The RdxA oxygen insensitive nitroreductase of the human gastric pathogen Helicobacter pylori is responsible for the susceptibility of this organism to the redox active prodrug metronidazole (MTZ). Loss-of-function mutations in rdxA are primarily responsible for resistance to this therapeutic. RdxA exhibits potent NADPH oxidase activity under aerobic conditions and metronidazole reductase activity under strictly anaerobic conditions. Here we report the crystal structure of RdxA, which is a homodimer exhibiting domain swapping and containing two molecules of FMN bound at the dimer interface. We have found a gap between the side chain of Tyr47 and the isoalloxazine ring of FMN that seems appropriate for substrate binding. The structure does not include residues 97–128, which corresponds to a locally unstable part of the NTR from E. coli, and might be involved in cofactor binding. Comparison of H pylori RdxA to other oxidoreductases of known structure suggests RdxA may belong to a new subgroup of oxidoreductases in which a cysteine sidechain close to the FMN cofactor could be involved in the reductive activity. In this respect, mutation of C159 to A or S (C159A/S) has resulted in loss of MTZ reductase activity, but not NADPH oxidase activity. The RdxA structure allows interpretation of the many loss-of-function mutations previously described, including those affecting C159, a residue whose interaction with FMN is required for nitroreduction of MTZ. Our studies provide unique insights into the redox behavior of the flavin in this key enzyme for metronidazole activation, and with potential use in gene therapy.
nitroreductase; metronidazole; Helicobacter pylor; prodrug; flavoprotein; antibiotic resistance
The pancreatic serine protease chymotrypsin C (CTRC) cleaves the Leu81-Glu82 peptide bond in the calcium binding loop of human cationic trypsinogen and thereby promotes its degradation. This serves as a protective mechanism against ectopic trypsinogen activation in the pancreas. In the present study we demonstrate that cleavage of the Leu81-Glu82 peptide bond by CTRC is highly specific and other human pancreatic chymotrypsins (CTRB1, CTRB2, CTRL1) and elastases (ELA2A, ELA3A, ELA3B) do not catalyze this reaction. To elucidate the mechanistic basis for CTRC specificity, we surveyed the primary (P1) cleavage preference of these pancreatic proteases on peptide substrates. We found that CTRC cleaved after a P1 Leu with at least 10-fold higher catalytic efficiency than other enzymes tested. To assess extended subsite interactions, we introduced Ala-mutations into human cationic trypsinogen at the P3, P1' P3' and P4' amino-acid positions, where P1-P1' corresponds to Leu81-Glu82. Interestingly, CTRC mediated cleavage was stimulated 3-fold by mutation E82A and unaffected by mutations E79A and N84A, but all three mutations compromised specificity and resulted in increased cleavage by ELA2A. Mutation E85A decreased CTRC cleavage by 2-fold. Remarkably, other chymotrypsins and elastases did not cleave human cationic trypsinogen even with the L81F or L81A mutations, which introduced favorable P1 residues for these enzymes. We conclude that specific cleavage of the Leu81-Glu82 peptide bond in human cationic trypsinogen by CTRC is primarily determined by its distinctively high activity on leucyl peptide bonds, whereas the P1' Glu82, P3' Asn84 and P4' Glu85 residues serve as additional specificity determinants.
chymotrypsin; elastase; trypsinogen; pancreas; serine protease
Ribonuclease H1 is a conserved enzyme that cleaves the RNA strand of RNA•DNA heteroduplexes, and has important functions in the nuclear and mitochondrial compartments. The therapeutic action of antisense oligodeoxynucleotides involves the recruitment of RNase H1 to cleave disease-relevant RNA targets. Recombinant human(Hs)-RNase H1 was purified from a bacterial expression host, and conditions were identified that provided optimal oligonucleotide-directed RNA cleavage in vitro. Hs-RNase H1 exhibits optimal catalytic activity in pH 7.5 HEPES buffer, and a salt (KCl) concentration of ~100–150 mM. Mg2+ best supports Hs-RNase H1, with an optimal concentration of 10 mM, but at higher concentrations inhibits enzyme activity. Mn2+ and Co2+ also support catalytic activity, while Ni2+ and Zn2+ exhibit only modest activities as cofactors. The optimized assay was used to show that an antisense oligonucleotide, added in substoichiometric amounts to initiate RNA cleavage, supports up to thirty rounds of reaction in 30 minutes. Mutation to alanine of the conserved histidine at position 264 causes a ~100-fold decrease in kcat under multiple-turnover conditions, but does not alter the Km. Under single-turnover conditions, the H264A mutant exhibits a 12-fold higher exponential time constant for substrate cleavage. The defective activity of the H264A mutant is not rescued in either assay condition by higher Mg2+ concentrations. These data implicate the H264 side chain in phosphodiester hydrolysis as well as in product release, and are consistent with a proposed model in which the H264 side chain interacts with a divalent metal ion to support catalysis.
