Modular domain structure in the Candida glabrata adhesin Epa1p,a beta1,6 glucan-cross-linked cell wall protein

The yeast pathogen Candida glabrata adheres avidly to cultured human epithelial cells. This interaction depends on the expression of EPA1, which encodes a lectin belonging to a large family of GPI-anchored glucan-cross-linked cell wall proteins (GPI-CWPs) found in diverse fungal species. To understand the relationship between different domains of EPA1 and its function, we have mapped functional domains of Epa1p and analysed their contribution to Epa1p function. We found that the N-terminal third of the protein contains the ligand-binding domain, and that the GPI anchor is essential both for cross-linking in the cell wall and for Epa1p-mediated adherence. We also found that the C-terminal Ser/Thr-rich domain, characteristic of many GPI-CWPs, was absolutely essential for function. Although Epa1p derivatives lacking the Ser/Thr domain were expressed abundantly in the cell wall, they were localized to internal layers of the cell wall; such constructs were unable to mediate adherence. The outer layer of the yeast cell wall is known to act as a permeability barrier; we found that the C-terminal Ser/Thr-rich region was absolutely required to project the N-terminal domain of Epa1p through this permeability barrier and into the external environment. Thus, the Ser/Thr-rich domain of Epa1p and, presumably, of other related GPI-CWPs serves an essential structural role in localization of the protein at the external surface of the yeast cell where it can interact with its ligand. In conclusion, Epa1p has a modular structure, with each domain serving a distinct and essential role in the function of the adhesin.     

HIV-EP2, a new member of the gene family encoding the human immunodeficiency virus type 1 enhancer-binding protein. Comparison with HIV-EP1/PRDII-BF1/MBP-1

At least two different types of proteins, NF-kappa B/KBF1 and HIV-EP1/PRDII-BF1/MBP-1, which are members of a family of rel oncoproteins and metal-finger proteins, respectively, bind to the human immunodeficiency virus type (HIV-1) enhancer. As a new member of a HIV-EP1 family that is expressed at a high level in T cells, we have isolated cDNA clones of HIV-EP2 by cross-hybridization with HIV-EP1 cDNA. HIV-EP2 protein consists of 1,833 amino acids and has a molecular weight of 211,000. HIV-EP2 protein is highly homologous with HIV-EP1/PRDII-BF1/MBP-1 in three regions. These three regions contain the potential nuclear localization signal followed by a Ser/Thr-rich region, the DNA-binding domain consisting of a metal-finger structure, and a cluster of acidic amino acids. The DNA-binding property of HIV-EP2 was similar to that of HIV-EP1. Northern blot analysis of HIV-EP2 mRNA indicated relatively high expression in the T cell line Molt-4 and in some tumor cell lines. Furthermore, like HIV-EP1, expression of HIV-EP2 mRNA was greatly induced by mitogen and phorbol ester treatment of Jurkat T cells, suggesting that HIV-EP2 acts in HIV production from latently infected T cells.     

Extracellular glycosylphosphatidylinositol-anchored mannoproteins and proteases of Cryptococcus neoformans

Extracellular proteins of Cryptococcus neoformans are involved in the pathogenesis of cryptococcosis, and some are immunoreactive antigens that may potentially serve as candidates for vaccine development. To further study the extracellular proteome of the human fungal pathogen Cry. neoformans, we conducted a proteomic analysis of secreted and cell wall-bound proteins with an acapsular strain of Cry. neoformans. Proteins were identified from both intact cells and cell walls. In both cases, extracellular proteins were removed with trypsin or beta-glucanase, and then all proteins/peptides were purified by solid-phase extraction, spin dialysis, and HPLC, and identified by liquid chromatography-mass spectrometry. This study identified 29 extracellular proteins with a predicted N-terminal signal sequence and also a predicted glycosylphosphatidylinositol anchor motif in more than half. Among the novel proteins identified were five glycosylphosphatidylinositol-anchored proteins with extensive Ser/Thr-rich regions but no apparent functional domains, a glycosylphosphatidylinositol-anchored aspartic protease, and a metalloprotease with structural similarity to an elastinolytic metalloprotease of Aspergillus fumigatus. This study suggests that Cry. neoformans has the machinery required to target glycosylphosphatidylinositol-anchored proteins to the cell wall, and it confirms the extracellular proteolytic ability of Cry. neoformans.     

