N2-guanine specific transfer RNA methyltransferase II from rat liver

An enzyme was purified from rat liver and leukemic rat spleen which methylates guanosine residues in tRNA to N²-methylguanosine. By sequence analysis of bulk E. coli tRNA methylated with crude extracts it was shown that the enzyme is responsible for about 50% of total m²G formed invitro. The extent of methylation of a number of homogenous tRNA species was measured using the purified enzyme from both sources. Among tested E. coli tRNAs only tRNA^Arg, tRNA^Phe, and tRNA^Val yielded significantly more m²G than the bulk tRNA. The K_m for tRNA^Arg in the methylation reaction with enzymes from either tissue was 7.8 × 10⁻⁷ M as compared to the value 1 × 10⁻⁵ M obtained for the bulk tRNA. In a pancreatic RNase digest of bulk tRNA as well as of pure tRNA^Arg, tRNA^Phe, and tRNA^Val, A-m²G-Cp was found to be the only sequence methylated. Thus, the mammalian methyltransferase specifically recognizes the guanylate residue at position 10 from the 5′-end contained in a sequence (s⁴)U-A-G-Cp. Furthermore, there is no change between the enzyme from normal liver and leukemic spleen in the affinity for tRNA, the methylating capacity, and tRNA site and sequence recognition specificity.     

Human ISCA1 Interacts with IOP1/NARFL and Functions in Both Cytosolic and Mitochondrial Iron-Sulfur Protein Biogenesis

Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.     

Properties of a Bacillus subtilis strain lacking DNA polymerase I

We have isolated a mutant of Bacillussubtilis deficient in DNA polymerase I, denominated polA42, which shows a reduced ability to repair the damage to DNA by UV radiation, MMS and mitomycin C;the ability to perform recombination is not appreciably impaired.DEAE cellulose chromatography allows the separation of polymerases I and II from the parental strain;a simple procedure is also described which allows to separate rapidly the polymerases II and III of the mutant strain. The three separated polymerases have similar catalytic properties but they can be distinguished for their sensitivity to inhibitors: PCMB inhibits polymerases II and III but not polymerase I; HPUra inhibits only polymerase III. All three enzymes are unaffected by nalidixate. The DNA synthesis occurring in cells of the polA42 strain permeabilized with toluene is inhibited by nalidixate, whereas the synthesis occurring in polA⁺ toluenized cells is unaffected by the drug. The polA gene has been mapped by transduction and localized between the phe₁₂ and argA₃ genes.     

Ubiquitin Regulates GGA3-mediated Degradation of BACE1

BACE1 (β-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of β-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1–3 (Golgi-localized γ-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and β-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.     

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties

The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.     

Collagen-binding Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM) of Gram-positive Bacteria Inhibit Complement Activation via the Classical Pathway

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r₂C1s₂ was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens.     

Transposon Assisted Gene Insertion Technology (TAGIT): A Tool for Generating Fluorescent Fusion Proteins

We constructed a transposon (transposon assisted gene insertion technology, or TAGIT) that allows the random insertion of gfp (or other genes) into chromosomal loci without disrupting operon structure or regulation. TAGIT is a modified Tn5 transposon that uses Kan^R to select for insertions on the chromosome or plasmid, β-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. The resulting gfp insertions maintain target gene reading frame (to the 5′ and 3′ of gfp) and are integrated at the native chromosomal locus, thereby maintaining native expression signals. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. Several of these latter GFP insertions localize to lacO arrays integrated in the E. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. TAGIT thereby faciliates the isolation of fully functional insertions of fluorescent proteins into target proteins expressed from the native chromosomal locus as well as potentially useful partially functional proteins.     

Differential Contributions of the Outer Membrane Receptors PhuR and HasR to Heme Acquisition in Pseudomonas aeruginosa

Pseudomonas aeruginosa PAO1 encodes two outer membrane receptors, PhuR (Pseudomonas heme uptake) and HasR (heme assimilation system). The HasR and PhuR receptors have distinct heme coordinating ligands and substrate specificities. HasR is encoded in an operon with a secreted hemophore, HasAp. In contrast the non-hemophore-dependent PhuR is encoded within an operon along with proteins required for heme translocation into the cytoplasm. Herein we report on the contributions of the HasR and PhuR receptors to heme uptake and utilization. Employing bacterial genetics and isotopic [¹³C]heme labeling studies we have shown both PhuR and HasR are required for optimal heme utilization. However, the unique His-Tyr-ligated PhuR plays a major role in the acquisition of heme. In contrast the HasR receptor plays a primary role in the sensing of extracellular heme and a supplementary role in heme uptake. We propose PhuR and HasR represent non-redundant heme receptors, capable of accessing heme across a wide range of physiological conditions on colonization of the host.     