Ribonuclease H1; Antisense oligonucleotides; Metal-dependent phosphodiester hydrolysis; Conserved histidine function; RNA-DNA heteroduplex
Nuclear factor erythroid-derived 2-related factor 1 (Nrf1) regulates cellular stress response genes, and has also been implicated to have a role in other cellular processes. We previously demonstrated that hepatocyte-specific deletion of Nrf1 in mice resulted in spontaneous apoptosis, inflammation, and development of liver tumors. Here, we showed that both fibroblasts derived from Nrf1-null mouse embryos, and fibroblasts bearing a conditional Nrf1 allele exhibited increased micronuclei and formation of abnormal nuclei. Lentiviral shRNA-mediated knockdown of Nrf1 in SAOS-2 cells also resulted in increased micronuclei, abnormal mitosis and multinucleated cells. Metaphase analyses showed increased aneuploidy in Nrf1-/- embryonic fibroblasts. Nuclear defects in Nrf1-deficient cells were associated with decreased expression of various genes encoding kinetochore and mitotic checkpoint proteins. Our findings suggest that Nrf1 may serve a function in maintaining genomic integrity, and Nrf1 dysregulation can induce tumorigenesis.
On the surface heat shock protein 90 (Hsp90) is an unlikely drug target for the treatment of any disease, let alone cancer. Hsp90 is highly conserved and ubiquitously expressed in all cells. There are two major isoforms α and β encoded by distinct genes and together they may constitute 1%–3% of the cellular protein. Deletion of the protein is embryonic lethal and there are no recognized polymorphisms suggesting an association or causal relationship with any human disease. With respect to cancer, the proteins absence from two recent high profile articles, ‘Hallmarks of cancer: the next generation’ [Hanahan & Weinberg (2011) Cell
144, 646–674] and ‘Comprehensive molecular portraits of human breast tumours’ [Koboldt et al. (2012) Nature] underlines the perception that it is an unlikely bona fide target to treat this disease. Yet, to date, there are 17 distinct Hsp90 inhibitors in clinical trials for multiple indications in cancer. The protein has been championed for over 20 years by the National Cancer Institute (Bethesda, MD, USA) as a cancer target since the discovery of the antitumor activity of the natural product geldanamycin. This review aims to look at the conundrum of why Hsp90 can even be considered a druggable target for the treatment of cancer. We propose that in contrast to the majority of chemotherapeutics our growing armamentarium of investigational Hsp90 drugs represents an elegant choice that offers real hope in the long-term treatment of certain cancers.
cancer; chaperone; combination therapy; drug discovery; extracellular Hsp90; Hsp90; protein activation
A major factor contributing to the high mutation rate of HIV-1 RT is its high propensity for misincorporation. Misincorporation requires both dNTP misinsertion and the subsequent extension of the mismatched terminus thus formed. We hypothesized that Lys66 is a determinant of mismatch extension based on its position near the primer terminus. This hypothesis was tested by steady-state kinetic studies using wild type HIV-1 RT and four Lys66 substitution mutants: Lys66Arg, Lys66Ala, Lys66Asn, and Lys66Thr. Efficiency of mismatch extension (fext) was reduced for all mutants, with Lys66Ala, Lys66Asn, and Lys66Thr showing 4 to 6-fold reduction compared to wild type. Surprisingly, the non-conservative substitutions also lead to large decreases in misinsertion efficiency (fins) ranging from as low as 3-fold to values much higher than 23-fold. Thus, Lys66Arg mutant was akin to wild type, while all non-conservative mutants displayed significantly decreased efficiency for both events. Our results suggest that Lys66, much like Lys65, is a determinant of both dNTP misinsertion and mismatch extension efficiency. While Lys65 is known to contact γ-phosphate of incoming dNTP, the Lys 66 side chain is in the vicinity of primer terminus. However, our results suggest that both residues have similar influence on dNTP misinsertion and mispair extension efficiencies of HIV-1 RT. When we tested the mutants for susceptibility to selected nucleoside analog and non-nucleoside analog drugs, similar to Lys65Arg, the Lys66Ala and Lys66Asn mutants displayed mild resistance to nucleoside analog drug AZTTP.
