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Biomedical subjects

A Valencia

Publications and source records attributed to A Valencia.

At least 109 records · Page 6Linked to original sources
PubMed1994

Correlated mutations and residue contacts in proteins.

The maintenance of protein function and structure constrains the evolution of amino acid sequences. This fact can be exploited to interpret correlated mutations observed in a sequence family as an indication of probable physical contact in three dimensions. Here we present a simple and general method to analyze correlations in mutational behavior between different positions in a multiple sequence alignment. We then use these correlations to predict contact maps for each of 11 protein families and compare the result with the contacts determined by crystallography. For the most strongly correlated residue pairs predicted to be in contact, the prediction accuracy ranges from 37 to 68% and the improvement ratio relative to a random prediction from 1.4 to 5.1. Predicted contact maps can be used as input for the calculation of protein tertiary structure, either from sequence information alone or in combination with experimental information.

Amino Acid Sequence↗
PubMed1994

A conserved loop in the ATPase domain of the DnaK chaperone is essential for stable binding of GrpE.

The activity of DnaK (Hsp70) chaperones in assisting protein folding relies on DnaK binding and ATP-controlled release of protein substrates. The ATPase activity of DnaK is tightly controlled by the nucleotide exchange factor GrpE. We find that GrpE interacts stably with the amino-terminal ATPase domain of DnaK. Analysis of the mutant DnaK756 protein, which has a lower affinity for GrpE, reveals a role for residue Gly 32 in GrpE binding. Gly 32 is located in an exposed loop near the nucleotide binding site of DnaK. Deletion of this loop prevents stable GrpE binding, ATPase stimulation by GrpE, and DnaK chaperone activity. Conservation of this loop within the Hsp70 family suggests that cooperation between Hsp70 and GrpE-like proteins may be a general feature of this class of chaperone.

Adenosine Triphosphatases↗
PubMed1994

Cloning and subcellular localization of novel rab proteins reveals polarized and cell type-specific expression.

Small GTPases of the rab subfamily are specific regulators of vesicular transport. The intracellular localization of these proteins has been mostly investigated in cultured cells where they have been found associated with distinct compartments of the exocytic and endocytic pathways. Using a PCR-based cloning approach we have recently identified several novel rab proteins, extending the total number of this family to more than 30 members. Here, we have investigated the mRNA expression in different tissues and the intracellular localization in organ cryosections of two rab proteins, rab18 and rab20. Both northern blot analysis and confocal immunofluorescence microscopy demonstrated that these proteins are expressed in a tissue- and cell type-dependent manner. Despite their presence in non-polarized cells and polarized cells, both proteins are highly expressed on the apical side of kidney tubule epithelial cells. Electron microscopic studies revealed that rab18 and rab20 are located in apical dense tubules, endocytic structures underlying the apical plasma membrane, suggesting that they play a role in apical endocytosis/recycling. In intestinal epithelial cells as well, both proteins were localized apically, but, in addition, rab18 was found associated with the basolateral domain, suggesting that this protein is not restricted to the apical transport machinery of polarized epithelial cells. The results demonstrate that, depending on the epithelial cell type, rab proteins that are also expressed in non-polarized cells may be enriched in one or both surface domains. Together with the observed tissue- and cell type-dependent variation in the expression of the rab proteins, this suggests that the large number of mammalian rab proteins might reflect the specific requirements in the organization of membrane traffic encountered by different cell types.

Amino Acid Sequence↗
PubMed1994

GeneQuiz: a workbench for sequence analysis.