Cdc37 (Cell Division Cycle 37) Restricts Hsp90 (Heat Shock Protein 90) Motility by Interaction with N-terminal and Middle Domain Binding Sites

The ATPase-driven dimeric molecular Hsp90 (heat shock protein 90) and its cofactor Cdc37 (cell division cycle 37 protein) are crucial to prevent the cellular depletion of many protein kinases. In complex with Hsp90, Cdc37 is thought to bind an important lid structure in the ATPase domain of Hsp90 and inhibit ATP turnover by Hsp90. As different interaction modes have been reported, we were interested in the interaction mechanism of Hsp90 and Cdc37. We find that Cdc37 can bind to one subunit of the Hsp90 dimer. The inhibition of the ATPase activity is caused by a reduction in the closing rate of Hsp90 without obviously bridging the two subunits or affecting nucleotide accessibility to the binding site. Although human Cdc37 binds to the N-terminal domain of Hsp90, nematodal Cdc37 preferentially interacts with the middle domain of CeHsp90 and hHsp90, exposing two Cdc37 interaction sites. A previously unreported site in CeCdc37 is utilized for the middle domain interaction. Dephosphorylation of CeCdc37 by the Hsp90-associated phosphatase PPH-5, a step required during the kinase activation process, proceeds normally, even if only the new interaction site is used. This shows that the second interaction site is also functionally relevant and highlights that Cdc37, similar to the Hsp90 cofactors Sti1 and Aha1, may utilize two different attachment sites to restrict the conformational freedom and the ATP turnover of Hsp90.     

The Urokinase Receptor Homolog Haldisin Is a Novel Differentiation Marker of Stratum Granulosum in Squamous Epithelia

Several members of the Ly-6/uPAR (LU)-protein domain family are differentially expressed in human squamous epithelia. In some cases, they even play important roles in maintaining skin homeostasis, as exemplified by the secreted single domain member, SLURP-1, the deficiency of which is associated with the development of palmoplantar hyperkeratosis in the congenital skin disorder Mal de Meleda. In the present study, we have characterized a new member of the LU-protein domain family, which we find to be predominantly expressed in the stratum granulosum of human skin, thus resembling the expression of SLURP-1. In accordance with its expression pattern, we denote this protein product, which is encoded by the LYPD5 gene, as Haldisin (human antigen with LU-domains expressed in skin). Two of the five human glycolipid-anchored membrane proteins with multiple LU-domains characterized so far are predominantly confined to squamous epithelia (i.e., C4.4A), to stratum spinosum, and Haldisin to stratum granulosum under normal homeostatic conditions. Whether Haldisin is a prognostic biomarker for certain epithelial malignancies, like C4.4A and SLURP-1, remains to be explored.     

Phosphorylation of the Cool-1/β-Pix Protein Serves as a Regulatory Signal for the Migration and Invasive Activity of Src-transformed Cells

Previously we showed that Cool-1 (Cloned out of library-1)/β-Pix (Pak-interactive exchange factor) is phosphorylated at a specific tyrosine residue (Tyr-442) in a Src-dependent manner and serves as a dual function guanine nucleotide exchange factor (GEF)/signaling-effector for Cdc42 that is essential for transformation by Src. Here, we show that knocking-down Cool-1 or overexpressing a Cool-1 mutant that contains substitutions within its Dbl homology domain and is defective for GEF activity, inhibits Src-promoted cell migration. Similarly, the expression of a Cool-1 mutant containing a tyrosine to phenylalanine substitution at position 442, making it incapable of being phosphorylated in response to serum, epidermal growth factor (EGF), or Src, also causes a significant inhibition of the migration and invasive activity of cells expressing oncogenic Src. We further demonstrate that the phosphorylation of Cool-1 at Tyr-442 weakens its ability to bind to one of its primary interaction-partners, Cat-1 (Cool-associated tyrosine phosphosubstrate-1)/Git-1 (G protein-coupled receptor kinase-interactor-1), thus making Cat more accessible for binding to paxillin. This enables cells to alternate between states where they contain large numbers of focal complexes (i.e. conditions favoring Cool-1-Cat interactions) versus reduced numbers of focal complexes (conditions favoring Cat-paxillin interactions). Overall, these findings show that the phosphorylation-dephosphorylation cycle of Cool-1 at Tyr-442 can serve as a key regulatory signal for focal complex assembly-disassembly, and consequently, for the migration and invasive activity of Src-transformed cells.     