Massive autophosphorylation of the Ser/Thr-rich domain controls protein kinase activity of TRPM6 and TRPM7

TRPM6 and TRPM7 are bifunctional proteins expressing a TRP channel fused to an atypical alpha-kinase domain. While the gating properties of TRPM6 and TRPM7 channels have been studied in detail, little is known about the mechanisms regulating kinase activity. Recently, we found that TRPM7 associates with its substrate myosin II via a kinase-dependent mechanism suggesting a role for autophosphorylation in substrate recognition. Here, we demonstrate that the cytosolic C-terminus of TRPM7 undergoes massive autophosphorylation (32+/-4 mol/mol), which strongly increases the rate of substrate phosphorylation. Phosphomapping by mass spectrometry indicates that the majority of autophosphorylation sites (37 out of 46) map to a Ser/Thr-rich region immediately N-terminal of the catalytic domain. Deletion of this region prevents substrate phosphorylation without affecting intrinsic catalytic activity suggesting that the Ser/Thr-rich domain contributes to substrate recognition. Surprisingly, the TRPM6-kinase is regulated by an analogous mechanism despite a lack of sequence conservation with the TRPM7 Ser/Thr-rich domain. In conclusion, our findings support a model where massive autophosphorylation outside the catalytic domain of TRPM6 and TRPM7 may facilitate kinase-substrate interactions leading to enhanced phosphorylation of those substrates.     

Ser/Thr-rich repetitive motifs as targets for phosphoglycan modifications in Leishmania mexicana secreted acid phosphatase.

The insect stage of the protozoan parasite Leishmania mexicana secretes a phosphomonoesterase in the form of a filamentous complex. The polypeptide subunits of this polymer are modified by phosphoglycans and/or oligomannosyl residues linked to phosphoserine. Based on peptide sequence data of a predominant 100 kDa protein of the filamentous complex, two tandemly arranged, single copy genes, lmsap1 and lmsap2, were cloned and sequenced. lmsap1 predicts a protein with features characteristic of acid phosphatases and a remarkable serine- and threonine-rich region of 32 amino acids close to the C-terminus. In the otherwise identical lmsap2 product, this region is extended to 383 amino acids and is composed of short Ser/Thr-rich repeats. Deletion analysis demonstrates that lmsap1 encodes the major 100 kDa protein of the complex while a minor 200 kDa component is derived from the lmsap2 gene. Null mutants of either gene retain the ability to secrete acid phosphatase filaments, while a deletion of both genes results in Leishmania defective in enzyme formation. The Ser/Thr-rich domains are the targets for phosphoglycan modifications as shown by the expression of secreted fusion proteins composed of these C-terminal regions and the N-terminal domain of a lysosomal acid phosphatase.     

The structure of interrupted human AC microsatellites

Microsatellite lengths change over evolutionary time through a process of replication slippage. A recently proposed model of this process holds that the expansionary tendencies of slippage mutation are balanced by point mutations breaking longer microsatellites into smaller units and that this process gives rise to the observed frequency distributions of uninterrupted microsatellite lengths. We refer to this as the slippage/point-mutation theory. Here we derive the theory's predictions for interrupted microsatellites comprising regions of perfect repeats, labeled segments, separated by dinucleotide interruptions containing point mutations. These predictions are tested by reference to the frequency distributions of segments of AC microsatellite in the human genome, and several predictions are shown not to be supported by the data, as follows. The estimated slippage rates are relatively low for the first four repeats, and then rise initially linearly with length, in accordance with previous work. However, contrary to expectation and the experimental evidence, the inferred slippage rates decline in segments above 10 repeats. Point mutation rates are also found to be higher within microsatellites than elsewhere. The theory provides an excellent fit to the frequency distribution of peripheral segment lengths but fails to explain why internal segments are shorter. Furthermore, there are fewer microsatellites with many segments than predicted. The frequencies of interrupted microsatellites decline geometrically with microsatellite size measured in number of segments, so that for each additional segment, the number of microsatellites is 33.6% less. Overall we conclude that the detailed structure of interrupted microsatellites cannot be reconciled with the existing slippage/point-mutation theory of microsatellite evolution, and we suggest that microsatellites are stabilized by processes acting on interior rather than on peripheral segments.     