We present the prototype of a software system, called GeneQuiz, for large-scale biological sequence analysis. The system was designed to meet the needs that arise in computational sequence analysis and our past experience with the analysis of 171 protein sequences of yeast chromosome III. We explain the cognitive challenges associated with this particular research activity and present our model of the sequence analysis process. The prototype system consists of two parts: (i) the database update and search system (driven by perl programs and rdb, a simple relational database engine also written in perl) and (ii) the visualization and browsing system (developed under C++/ET++). The principal design requirement for the first part was the complete automation of all repetitive actions: database updates, efficient sequence similarity searches and sampling of results in a uniform fashion. The user is then presented with "hit-lists" that summarize the results from heterogeneous database searches. The expert's primary task now simply becomes the further analysis of the candidate entries, where the problem is to extract adequate information about functional characteristics of the query protein rapidly. This second task is tremendously accelerated by a simple combination of the heterogeneous output into uniform relational tables and the provision of browsing mechanisms that give access to database records, sequence entries and alignment views. Indexing of molecular sequence databases provides fast retrieval of individual entries with the use of unique identifiers as well as browsing through databases using pre-existing cross-references. The presentation here covers an overview of the architecture of the system prototype and our experiences on its applicability in sequence analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Effects of estradiol on the development of sexual dimorphism in the bed nucleus of the accessory olfactory tract in the rat.

Orchidectomized males injected with a single dose of estradiol benzoate (EB) on the day of birth (D1) showed a volume and neuron number in the nucleus of the accessory olfactory tract (BAOT) similar to that of control males. However, orchidectomized males and those orchidectomized and given a single dose of DHT on D1 showed a decrease in BAOT volume and neuron number with respect to control males. These results support the notion that estradiol induces the morphological masculinization of this structure. The inability of DHT in counteracting the effect of orchidectomy is addressed taking into account the inhibitory action of androgens.

Animals↗
PubMed1993

How does the switch II region of G-domains work?

The transition of guanine nucleotide binding proteins between the 'on' (GTP-bound) and 'off' (GDP-bound) states has become a paradigm of molecular switching after a chemical reaction. The mechanism by which the switch signal is transmitted to the downstream recipients in the intracellular signal pathway has been extensively studied by biochemical, biophysical and genetic methods, but a clear picture of this process has yet to emerge. Based on the similarities of ras-p21 and elongation factor Tu we propose here a model of the GDP state of ras-p21 that is in agreement with all relevant experimental evidence. The model provides important clues about: (1) a possible molecular mechanism for signal transmission from the site of GTP hydrolysis to downstream effectors; (2) a major conformational change during signal generation and a key residue involved in this process (Tyr-64); and (3) regions in ras-p21 that can be differentially recognized by binding to external partners in a GTP/GDP state dependent fashion, most notably residues D69, Q70, R73, T74, R102, K104, D105 at the end of the alpha-helices 2 and 3.

Amino Acid Sequence↗
PubMed1993

Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases.

Kinases that catalyze phosphorylation of sugars, called here sugar kinases, can be divided into at least three distinct nonhomologous families. The first is the hexokinase family, which contains many prokaryotic and eukaryotic sugar kinases with diverse specificities, including a new member, rhamnokinase from Salmonella typhimurium. The three-dimensional structure of hexokinase is known and can be used to build models of functionally important regions of other kinases in this family. The second is the ribokinase family, of unknown three-dimensional structure, and comprises pro- and eukaryotic ribokinases, bacterial fructokinases, the minor 6-phosphofructokinase 2 from Escherichia coli, 6-phosphotagatokinase, 1-phosphofructokinase, and, possibly, inosine-guanosine kinase. The third family, also of unknown three-dimensional structure, contains several bacterial and yeast galactokinases and eukaryotic mevalonate and phosphomevalonate kinases and may have a substrate binding region in common with homoserine kinases. Each of the three families of sugar kinases appears to have a distinct three-dimensional fold, since conserved sequence patterns are strikingly different for the three families. Yet each catalyzes chemically equivalent reactions on similar or identical substrates. The enzymatic function of sugar phosphorylation appears to have evolved independently on the three distinct structural frameworks, by convergent evolution. In addition, evolutionary trees reveal that (1) fructokinase specificity has evolved independently in both the hexokinase and ribokinase families and (2) glucose specificity has evolved independently in different branches of the hexokinase family. These are examples of independent Darwinian adaptation of a structure to the same substrate at different evolutionary times. The flexible combination of active sites and three-dimensional folds observed in nature can be exploited by protein engineers in designing and optimizing enzymatic function.

Amino Acid Sequence↗
PubMed1993

A new ATP-binding fold in actin, hexokinase and Hsc70.

