Jarkko Valjakka

Biochemical characterization of CA IX: one of the most active carbonic anhydrase isozymes

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys119-Cys299) and a unique N-linked glycosylation site (Asn309) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr78) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.

Zinc Coordination Spheres in Protein Structures

Zinc metalloproteins are one of the most abundant and structurally diverse proteins in nature. In these proteins, the Zn(II) ion possesses a multifunctional role as it stabilizes the fold of small zinc fingers, catalyzes essential reactions in enzymes of all six classes, or assists in the formation of biological oligomers. Previously, a number of database surveys have been conducted on zinc proteins to gain broader insights into their rich coordination chemistry. However, many of these surveys suffer from severe flaws and misinterpretations or are otherwise limited. To provide a more comprehensive, up-to-date picture on zinc coordination environments in proteins, zinc containing protein structures deposited in the Protein Data Bank (PDB) were analyzed in detail. A statistical analysis in terms of zinc coordinating amino acids, metal-to-ligand bond lengths, coordination number, and structural classification was performed, revealing coordination spheres from classical tetrahedral cysteine/histidine binding sites to more complex binuclear sites with carboxylated lysine residues. According to the results, coordination spheres of hundreds of crystal structures in the PDB could be misinterpreted due to symmetry-related molecules or missing electron densities for ligands. The analysis also revealed increasing average metal-to-ligand bond length as a function of crystallographic resolution, which should be taken into account when interrogating metal ion binding sites. Moreover, one-third of the zinc ions present in crystal structures are artifacts, merely aiding crystal formation and packing with no biological significance. Our analysis provides solid evidence that a minimal stable zinc coordination sphere is made up by four ligands and adopts a tetrahedral coordination geometry.

DNA-Binding and -Bending Activities of SAP30L and SAP30 Are Mediated by a Zinc-Dependent Module and Monophosphoinositides

ABSTRACT Deacetylation of histones is carried out by a corepressor complex in which Sin3A is an essential scaffold protein. Two proteins in this complex, the Sin3A-associated proteins SAP30L and SAP30, have previously been suggested to function as linker molecules between various corepressors. In this report, we demonstrate new functions for human SAP30L and SAP30 by showing that they can associate directly with core histones as well as naked DNA. A zinc-coordinating structure is necessary for DNA binding, one consequence of which is bending of the DNA. We provide evidence that a sequence motif previously shown to be a nuclear localization signal is also a phosphatidylinositol (PI)-binding element and that binding of specific nuclear monophosphoinositides regulates DNA binding and chromatin association of SAP30L. PI binding also decreases the repression activity of SAP30L and affects its translocation from the nucleus to the cytoplasm. Our results suggest that SAP30L and SAP30 play active roles in recruitment of deacetylating enzymes to nucleosomes, and mediate key protein-protein and protein-DNA interactions involved in chromatin remodeling and transcription.

Analysis of a shortened form of human carbonic anhydrase VII expressed in vitro compared to the full-length enzyme

Carbonic anhydrase (CA) enzymes are expressed in all organs of the mammalian body where they participate in important physiological functions. CA VII is a cytosolic isozyme which may be expressed as two forms according to the recent GenBank data. We designed a present study to express and characterize the human CA VII forms: full-length CA VII and short form (predicted to lack 56 residues from the N-terminus). Reverse transcriptase PCR analysis revealed mRNAs for both CA VII forms in the human brain. These different forms were expressed as recombinant proteins to investigate their biochemical properties. The full-length CA VII was used to raise a polyclonal antiserum in a rabbit, and the antiserum was then employed in western blot analyses and immunohistochemistry of mouse tissues. Data from mass spectrometry and comparative modeling showed that CA VII protein contains a single intramolecular disulfide bridge (Cys-56 to Cys-180) which is lacking in the short form. The computer model suggested distinctly different folding for the different forms. The more exposed structure and the absence of the disulfide bridge in the short form could make this protein more susceptible to degradation. In fact, this was realized in several protein purification efforts in which the short form readily degraded during the experimental procedures. From these results, we conclude that the full-length CA VII is a predominant active form in human brain and also in other tissues. In addition to the brain, CA VII is expressed in several other organs including the stomach, duodenum, colon, liver, and skeletal muscle. The distribution pattern suggests multiple functions for CA VII in different organs.

Acetaldehyde-derived modifications on cytosolic human carbonic anhydrases

Acetaldehyde can generate modifications in several proteins, such as carbonic anhydrase (CA) II. In this study, we extended in vitro investigations on acetaldehyde adduct formation by focusing on the other human cytosolic CA enzymes I, III, VII, and XIII. High-resolution mass spectrometric analysis indicated that acetaldehyde most efficiently formed covalent adducts with CA II and XIII. The binding of up to 19 acetaldehydes in CA II is probably attributable to the high number of lysine residues (n = 24) located mainly on the surface of the enzyme molecule. CA XIII formed more adducts (up to 25) than it contains lysine residues (n = 16) in its primary structure. Acetaldehyde treatment induced only minor changes in CA catalytic activity in most cases. The present study provides the first evidence that acetaldehyde can bind to several cytosolic CA isozymes. The functional consequences of such modifications will be further investigated in vivo by using animal models.