HIV-1; reverse transcriptase; polymerase fidelity; misinsertion efficiency; mismatch extension efficiency
Cysteine residues with depressed pKa values are critical for the functions of many proteins. Several types of interactions can stabilize cysteine thiolate anions, including hydrogen bonds between thiol(ate)s and nearby residues as well as electrostatic interactions involving charged residues or dipoles. Dipolar stabilization of thiolates by peptide groups has been suggested to play a particularly important role near the N-termini of α-helices. Using a combination of X-ray crystallography, site-directed mutagenesis, and spectroscopic methods, we show that the reactive cysteine residue (Cys111) in Schizosaccharomyces pombe DJ-1 experiences a 0.6 unit depression of its thiol pKa as a consequence of a hydrogen bond donated by a threonine sidechain (Thr114) to a nearby peptide carbonyl oxygen at the N-terminus of an α-helix. This extended hydrogen bonded interaction is consistent with a sum of dipoles model whereby the distal hydrogen bond polarizes and strengthens the direct hydrogen bond between the proximal amide hydrogen and the cysteine thiol(ate). Therefore, our results suggest that the local dipolar enhancement of hydrogen bonds can appreciably stabilize cysteine thiolate formation. However, the substitution of a valine residue with a proline at the i+3 position has only a minor effect (0.3 units) on the pKa of Cys111. As proline has a reduced peptide dipole moment, this small effect suggests that a more extended helix macrodipolar effect does not play a major role in this system.
cysteine pKa; peptide dipole; DJ-1 superfamily; X-ray crystallography; redox biochemistry
Prion diseases are fatal neurodegenerative disorders caused by proteinaceous infectious pathogens termed prions (PrPSc). To date, there is no prophylaxis or therapy available for these transmissible encephalopathies. Passive immunization with monclonal antibodies recognizing the normal host-encoded prion protein (PrPC) has been reported to abolish PrPSc infectivity and to delay onset of disease. Because of established immunologic tolerance against the widely expressed PrPC, active immunization appears to be difficult to achieve. To overcome this limitation, papillomavirus-like particles were generated that display a nine amino acid B-cell epitope, DWEDRYYRE, of the murine/rat prion protein in an immunogenic capsid surface loop, by insertion into the L1 major capsid protein of bovine papillomavirus type 1. The PrP peptide was selected on the basis of its previously suggested central role in prion pathogenesis. Immunization with PrP–virus-like particles induced high-titer antibodies to PrP in rabbit and in rat, without inducing overt adverse effects. As determined by peptide-specific ELISA, rabbit immune sera recognized the inserted murine/rat epitope and also cross-reacted with the homologous rabbit/human epitope differing in one amino acid residue. In contrast, rat immune sera recognized the murine/rat peptide only. Sera of both species reacted with PrPC in its native conformation in mouse brain and on rat pheochromocytoma cells, as determined by immunoprecipitation and fluorescence-activated cell sorting analysis. Importantly, rabbit anti-PrP serum contained high-affinity antibody that inhibited de novo synthesis of PrPSc in prion-infected cells. If also effective in vivo, PrP–virus-like particle vaccination opens a unique possibility for immunologic prevention of currently fatal and incurable pri-on-mediated diseases.
immunotherapy; papillomavirus-like particles; prion
HOXB9 is a homeobox containing gene and is critical for the development of mammary gland and sternum. HOXB9 is also regulated by estrogen and is critical for angiogenesis. Herein, we investigated the biochemical roles of HOXB9 and its homeodomain in cell cycle progression and tumorigenesis. Our studies demonstrated that HOXB9 is overexpressed in breast cancer tissue. HOXB9 overexpression stimulated three-dimensional colony formation in soft-agar assay. HOXB9 binds to the promoters of various tumor growth and angiogenic factors and regulates their expression. Homeodomain of HOXB9 plays crucial roles in transcriptional regulation of tumor growth factors and also in three dimensional colony formation indicating crucial roles of HOXB9 homeodomain in tumorigenesis. Overall, we demonstrated that HOXB9 is critical regulators of tumor growth factors and is associated with tumorigenesis.