One of a cell biologist's favourite occupations is to discover the proteins that perform newly described functions in the cell. Very often lately, this has resulted in the identification of protein families whose related amino acid sequences reflect similar functions, but can proteins with totally unrelated sequences have similar structures and functions? In this review, Ken Holmes, Chris Sander and Alfonso Valencia describe the structural similarities between three well-known proteins that have no readily detectable primary sequence similarities but for which X-ray crystallography has revealed very similar structures. A comparison of their structures provides insights into their common mechanisms of action and into protein evolution, and has been used to detect related proteins in sequence data bases.

Journal Article↗
PubMed1993

Rab17, a novel small GTPase, is specific for epithelial cells and is induced during cell polarization.

The rab subfamily of small GTPases has been demonstrated to play an important role in the regulation of membrane traffic in eukaryotic cells. Compared with nonpolarized cells, epithelial cells have distinct apical and basolateral transport pathways which need to be separately regulated. This raises the question whether epithelial cells require specific rab proteins. However, all rab proteins identified so far were found to be equally expressed in polarized and nonpolarized cells. Here we report the identification of rab17, the first epithelial cell-specific small GTPase. Northern blot analysis on various mouse organs, revealed that the rab17 mRNA is present in kidney, liver, and intestine but not in organs lacking epithelial cells nor in fibroblasts. To determine whether rab17 is specific for epithelial cells we studied its expression in the developing kidney. We found that rab17 is absent from the mesenchymal precursors but is induced upon their differentiation into epithelial cells. In situ hybridization studies on the embryonic kidney and intestine revealed that rab17 is restricted to epithelial cells. By immunofluorescence and immunoelectron microscopy on kidney sections, rab17 was localized to the basolateral plasma membrane and to apical tubules. Rab proteins associated with two distinct compartments have been found to regulate transport between them. Therefore, our data suggest that rab17 might be involved in transcellular transport.

Amino Acid Sequence↗
PubMed1992

An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins.

The functionally diverse actin, hexokinase, and hsp70 protein families have in common an ATPase domain of known three-dimensional structure. Optimal superposition of the three structures and alignment of many sequences in each of the three families has revealed a set of common conserved residues, distributed in five sequence motifs, which are involved in ATP binding and in a putative interdomain hinge. From the multiple sequence alignment in these motifs a pattern of amino acid properties required at each position is defined. The discriminatory power of the pattern is in part due to the use of several known three-dimensional structures and many sequences and in part to the "property" method of generalizing from observed amino acid frequencies to amino acid fitness at each sequence position. A sequence data base search with the pattern significantly matches sugar kinases, such as fuco-, glucono-, xylulo-, ribulo-, and glycerokinase, as well as the prokaryotic cell cycle proteins MreB, FtsA, and StbA. These are predicted to have subdomains with the same tertiary structure as the ATPase subdomains Ia and IIa of hexokinase, actin, and Hsc70, a very similar ATP binding pocket, and the capacity for interdomain hinge motion accompanying functional state changes. A common evolutionary origin for all of the proteins in this class is proposed.

Actins↗
PubMed1992

Postnatal administration of dihydrotestosterone to the male rat abolishes sexual dimorphism in the accessory olfactory bulb: a volumetric study.

The regulatory action of the non-aromatizable androgen dihydrotestosterone (DHT) on sexual differentiation of the volume of the rat accessory olfactory bulb (AOB) was studied. Postnatal treatment with DHT (180 micrograms/day) carried out daily between days 6 and 20 produced a drastic reduction in overall AOB size and that of its constituent neural layers in genetic males with respect to intact and control males. The volumetric measures found in DHT-treated males did not differ from those shown by the intact females. These results, which indicate a demasculinization and a feminization of the AOB volume in gonadally intact male rats induced by DHT, are discussed in relation to the presumably regulatory role of DHT on neuron populations during the sexual organizational process of the brain.

Animals↗
PubMed1992

Female's DHT controls sex differences in the rat bed nucleus of the accessory olfactory tract.