Insight into role of selection in the evolution of polyglutamine tracts in humans

Glutamine tandem repeats are common in eukaryotic proteins. Although some studies have proposed that replication slippage plays an important role in shaping these repeats, the role of natural selection in glutamine tandem repeat evolution is somewhat unclear. In this study, we identified all of the glutamine tandem repeats containing four or more glutamines in human proteins and then estimated the nonsynonymous (d(N)) and synonymous (d(S)) substitution rates for the regions flanking the glutamine tandem repeats and the proteins containing them. The results indicated that most of the proteins containing polyglutamine (polyQ) tracts of four or more glutamines have undergone purifying selection, and that the purifying selection for the regions flanking the repeats is weaker. Additionally, we observed that the conserved repeats were under stronger selection constraints than the nonconserved repeats. Interestingly, we found that there was a higher level of purifying selection for the regions flanking the polyQ tracts encoded by pure CAG codons compared with those encoded by mixed codons. Based on our findings, we propose that selection has played a more important role than was previously speculated in constraining the expansion of polyQ tracts encoded by pure codons.     

A Phylogenetic Perspective on Sequence Evolution in Microsatellite Loci

We examined the evolution of the repeat regions of three noncoding microsatellite loci in 58 species of the Polistinae, a subfamily of wasps that diverged over 140 million years ago. A phylogenetic approach allows two new kinds of approaches to studying microsatellite evolution: character mapping and comparative analysis. The basic repeat structure of the loci was highly conserved, but was often punctuated with imperfections that appear to be phylogenetically informative. Repeat numbers evolved more rapidly than other changes in the repeat region. Changes in number of repeats among species seem consistent with the stepwise mutation model, which is based on slippage during replication as the main source of mutations. Changes in repeat numbers can occur even when there are very few tandem repeats but longer repeats, especially perfect repeats led to greater rates of evolutionary change. Species phylogenetically closer to the one from which we identified the loci had longer stretches of uninterrupted repeats and more different motifs, but not longer total repeat regions. The number of perfect repeats increased more often than it decreased. However, there was no evidence that some species have consistently greater numbers of repeats across loci than other species have, once ascertainment bias is eliminated. We also found no evidence for a population size effect posited by one form of the directionality hypothesis. Overall, phylogenetic variation in repeat regions can be explained by adding neutral evolution to what is already known about the mutation process. The life cycle of microsatellites appears to reflect a balance between growth by slippage and degradation by an essentially irreversible accumulation of imperfections. Received: 13 April 1999 / Accepted: 8 September 1999     

Replication slippage versus point mutation rates in short tandem repeats of the human genome

Short tandem repeats (STRs) are subjected to two kinds of mutational modifications: point mutations and replication slippages. The latter is found to be the more frequent cause of STR modifications, but a satisfactory quantitative measure of the ratio of the two processes has yet to be determined. The comparison of entire genome sequences of closely enough related species enables one to obtain sufficient statistics by counting the differences in the STR regions. We analyzed human–chimpanzee DNA sequence alignments to obtain the counts of point mutations and replication slippage modifications. The results were compared with the results of a computer simulation, and the parameters quantifying the replication slippage probability as well as the probabilities of point mutations within the repeats were determined. It was found that within the STRs with repeated units consisting of one, two or three nucleotides, point mutations occur approximately twice as frequently as one would expect on the basis of the 1.2% difference between the human and chimpanzee genomes. As expected, the replication slippage probability is negligible below a 10-bp threshold and grows above this level. The replication slippage events outnumber the point mutations by one or two orders of magnitude, but are still lower by one order of magnitude relative to the mutability of the markers that are used for genotyping purposes.     

Molecular evolution of minisatellites in hemiascomycetous yeasts

Minisatellites are DNA tandem repeats exhibiting size polymorphism among individuals of a population. This polymorphism is generated by two different mechanisms, both in human and yeast cells, "replication slippage" during S-phase DNA synthesis and "repair slippage" associated to meiotic gene conversion. The Saccharomyces cerevisiae genome contains numerous natural minisatellites. They are located on all chromosomes without any obvious distribution bias. Minisatellites found in protein-coding genes have longer repeat units and on the average more repeat units than minisatellites in noncoding regions. They show an excess of cytosines on the coding strand, as compared to guanines (negative GC skew). They are always multiples of three, encode serine- and threonine-rich amino acid repeats, and are found preferably within genes encoding cell wall proteins, suggesting that they are positively selected in this particular class of genes. Genome-wide, there is no statistically significant association between minisatellites and meiotic recombination hot spots. In addition, minisatellites that are located in the vicinity of a meiotic hot spot are not more polymorphic than minisatellites located far from any hot spot. This suggests that minisatellites, in S. cerevisiae, evolve probably by strand slippage during replication or mitotic recombination. Finally, evolution of minisatellites among hemiascomycetous yeasts shows that even though many minisatellite-containing genes are conserved, most of the time the minisatellite itself is not conserved. The diversity of minisatellite sequences found in orthologous genes of different species suggests that minisatellites are differentially acquired and lost during evolution of hemiascomycetous yeasts at a pace faster than the genes containing them.     