HOXB9; Breast cancer; tumor growth; colony formation; gene regulation
Soluble, tetrameric, plasma butyrylcholinesterase from horse has previously been shown to include a non-covalently attached polyproline peptide in its structure. The polyproline peptide matched the polyproline rich region of human lamellipodin. Our goal was to examine the tetramer organizing peptides of horse butyrylcholinesterase in more detail. Horse butyrylcholinesterase was denatured by boiling, thus releasing a set of polyproline peptides ranging in mass from 1173 to 2098 Da. The peptide sequences were determined by fragmentation in the MALDI-TOF-TOF and LTQ-Orbitrap mass spectrometers. Twenty-seven polyproline peptides grouped into 13 families were identified. Peptides contained a minimum of 11 consecutive proline residues and as many as 21. Many of the peptides had a non-proline amino acid at the N-terminus. A search of the protein databanks matched peptides to 9 proteins, though not all peptides matched a known protein. It is concluded that polyproline peptides of various lengths and sequences are included in the tetramer structure of horse BChE. The function of these polyproline peptides is to serve as tetramer organizing peptides.
mass spectrometry; serum butyrylcholinesterase; polyproline peptide
Ricin A chain (RTA) depurinates the sarcin/ricin loop (SRL) of 28S ribosomal RNA and inhibits protein synthesis in mammalian cells. In yeast, the ribosomal stalk facilitates the interaction of RTA with the ribosome and subsequent depurination. Despite homology between the stalk structures from yeast and humans there are notable differences. The human ribosomal stalk contains two identical heterodimers of P1/P2 bound to P0, while the yeast stalk consists of two different heterodimers, P1α/P2β and P2α/P1β, bound to P0. RTA exhibits higher activity towards mammalian ribosomes than ribosomes from other organisms, suggesting that the mode of interaction with ribosomes may vary. Here we examine whether the human ribosomal stalk proteins facilitate the interaction of RTA with human ribosomes and subsequent depurination of the SRL. Using siRNA-mediated knockdown of P1/P2 expression in human cells, we demonstrate that the depurination activity of RTA is lower when P1 and P2 protein levels are reduced. Ribosomes from P1/P2-depleted cells have a reduced ability to bind RTA by Biacore analysis, which correlates with reduced depurination activity both in vitro and inside cells. RTA interacts directly with recombinant human P1/P2 dimer, further demonstrating the importance of the human P1/P2 proteins in enabling RTA to bind and depurinate human ribosomes.
ricin; ribosome; P protein; sarcin/ricin loop; ribosomal stalk
Signal transducer and activator of transcription 3 (STAT3) is a key mediator of the inflammatory response by macrophages and other immune cell types. The naturally occurring polyphenol resveratrol is associated with anti-proliferative and anti-inflammatory properties via mechanisms implicating inhibition of STAT3 signaling. Here, we report that the small-molecule analogs of resveratrol, RSVA314 and RSVA405, are potent inhibitors of STAT3. RSVA314 and RSVA405 inhibited both constitutive and stimulated STAT3 in HEK293 cells and lipopolysaccharide (LPS)-activated RAW 264.7 macrophages, respectively. The small-molecule analogs inhibited STAT3 nearly 50 times more potently than did resveratrol (apparent IC50 ~ 0.5 μM). We further show that RSVA405 interfered with the inflammatory response by RAW 264.7 cells upon LPS stimulation by inhibiting IKK and IκBα phosphorylation and by decreasing the expression of several cytokines, including the NF-κB target genes, tumor necrosis factor-α and interleukin-6. Downstream activation of STAT1 upon LPS stimulation was also inhibited by RSVA405. Consequently, RSVA405 significantly interfered with the phagocytotic activity and proliferation of LPS-activated RAW 264.7 macrophages. Finally, we found that the effect of the two small-molecule analogs on STAT3 phosphorylation could be prevented by inhibitors of protein tyrosine phosphatases (PTPs), indicating that the small-molecules acted by promoting the dephosphorylation of STAT3 by PTPs.