Structure-function analysis indicates that sumoylation modulates DNA-binding activity of STAT1

BackgroundSTAT1 is an essential transcription factor for interferon-γ-mediated gene responses. A distinct sumoylation consensus site (ψKxE) 702IKTE705 is localized in the C-terminal region of STAT1, where Lys703 is a target for PIAS-induced SUMO modification. Several studies indicate that sumoylation has an inhibitory role on STAT1-mediated gene expression but the molecular mechanisms are not fully understood.ResultsHere, we have performed a structural and functional analysis of sumoylation in STAT1. We show that deconjugation of SUMO by SENP1 enhances the transcriptional activity of STAT1, confirming a negative regulatory effect of sumoylation on STAT1 activity. Inspection of molecular model indicated that consensus site is well exposed to SUMO-conjugation in STAT1 homodimer and that the conjugated SUMO moiety is directed towards DNA, thus able to form a sterical hindrance affecting promoter binding of dimeric STAT1. In addition, oligoprecipitation experiments indicated that sumoylation deficient STAT1 E705Q mutant has higher DNA-binding activity on STAT1 responsive gene promoters than wild-type STAT1. Furthermore, sumoylation deficient STAT1 E705Q mutant displayed enhanced histone H4 acetylation on interferon-γ-responsive promoter compared to wild-type STAT1.ConclusionsOur results suggest that sumoylation participates in regulation of STAT1 responses by modulating DNA-binding properties of STAT1.

Characterization of Non-Specific Cytotoxic Cell Receptor Protein 1: A New Member of the Lectin-Type Subfamily of F-Box Proteins

Our previous microarray study showed that the non-specific cytotoxic cell receptor protein 1 (Nccrp1) transcript is significantly upregulated in the gastric mucosa of carbonic anhydrase IX (CA IX)-deficient (Car9−/−) mice. In this paper, we aimed to characterize human NCCRP1 and to elucidate its relationship to CA IX. Recombinant NCCRP1 protein was expressed in Escherichia coli, and a novel polyclonal antiserum was raised against the purified full-length protein. Immunocytochemistry showed that NCCRP1 is expressed intracellularly, even though it has previously been described as a transmembrane protein. Using bioinformatic analyses, we identified orthologs of NCCRP1 in 35 vertebrate genomes, and up to five paralogs per genome. These paralogs are FBXO genes whose protein products are components of the E3 ubiquitin ligase complexes. NCCRP1 proteins have no signal peptides or transmembrane domains. NCCRP1 has mainly been studied in fish and was thought to be responsible for the cytolytic function of nonspecific cytotoxic cells (NCCs). Our analyses showed that in humans, NCCRP1 mRNA is expressed in tissues containing squamous epithelium, whereas it shows a more ubiquitous tissue expression pattern in mice. Neither human nor mouse NCCRP1 expression is specific to immune tissues. Silencing CA9 using siRNAs did not affect NCCRP1 levels, indicating that its expression is not directly regulated by CA9. Interestingly, silencing NCCRP1 caused a statistically significant decrease in the growth of HeLa cells. These studies provide ample evidence that the current name, “non-specific cytotoxic cell receptor protein 1,” is not appropriate. We therefore propose that the gene name be changed to FBXO50.

Does the cis/trans configuration of peptide bonds in bioactive tripeptides play a role in ACE-1 enzyme inhibition?

Background The milk casein-derived bioactive tripeptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP) have been shown to prevent development of hypertension in animal models and to lower blood pressure in moderately hypertensive subjects in most but not all clinical trials. Inhibition of angiotensin-converting enzyme 1 (ACE-1) has been suggested as the explanation for these antihypertensive and beneficial vascular effects. Previously, human umbilical vein endothelial cells (HUVEC) have not been used to test ACE-1 inhibiting properties of casein derived tripeptides in vasculature. Purpose We focused on the cis/trans configurations of the peptide bonds in proline-containing tripeptides in order to discover whether the different structural properties of these peptides influence their activity in ACE-1 inhibition. We hypothesized that the configuration of proline-containing peptides plays a significant role in enzyme inhibition. Methods AutoDock 4.2 docking software was used to predict suitable peptide bond configurations of the tripeptides. Besides modeling studies, we completed ACE-1 activity measurements in vitro using HUVEC cultures. Results In HUVEC cells, both IPP and VPP inhibited ACE-1. Based on molecular docking studies, we propose that in ACE-1 inhibition IPP and VPP share a similar cis configuration between the first aliphatic (isoleucine or valine) and the second (proline) amino acid residues and more different configurations between two proline residues. In vivo experiments are needed to validate the significance of the present findings.

Production of l-xylose from l-xylulose using Escherichia coli l-fucose isomerase

L-Xylulose was used as a raw material for the production of L-xylose with a recombinantly produced Escherichia coli L-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for L-xylulose with a K(m) of 41 mM and a V(max) of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6h 50 min and 1h 31 min, respectively. The reaction equilibrium between L-xylulose and L-xylose was 15:85 at 35 °C and thus favored the formation of L-xylose. Contrary to the L-rhamnose isomerase catalyzed reaction described previously [14]L-lyxose was not detected in the reaction mixture with L-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to L-xylulose the inhibition did not reach 50%.

Structure of 20K endoglucanase from Melanocarpus albomyces at 1.8 Å resolution

The crystal structure of the 20K endoglucanase from the thermophilic fungus Melanocarpus albomyces (Ma20k) has been determined. The structure was refined to 1.8 A resolution using data obtained at 120 K. Ma20k belongs to glycoside hydrolase family 45. The three-dimensional structures of endoglucanase V (EGV) from the fungus Humicola insolens and of an endoglucanase from H. grisea var. thermoidea have previously been determined. The overall structure of Ma20k consists of a six-stranded beta-barrel domain similar to that found previously in family 45 endoglucanases. The flexible loop between strands V and VI, which was disordered in the uncomplexed structures of the Humicola endoglucanases but was ordered in complexed structures of EGV, is found to be well ordered in the native structure of Ma20k. The structure of Ma20k allows comparison between thermophilic and mesophilic proteins of family 45 and different principles for thermostability are discussed.