Structure of the Legionella Effector,lpg1496, Suggests a Role in Nucleotide Metabolism

Pathogenic Gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and biochemical processes for the benefit of the pathogen. lpg1496 is a previously uncharacterized effector of Legionella pneumophila, the causative agent of Legionnaires disease. Here, we crystallized three nucleotide binding domains from lpg1496. The C-terminal domain, which is conserved among the SidE family of effectors, is formed of two largely α-helical lobes with a nucleotide binding cleft. A structural homology search has shown similarity to phosphodiesterases involved in cleavage of cyclic nucleotides. We have also crystallized a novel domain that occurs twice in the N-terminal half of the protein that we term the KLAMP domain due to the presence of homologous domains in bacterial histidine kinase-like ATP binding region-containing proteins and S-adenosylmethionine-dependent methyltransferase proteins. Both KLAMP structures are very similar but selectively bind 3′,5′-cAMP and ADP. A co-crystal of the KLAMP1 domain with 3′,5′-cAMP reveals the contribution of Tyr-61 and Tyr-69 that produces π-stacking interactions with the adenine ring of the nucleotide. Our study provides the first structural insights into two novel nucleotide binding domains associated with bacterial virulence.     

Nucleotide sequences of the 5' termini of phi X174 mRNAs synthesized in vitro

When using X174 RFI DNA as a template, in vitro, E. coli RNA polymerase synthesizes four major purine triphosphate-containing 5′ end sequences. RNase A digests of α³²P labeled RNA were further digested with spleen exonuclease to remove the bulk of the oligonucleotides with 5′ hydroxyls and then chromatographed on DEAE cellulose to resolve the remaining 5′ terminal oligonucleotides. By application of standard separation and sequence techniques, the major 5′ end sequences were shown to be: pppApUp(Cp), pppApApApUp(Cp), pppApApApApUp(Cp), and pppGpApUp(Gp).     

Structure/Function Analysis of PARP-1 in Oxidative and Nitrosative Stress-Induced Monomeric ADPR Formation

Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2^nd messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1′s structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1′s BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2^nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.     

Mycobacterium tuberculosis Prokaryotic Ubiquitin-like Protein-deconjugating Enzyme Is an Unusual Aspartate Amidase

Deamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique to bacteria, providing an ideal target for the development of selective chemotherapies. We used a combination of genetics and chemical biology to characterize the mechanism of depupylation. We identified an aspartate as a potential nucleophile in the active site of Dop, suggesting a novel protease activity to target for inhibitor development.     

BRIZ1 and BRIZ2 Proteins Form a Heteromeric E3 Ligase Complex Required for Seed Germination and Post-germination Growth in Arabidopsis thaliana

Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in a severe phenotype. Heterozygous parents produce progeny that segregate 3:1 for wild-type:growth-arrested seedlings. Homozygous T-DNA insertion lines are recovered for BRIZ1 and BRIZ2 after introduction of a transgene containing the respective coding sequence, demonstrating that disruption of BRIZ1 or BRIZ2 in the T-DNA insertion lines is responsible for the observed phenotype. Both proteins have multiple predicted domains in addition to the RING domain as follows: a BRAP2 (BRCA1-Associated Protein 2), a ZnF UBP (Zinc Finger Ubiquitin Binding protein), and a coiled-coil domain. In vitro, both BRIZ1 and BRIZ2 are active as E3 ligases but only BRIZ2 binds ubiquitin. In vitro synthesized and purified recombinant BRIZ1 and BRIZ2 preferentially form hetero-oligomers rather than homo-oligomers, and the coiled-coil domain is necessary and sufficient for this interaction. BRIZ1 and BRIZ2 co-purify after expression in tobacco leaves, which also requires the coiled-coil domain. BRIZ1 and BRIZ2 coding regions with substitutions in the RING domain are inactive in vitro and, after introduction, fail to complement their respective mutant lines. In our current model, BRIZ1 and BRIZ2 together are required for formation of a functional ubiquitin E3 ligase in vivo, and this complex is required for germination and early seedling growth.     

A Fungal Sarcolemmal Membrane-Associated Protein (SLMAP) Homolog Plays a Fundamental Role in Development and Localizes to the Nuclear Envelope,Endoplasmic Reticulum,and Mitochondria

Sarcolemmal membrane-associated protein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved striatin-interacting phosphatase and kinase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora, a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its forkhead-associated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution structured-illumination microscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.