STAT3; Inflammation; RSVA314; RSVA405; Resveratrol
Metacaspases are cysteine peptidases found only in yeast, plants and lower eukaryotes, including the protozoa. To investigate the extended substrate specificity and effects of Ca2+ on the activation of these enzymes, detailed kinetic, biochemical and structural analyses were carried out on metacaspase 2 from Trypanosoma brucei (TbMCA2). These results reveal that TbMCA2 has is an unambiguous preference for basic amino acids at the P1 position of peptide substrates and that this is most likely a result of hydrogen bonding from the P1 residue to Asp95 and Asp211 in TbMCA2. In addition, TbMCA2 also has a preference for charged residues at the P2 and P3 positions and for small residues at the prime-side of a peptide substrate. Studies into the effects of Ca2+ on the enzyme revealed the presence of two calcium-binding sites and a reversible structural modification of the enzyme upon Ca2+-binding. In addition, the concentration of Ca2+ used for activation of TbMCA2 was found to produce a differential effect on the activity of TbMCA2, but only when a series of peptides that differed in P2 were examined, suggesting that Ca2+ activation of TbMCA2 has a structural effect on the enzyme in the vicinity of the S2 binding pocket. Collectively, these data give new insights into the substrate specificity, and Ca2+ activation of TbMCA2. This provides important functional details and leads to a better understanding of metacaspases, which are known to play an important role in trypanosomes, and make attractive drug targets due to their absence in humans.
Metacaspase; substrate specificity; structural modification; kinetic parameters; calcium binding
Lens epithelium-derived growth factor (LEDGF), a ubiquitously expressed nuclear protein, acts by interacting with DNA and protein and is involved in widely varying cellular functions. Despite its importance, the mechanism(s) that regulate naturally occurring LEDGF activity are unidentified. Here we report that LEDGF is constitutively Sumoylated, and that the dynamical regulatory mechanism(s), Sumoylation and deSumoylation act as a molecular switch in modulating DNA binding and transcriptional activity of LEDGF with the functional consequences. Using bioinformatics analysis coupled with in vitro and in vivo Sumoylation assays, we found that lysine (K) 364 of LEDGF was Sumoylated, repressing its transcriptional activity. Conversely, mutation of K364 to arginine (R) or deSumoylation by Senp-1, a nuclear deSumoylase, enhanced the transactivation capacity of LEDGF and its cellular abundance. The enhancements were directly correlated with an increase in LEDGF’s DNA binding activity and small heat shock protein (Hsps) transcription, while the process was reversed in cells overexpressing Sumo1. Interestingly, cells expressing Sumoylation-deficient pEGFP-K364R protein showed increased cellular survival compared with the wild-type LEDGF protein. The findings provide insights into regulation and regulatory functions of LEDGF in Sumoylation-dependent transcriptional control that may be essential for modifying the physiology of cells to maintain cellular homeostasis. These studies also provide new evidence of the important role of post-translational modification in controlling LEDGF function.
LEDGF; Senp-1; Sumo1; Sp1; Hsp27
MUC1 and other membrane-associated mucins harbor long, up to a micrometer, extended highly glycosylated mucin domains and SEA domains situated on their extracellular parts. These mucins line luminal tracts and organs, and are anchored to the apical cell membrane by a transmembrane domain. The SEA domain is a highly conserved domain that undergoes a molecular strain-dependent autocatalytic cleavage during folding in the endoplasmic reticulum, a process required for apical plasma membrane expression. So far no specific function has been designated for the SEA domain. Here, we constructed a recombinant protein consisting of three SEA domains in tandem and used force spectroscopy to assess the dissociation force required to unfold individual, folded SEA domains. Force-distance curves revealed three peaks, each representing unfolding of a single SEA domain. Fitting the observed unfolding events to a worm-like chain model yielded an average contour length of 32 nm per SEA domain. Analysis of forces applied on the recombinant protein revealed an average unfolding force of 168 pN for each SEA domain at a loading rate of 25 nNs−1. Thus, the SEA domain may act as a breaking point that can dissociate before the plasma membrane is breached when mechanical forces are applied to cell surfaces.