Creation of a chloroplast microsatellite reporter for detection of replication slippage in Chlamydomonas reinhardtii

Microsatellites are composed of short tandem direct repeats; deletions or duplications of those repeats through the process of replication slippage result in microsatellite instability relative to other genomic loci. Variation in repeat number occurs so frequently that microsatellites can be used for genotyping and forensic analysis. However, an accurate assessment of the rates of change can be difficult because the presence of many repeats makes it difficult to determine whether changes have occurred through single or multiple events. The current study was undertaken to experimentally assess the rates of replication slippage that occur in vivo in the chloroplast DNA of Chlamydomonas reinhardtii. A reporter construct was created in which a stretch of AAAG repeats was inserted into a functional gene to allow changes to be observed when they occurred at the synthetic microsatellite. Restoration of the reading frame occurred through replication slippage in 15 of every million viable cells. Since only one-third of the potential insertion/deletion events would restore the reading frame, the frequency of change could be deduced to be 4.5 x 10(-5). Analysis of the slippage events showed that template slippage was the primary event, resulting in deletions rather than duplications. These findings contrasted with events observed in Escherichia coli during maintenance of the plasmid, where duplications were the rule.     

Crystal structure of protein isoaspartyl methyltransferase: a catalyst for protein repair

BACKGROUND: Formation of isoaspartyl residues is one of several processes that damage proteins as they age. Protein L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) is a conserved and nearly ubiquitous enzyme that catalyzes the repair of proteins damaged by isoaspartyl formation. RESULTS: We have determined the first structure of a PIMT from crystals of the T. maritima enzyme complexed to S-adenosyl-L-homocysteine (AdoHcy) and refined it to 1.8 A resolution. Although PIMT forms one structural unit, the protein can be divided functionally into three subdomains. The central subdomain closely resembles other S-adenosyl-L-methionine-dependent methyltransferases but bears a striking alteration of topological connectivity, which is not shared by any other member of this family. Rather than arranged as a mixed beta sheet with topology 6 upward arrow7 downward arrow5 upward arrow4 upward arrow1 upward arrow2 upward arrow3 upward arrow, the central sheet of PIMT is reorganized to 7 upward arrow6 downward arrow5 upward arrow4 upward arrow1 upward arrow2 upward arrow3 upward arrow. AdoHcy is largely buried between the N-terminal and central subdomains by a conserved and largely hydrophobic loop on one rim of the binding cleft, and a conserved Ser/Thr-rich beta strand on the other. The Ser/Thr-rich strand may provide hydrogen bonds for specific interactions with isoaspartyl substrates. The side chain of Ile-206, a conserved residue, crosses the cleft, restricting access to the donor methyl group to a deep well, the putative isoaspartyl methyl acceptor site. CONCLUSIONS: The structure of PIMT reveals a unique modification of the methyltransferase fold along with a site for specific recognition of isoaspartyl substrates. The sequence conservation among PIMTs suggests that the current structure should prove a reliable model for understanding the repair of isoaspartyl damage in all organisms.     

Graph-based modeling of tandem repeats improves global multiple sequence alignment

Tandem repeats (TRs) are often present in proteins with crucial functions, responsible for resistance, pathogenicity and associated with infectious or neurodegenerative diseases. This motivates numerous studies of TRs and their evolution, requiring accurate multiple sequence alignment. TRs may be lost or inserted at any position of a TR region by replication slippage or recombination, but current methods assume fixed unit boundaries, and yet are of high complexity. We present a new global graph-based alignment method that does not restrict TR unit indels by unit boundaries. TR indels are modeled separately and penalized using the phylogeny-aware alignment algorithm. This ensures enhanced accuracy of reconstructed alignments, disentangling TRs and measuring indel events and rates in a biologically meaningful way. Our method detects not only duplication events but also all changes in TR regions owing to recombination, strand slippage and other events inserting or deleting TR units. We evaluate our method by simulation incorporating TR evolution, by either sampling TRs from a profile hidden Markov model or by mimicking strand slippage with duplications. The new method is illustrated on a family of type III effectors, a pathogenicity determinant in agriculturally important bacteria Ralstonia solanacearum. We show that TR indel rate variation contributes to the diversification of this protein family.     

