staggered orientation. The iron atom moves from a position 0.30 A from the plane of the pyrrole nitrogen atoms in deoxy- to a position in the plane in oxy-leghaemoglobin while the Fe- bond distance remains constant at 2.02 A. The Fe-O-O angle is 152 degrees, as in human haemoglobin. The oxygen is hydrogen-bonded to the distal histidine at N epsilon 2-O1 and N epsilon 2-O2 distance of 2.95 A and 2.68 A, respectively. The porphyrin is ruffled equally in deoxy- and oxy-leghaemoglobins, due to rotations of the pyrrols about the N-Fe-N bonds, causing the methine bridges to deviate by up to 0.32 A from the mean porphyrin plane. The only feature capable of accounting for the high on-rate of the reaction with oxygen are the mobilities of the proximal histidine and distal histidine residues in deoxy-leghaemoglobin. The eclipsed positions of the proximal histidine in deoxy-leghaemoglobin maximize steric hindrance with the porphyrin nitrogen atoms and minimize pi-->p electron donation, while its staggered position in oxy-leghaemoglobin reverses both these effects. Together with the oscillation of the imidazole between the two orientations, these two factors may reduce the activation energy for the reaction of leghaemoglobin with oxygen. The distal histidine is in a fixed position in the haem pocket in the crystal, but must be swinging in and out of the pocket at a high rate in solution to allow the oxygen to enter.

A procedure for the automatic determination of hydrophobic cores in protein structures.

An algorithm is described for automatically detecting hydrophobic cores in proteins of known structure. Three pieces of information are considered in order to achieve this goal. These are: secondary structure, side-chain accessibility, and side-chain-side-chain contacts. Residues are considered to contribute to a core when they occur in regular secondary structure and have buried side chains that form predominantly nonpolar contacts with one another. This paper describes the algorithm's application to families of proteins with conserved topologies but low sequence similarities. The aim of this investigation is to determine the efficacy of the algorithm as well as to study the extent to which similar cores are identified within a common topology.

Entrapment of purified alpha-hemoglobin chains in normal erythrocytes as a model for human beta thalassemia.

Entrapment of purified alpha-hemoglobin chains within normal erythrocytes resulted in structural and functional changes very similar to those observed in human beta thalassemic erythrocytes (Table 1). Membrane proteins and reactive thiol groups were decreased in a pattern similar to that observed in vivo in beta thalassemia. In addition, the alpha-chain loaded cells exhibited evidence of enhanced oxidant stress. Functionally, entrapment of alpha-chains resulted in the loss of cellular and membrane deformability, an important pathologic characteristic of the beta thalassemic erythrocytes. These results also demonstrate that the loss of membrane proteins and thiols as well as the functional loss of cellular and membrane deformability characteristic of the beta thalassemic cell occur very rapidly in the presence of soluble alpha-chains. Utilizing this model of the thalassemic erythrocyte, it is now possible to directly investigate the mechanisms underlying the cellular pathophysiology induced by excess alpha-chains. An understanding of these mechanisms may allow for the development of therapeutic interventions that would improve effective erythropoiesis and prolong erythrocyte survival in the peripheral circulation of individuals with beta thalassemia. Successful therapeutic interventions would diminish the frequency and/or necessity of blood transfusions and chelation therapy in beta thalassemia.