Atomic Force Microscopy; AFM; Single-Molecule Force Spectroscopy; MUC1; SEA domain; Mucin
Agglutination of red blood cells, including chicken RBCs (cRBCs), have been used extensively to estimate viral titer, to screen glycan-receptor binding preference, and to assess the protective response of vaccines. While enjoying widespread use, it is known that some virus strains do not agglutinate RBCs. To address these underlying issues and increase the usefulness of cRBCs as tools to study viruses, such as influenza, we analyzed the cell surface N-glycans of cRBCs. Based on the results from complementary analytical strategies including mass spectroscopy, 1D and 2D-NMR spectroscopy, exoglycosidase digestions and HPLC profiling, we report here the major glycan structures present on cRBCs. By comparing the glycan structures of cBRCs to those of representative human upper respiratory cells, we offer a possible explanation for the fact that certain influenza strains do not agglutinate cRBCs, using specific human-adapted influenza hemagglutinins as examples. Finally, recent understanding of the role of various glycan structures in high affinity binding to influenza hemagglutinins provides context to our findings [1, 2]. These results illustrate that the field of glycomics can provide important information with regards to experimental systems used to characterize, detect, and study viruses.
Influenza; glycans; mass spectrometry; nuclear magnetic resonance
γS-crystallin (γS) is a highly conserved component of the eye lens. To gain insights into the functional role(s) of this protein, the mouse gene (Crygs) was deleted. Although mutations in γS can cause severe cataracts, loss of function of γS in knockout (KO) mice produced no obvious lens opacity, but was associated with focusing defects. Electron microscopy showed no major differences in lens cell organization, suggesting that the optical defects are primarily cytoplasmic in origin. KO lenses were also grossly normal by light microscopy but showed evidence of incomplete clearance of cellular organelles in maturing fiber cells. Phalloidin labeling showed an unusual distribution of F-actin in a band of mature fiber cells in KO lenses, suggesting a defect in the organization or processing of the actin cytoskeleton. Indeed, in wild-type lenses, γS and F-actin colocalize along the fiber cell plasma membrane. Relative levels of F-actin and G-actin in wild-type and KO lenses were estimated from fluorescent staining profiles and from isolation of actin fractions from whole lenses. Both methods showed a twofold reduction in the F-actin/G-actin ratio in KO lenses, whereas no difference in tubulin organization was detected. In vitro experiments showed that recombinant mouse γS can directly stabilize F-actin. This suggests that γS may have a functional role related to actin, perhaps in ‘shepherding’ filaments to maintain the optical properties of the lens cytoplasm and normal fiber cell maturation.
actin cytoskeleton; crystallins; knock-out mouse; organelles
Oligomerization of the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) is important for optimal ligand binding and internalization. M6P/IGF2R is a tumor suppressor gene that exhibits loss of heterozygosity and is mutated in several cancers. We tested the potential dominant-negative effects of two cancer-associated mutations that truncate the M6P/IGF2R in ectodomain repeats 9 and 14. Our hypothesis was that co-expression of the truncated receptors with wild-type/endogenous, full-length M6P/IGF2R would interfere with M6P/IGF2R function by heterodimer interference. Immunoprecipitation confirmed formation of heterodimeric complexes between full-length M6P/IGF2Rs and the truncated receptors, termed Rep9F and Rep14F. Remarkably, increasing expression of either Rep9F or Rep14F provoked decreased levels of full-length M6P/IGF2Rs in both cell lysates and plasma membranes, indicating a dominant-negative effect on receptor availability. Loss of full-length M6P/IGF2R was not due to increased proteasomal or lysosomal degradation, but instead arose from increased proteolytic cleavage of cell-surface M6P/IGF2Rs resulting in ectodomain release, by a mechanism that was inhibited by metal ion chelators. These data suggest that M6P/IGF2R truncation mutants may contribute to the cancer phenotype by decreasing availability of full-length M6P/IG2Rs to perform tumor-suppressive functions such as binding/internalization of receptor ligands like IGF-II.