In the present study the regulatory action of the non-aromatic androgen dihydrotestoterone (DHT) on the volume of the sexually dimorphic bed nucleus of the accessory olfactory tract (BAOT) was investigated. Postnatal treatment with DHT (180 micrograms day-1) between days 6 and 20 (D6-D20) induced, in gonadally intact male rats, a drastic reduction in the overall volume to levels typical in control females. Conversely, the postnatal administration of the anti-androgen cyproterone acetate (CA) to the females from D6-D20 produced an increment in the BAOT volume not dissimilar to that found in control males. These findings reveal that sexual organization in this vomeronasal structure is dependent on the presence of DHT in females during postnatal development.

Androgen Antagonists↗
PubMed1991

GTPase domains of ras p21 oncogene protein and elongation factor Tu: analysis of three-dimensional structures, sequence families, and functional sites.

GTPase domains are functional and structural units employed as molecular switches in a variety of important cellular functions, such as growth control, protein biosynthesis, and membrane traffic. Amino acid sequences of more than 100 members of different subfamilies are known, but crystal structures of only mammalian ras p21 and bacterial elongation factor Tu have been determined. After optimal superposition of these remarkably similar structures, careful multiple sequence alignment, and calculation of residue-residue interactions, we analyzed the two subfamilies in terms of structural conservation, sequence conservation, and residue contact strength. There are three main results. (i) A structure-based alignment of p21 and elongation factor Tu. (ii) The definition of a common conserved structural core that may be useful as the basis of model building by homology of the three-dimensional structure of any GTPase domain. (iii) Identification of sequence regions, other than the effector loop and the nucleotide binding site, that may be involved in the functional cycle: they are loop L4, known to change conformation after GTP hydrolysis; helix alpha 2, especially Arg-73 and Met-67 in ras p21; loops L8 and L10, including ras p21 Arg-123, Lys-147, and Leu-120; and residues located spatially near the N and C termini. These regions are candidate sites for interaction either with the GTP/GDP exchange factor, with a GTPase-affected function, or with a molecule delivered to a destination site with the aid of the GTPase domain.

Amino Acid Sequence↗
PubMed1991

Early postnatal diazepam exposure alters sex differences in the rat brain.

The volume and neuron number of the sexually dimorphic accessory olfactory bulb and locus coeruleus are altered by early postnatal exposure (from the day of birth to postnatal day 16) to diazepam. After diazepam treatment, both volume and neuron number were decreased in the male accessory olfactory bulb and in the female locus coeruleus. These results indicate that early postnatal diazepam administration can bear gender-dependent teratogenic effects upon sexually dimorphic nuclei and suggest that endogenous benzodiazepines may be involved in the sexual differentiation of the brain.

Aging↗
PubMed1990

Sexual dimorphism in the bed nucleus of the accessory olfactory tract in the rat.

This work investigates the existence of sex differences in the volume and number of neurons and glial cells in the bed nucleus of the accessory olfactory tract (BAOT). Males showed larger volume and number of cells than female rats. Early postnatal (day 1 after birth) orchidectomy in males, and androgenization in females, reversed these differences. No sex differences were found in BAOT glial cells. The sexual dimorphism found in the neuron/glial cell ratio reflects sex differences in neuron number. The existence of sexual dimorphism in the BAOT supports our earlier hypothesis which states that the vomeronasal system (VNS) is sexually dimorphic.

Animals↗
PubMed1990

Uterine estrogen sulfatase activity. Influence of steroid hormones and adenine nucleotides.

Steroid sulfatase enzymes participate greatly in reproductive events. To date, estrogen sulfatase seems to have a regulatory role in the control of free estrogen levels in target tissues. The present study evaluates the participation of some adenine nucleotides in estrogen sulfatase kinetics. Using ADP, ATP, NAD and the combination of ADP + NAD or ATP + NAD it was found that adding either of the combined cofactors, the enzymatic activity increased more than 2.0 times. In ovariectomized rats, the corresponding mean enzyme activity was found to be higher than in intact rats. It was also found, in ovariectomized rats treated with ovarian hormones, an inhibition that was higher with estradiol-17 beta than with progesterone treatment. This data suggests that the estrogen sulfatase, being a hormone-dependent enzyme, participates in a new control mechanism of estrogen levels in presence of some cofactors and free steroids.

Adenine Nucleotides↗