A general amphipathic alpha-helical motif for sensing membrane curvature

The Golgi-associated protein ArfGAP1 has an unusual membrane-adsorbing amphipathic alpha-helix: its polar face is weakly charged, containing mainly serine and threonine residues. We show that this feature explains the specificity of ArfGAP1 for curved versus flat lipid membranes. We built an algorithm to identify other potential amphipathic alpha-helices rich in serine and threonine residues in protein databases. Among the identified sequences, we show that three act as membrane curvature sensors. In the golgin GMAP-210, the sensor may serve to trap small vesicles at the end of a long coiled coil. In Osh4p/Kes1p, which transports sterol between membranes, the sensor controls access to the sterol-binding pocket. In the nucleoporin Nup133, the sensor corresponds to an exposed loop of a beta-propeller structure. Ser/Thr-rich amphipathic helices thus define a general motif used by proteins of various functions for sensing membrane curvature.     

The dysfunctional LDL receptor in a monensin-resistant mutant of Chinese hamster ovary cells lacks selected O-linked oligosaccharides

The Chinese hamster ovary (CHO) cell line Monr31, which is resistant to the cytotoxic ionophore monensin, produces a receptor for the low density lipoprotein (LDL) that has a lowered binding affinity for LDL and is approximately 5 kDa smaller in size than the receptor from parental CHO cells. It has been proposed that the reduced size and affinity for LDL are associated with a reduced level of O-glycosylation of Ser/Thr residues in the receptor. To examine this possibility in more detail, both parental CHO and Monr31 cells were metabolically radiolabeled with [3H]glucosamine, and the labeled LDL receptors were purified by immunoprecipitation and identified by SDS-PAGE-fluorography. The Ser/Thr-linked oligosaccharides in the receptors from both parental CHO and Monr31 cells are mono- and desialylated species having the common core structure Gal beta 1-3GalNAc. The receptor from Monr31 cells, however, contains about one-third fewer Ser/Thr-linked oligosaccharides than the receptor from parental CHO cells. Analysis of the glycopeptides derived from the Monr31 cell LDL receptors indicates that they contain Ser/Thr-linked oligosaccharides only in the clustered domain and are missing Ser/Thr-linked oligosaccharides in the unclustered regions of the protein. Additionally, analysis of a human LDL receptor lacking the domain for attachment of the clustered Ser/Thr-linked oligosaccharides and expressed in both parental CHO and Monr31 cells indicated that the truncated human receptor from Monr31 cells is devoid of Ser/Thr-linked oligosaccharides. In contrast, the truncated human receptor produced by parental CHO cells contains Ser/Thr-linked oligosaccharides contributing approximately 5 kDa to its apparent size. Collectively, these results demonstrate that the LDL receptor produced by the Monr31 cells contains Ser/Thr-linked oligosaccharides in the clustered domain but is missing Ser/Thr-linked oligosaccharides in the unclustered, NH2-terminal domains of the receptor.     

Genome organisation and chromatin structure in Escherichia coli

We have analysed the complete sequence of the Escherichia coli K12 isolate MG1655 genome for chromatin-associated protein binding sites, and compared the predicted location of predicted sites with experimental expression data from 'DNA chip' experiments. Of the dozen proteins associated with chromatin in E. coli, only three have been shown to have significant binding preferences: integration host factor (IHF) has the strongest binding site preference, and FIS sites show a weak consensus, and there is no clear consensus site for binding of the H-NS protein. Using hidden Markov models (HMMs), we predict the location of 608 IHF sites, scattered throughout the genome. A subset of the IHF sites associated with repeats tends to be clustered around the origin of replication. We estimate there could be roughly 6000 FIS sites in E. coli, and the sites tend to be localised in two regions flanking the replication termini. We also show that the regions upstream of genes regulated by H-NS are more curved and have a higher AT content than regions upstream of other genes. These regions in general would also be localised near the replication terminus.