mannose 6-phosphate receptor; truncation mutants; dimerization; dominant negative; ectodomain shedding
HMGN1 is a nuclear protein that binds to nucleosomes and alters the accessibility of regulatory factors to their chromatin targets. To elucidate its biological function and identify specific HMGN1 target genes, we generated Hmgn1−/− mice. DNA microarray analysis of Hmgn1+/+ and Hmgn1−/− embryonic fibroblasts identified N-cadherin as a potential HMGN1 gene target. RT-PCR and western blot analysis confirmed a linkage between HMGN1 expression and N-cadherin levels. In both transformed and primary mouse embryonic fibroblasts (MEFs), HMGN1 acted as negative regulator of N-cadherin expression. Likewise, the N-cadherin levels in early embryos of Hmgn1−/− mice were higher than those of their Hmgn1+/+ littermates. Loss of HMGN1 increased the adhesiveness, motility and aggregation potential of Hmgn1−/− MEFs, a phenotype consistent with increased levels of N-cadherin protein. Re-expression of wildtype HMGN1, but not of the mutant HMGN1 protein that does not bind to chromatin, in Hmgn1−/− MEFs, decreased the levels of N-cadherin and restored the Hmgn1+/+ phenotype. These studies demonstrate a role for HMGN1 in the regulation of specific gene expression. We suggest that in MEFs, and during early mouse development, the interaction of HMGN1 with chromatin down-regulates the expression of N-cadherin.
HMG protein; N-cadherin; chromatin; transcription
There is ample evidence to suggest that a dramatic decrease in mitochondrial Ca2+ retention may contribute to the cell death associated with stroke, excitotoxicity, ischemia and reperfusion, and neurodegenerative diseases. Mitochondria from all studied tissues can accumulate and store Ca2+, but the maximum Ca2+ storage capacity varies widely and exhibits striking tissue specificity. There is currently no explanation for this fact. Precipitation of Ca2+ and phosphate in the mitochondrial matrix has been suggested to be the major form of storage of accumulated Ca2+ in mitochondria. How this precipitate is formed is not known. The molecular identity of almost all proteins involved in Ca2+ transport, storage and formation of the permeability transition pore is also unknown. This review summarizes studies aimed at identifying these proteins, and describes the properties of a known mitochondrial protein that may be involved in Ca2+ transport and the structure of the permeability transition pore.
brain mitochondria; Ca2+ accumulation; Ca2+ and Pi precipitate; calciphorin; calcium uniporter; calvectin; dense granules; gC1qR; liver mitochondria; permeability transition pore
In search of molecules involved in the interaction of intestinal nematodes and mammalian mucosal host cells, we performed mass spectrometry to identify excretory/secretory proteins (ESP) from Strongyloides ratti. In addition to other peptides, we detected in the ESP of parasitic female stage peptides homologous to the Caenorhabditis elegans heat shock protein-17, named Sra-HSP-17.1 (~19 kDa) and Sra-HSP-17.2 (~ 18 kDa) with 49% amino acid identity. The full-length cDNAs (483 bp and 474 bp, respectively) were identified and the genomic organization analyzed. To allow further characterization, the proteins were recombinantly expressed and purified. Profiling of transcription by qRT-PCR and of protein by ELISA in various developmental stages revealed parasitic female-specific expression. The sequence analysis of both DNA and amino acid sequence showed two genes share a conserved alpha-crystallin domain and variable N-terminals. The Sra-HSP-17 proteins showed the highest homology to the deduced small heat-shock protein sequence of the human pathogen S. stercoralis. We observed strong immunogenicity of both proteins, leading to high IgG responses following infection of rats. Flow cytometric analysis indicated the binding of Sra-HSP-17s to the monocytes/macrophage lineage but not to peripheral lymphocytes or neutrophils. A rat intestinal epithelial cell line showed dose dependent binding to Sra-HSP-17.1, but not to Sra-HSP-17.2. Exposed monocytes released IL-10 but not TNF-alpha in response to Sra-HSP-17s, suggesting a possible involvement of secreted female proteins in host immune responses.
Heat shock proteins; HSP-17; Strongyloides; excretory/secretory proteins; immune response
Pathophysiologic responses in brain after stroke are highly complex. Thus far, a singular focus on saving neurons alone has not revealed any clinically effective neuroprotectants. To address this limitation, the concept of a neurovascular unit was developed. Within this conceptual framework, brain function and dysfunction are manifested at the level of cell–cell signaling between neuronal, glial and vascular elements. For stroke, coordinated responses at the neurovascular interface will mediate acute as well as chronic events in ischemic and hemorrhagic brain tissue. In this minireview, we briefly survey two representative examples of neurovascular responses in stroke. During the early acute phase of neurovascular injury, blood– brain barrier perturbations should predominate with key roles for various matrix proteases. During the delayed phase, brain angiogenesis may provide the critical neurovascular substrates for neuronal remodeling. In this minireview, we propose the hypothesis that the biphasic nature of neuro-vascular responses represents an endogenous attempt by damaged parenchyma to trigger brain angiogenesis and repair. This phenomenon may allow acute deleterious signals to transition into beneficial effects during stroke recovery. Understanding how neurovascular signals and substrates make the transition from initial injury to angiogenic recovery will be important if we are to find new therapeutic approaches for stroke.
angiogenesis; edema; endothelial progenitor cell; hemorrhage; ischemia; matrix metalloproteinase; neurogenesis; neurovascular unit; remodeling; stroke
The ryanodine receptor (RyR) is a large, homotetrameric sarcoplasmic reticulum (SR) membrane protein essential for calcium cycling in both skeletal and cardiac muscle. Genetic mutations in RyR1 are associated with severe conditions including malignant hyperthermia (MH) and central core disease (CCD). One phosphorylation site (Ser 2843) has been identified in a segment of RyR1 flanked by two RyR motifs, which are found exclusively in all RyR isoforms organized as closely-associated tandem (or paired) motifs, and named after the protein itself. These motifs also contain six known MH mutations. In this study we designed, expressed, and purified the tandem RyR motifs and show that this domain contains a putative binding site for the Ca2+/calmodulin-dependent protein kinase beta isoform. We present a 2.2Å resolution crystal structure of the RyR domain revealing a two-fold, symmetric, extended four-helix bundle stabilized by a β-sheet. Docking experiments with a tetrameric EM structure of native RyR1 suggest that this domain is localized in the RyR’s clamp region, which is absent in its cousin protein inositol 1,4,5-trisphosphate receptor
PMID: 22913516 CAMSID: cams2798
x-ray crystallography; excitation contraction; RyR
Detailed characterization of LRRK2 function may provide insight into the molecular basis of neurodegeneration in Parkinson’s disease (PD) since mutations in LRRK2 cause a phenotype with strong overlap to typical late-onset disease and LRRK2 mutations are responsible for significant proportions of PD in some populations. The complexity of large multi-domain protein kinases like LRRK2 challenge traditional functional approaches, although initial studies have successfully defined the basic mechanisms of enzyme activity with putative effects of pathogenic mutations on kinase activity. The role of LRRK2 in cells remains elusive, with potential function in MAP kinase pathways, protein translation control, programmed cell death pathways, and activity in cytoskeleton dynamics. The initial focus on LRRK2-kinase dependent phenomena places emphasis on the discovery of LRRK2 kinase substrates, although candidate substrates are yet confined to in vitro assays. Herein, hypothetical mechanisms for LRRK2-mediated cell death and kinase activation are proposed. As a promising target for neuroprotection strategies in PD, in vitro and in vivo models that accurately demonstrate LRRK2 function relevant to neurodegeneration will aide in the identification of molecules with the highest chance for success in the clinic.
Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2) during myocardial ischemia/reperfusion (I/R) injury has been established. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP-2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). If hearts were subjected to I/R in the presence of ML-7 (a myosin light chain kinase (MLCK) inhibitor) or doxycycline (a MMP inhibitor) an improved recovery of contractile function was seen compared to aerobic hearts and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP-2 for the phosphorylated form of MLC1 compared to non-phosphorylated MLC1. We conclude that MLC1 phosphorylation is important mechanism controlling the intracellular action of MMP-2 and promoting the degradation of MLC1. These results further support previous findings implicating posttranslational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia.
myosin light chain; phosphorylation; matrix metalloproteinase; ischemia-reperfusion; ML-7; doxycycline