Three members of a novel small gene-family from Arabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and “APS reductase” activity

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector λYES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5′-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35S-labeled substrate to acid-volatile 35S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel “APS reductase” enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of ≈1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.

Abrogation of the alternative complement pathway by targeted deletion of murine factor B

To investigate the role of complement protein factor B (Bf) and alternative pathway activity in vivo, and to test the hypothesized potential genetic lethal effect of Bf deficiency, the murine Bf gene was interrupted by exchange of exon 3 through exon 7 (including the factor D cleaving site) with the neor gene. Mice heterozygous for the targeted Bf allele were interbred, yielding Bf-deficient offspring after the F1 generation at a frequency suggesting that Bf deficiency alone has no major effect on fertility or fetal development. However, in the context of one or more genes derived from the 129 mouse strain, offspring homozygous for Bf deficiency were generated at less than expected numbers (P = 0.012). Bf-deficient mice showed no gross phenotypic difference from wild-type littermates. Sera from Bf-deficient mice lacked detectable alternative complement pathway activity; purified mouse Bf overcame the deficit. Classical pathway-dependent total hemolytic activity was lower in Bf-deficient than wild-type mice, possibly reflecting loss of the alternative pathway amplification loop. Lymphoid organ structure and IgG1 antibody response to a T-dependent antigen appeared normal in Bf-deficient mice. Sensitivity to lethal endotoxic shock was not significantly altered in Bf-deficient mice. Thus, deficiency of Bf and alternative complement activation pathway led to a less dramatic phenotype than expected. Nevertheless, these mice provide an excellent model for the assessment of the role of Bf and the alternative pathway in host defense and other functions in vivo.

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR–thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.

An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

A Rab2 Mutant with Impaired GTPase Activity Stimulates Vesicle Formation from Pre-Golgi Intermediates

Rab2 immunolocalizes to pre-Golgi intermediates (vesicular-tubular clusters [VTCs]) that are the first site of segregation of anterograde- and retrograde-transported proteins and a major peripheral site for COPI recruitment. Our previous work showed that Rab2 Q65L (equivalent to Ras Q61L) inhibited endoplasmic reticulum (ER)-to-Golgi transport in vivo. In this study, the biochemical properties of Rab2 Q65L were analyzed. The mutant protein binds GDP and GTP and has a low GTP hydrolysis rate that suggests that Rab2 Q65L is predominantly in the GTP-bound–activated form. The purified protein arrests vesicular stomatitis virus glycoprotein transport from VTCs in an assay that reconstitutes ER-to-Golgi traffic. A quantitative binding assay was used to measure membrane binding of β-COP when incubated with the mutant. Unlike Rab2 that stimulates recruitment, Rab2 Q65L showed a dose-dependent decrease in membrane-associated β-COP when incubated with rapidly sedimenting membranes (ER, pre-Golgi, and Golgi). The mutant protein does not interfere with β-COP binding but stimulates the release of slowly sedimenting vesicles containing Rab2, β-COP, and p53/gp58 but lacking anterograde grade-directed cargo. To complement the biochemical results, we observed in a morphological assay that Rab2 Q65L caused vesiculation of VTCs that accumulated at 15°C. These data suggest that the Rab2 protein plays a role in the low-temperature–sensitive step that regulates membrane flow from VTCs to the Golgi complex and back to the ER.

A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity

This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Gtx. Sequential deletions of the 5′ untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragments were tested, four had IRES activity. These observations are consistent with other cellular IRES analyses suggesting that some cellular IRESes are composed of segments (IRES modules) that independently and combinatorially contribute to overall IRES activity. We characterize a 9-nt IRES module from the Gtx 5′ UTR that is 100% complementary to the 18S rRNA at nucleotides 1132–1124. In previous work, we demonstrated that this mRNA segment could be crosslinked to its complement within intact 40S subunits. Here we show that increasing the number of copies of this IRES module in the intercistronic region of a dicistronic mRNA strongly enhances IRES activity in various cell lines. Ten linked copies increased IRES activity up to 570-fold in Neuro 2a cells. This level of IRES activity is up to 63-fold greater than that obtained by using the well characterized encephalomyocarditis virus IRES when tested in the same assay system. When the number of nucleotides between two of the 9-nt Gtx IRES modules was increased, the synergy between them decreased. In light of these findings, we discuss possible mechanisms of ribosome recruitment by cellular mRNAs, address the proposed role of higher order RNA structures on cellular IRES activity, and suggest parallels between IRES modules and transcriptional enhancer elements.

Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases.

Residue 225 in serine proteases is typically Pro or Tyr and specifies an important and unanticipated functional aspect of this class of enzymes. Proteases with Y225, like thrombin, are involved in highly specialized functions like blood coagulation and complement that are exclusively found in vertebrates. In these proteases, the catalytic activity is enhanced allosterically by Na+ binding. Proteases with P225, like trypsin, are typically involved in digestive functions and are also found in organisms as primitive as eubacteria. These proteases have no requirement for Na+ or other monovalent cations. The molecular origin of this physiologically important difference is remarkably simple and is revealed by a comparison of the Na+ binding loop of thrombin with the homologous region of trypsin. The carbonyl O atom of residue 224 makes a key contribution to the coordination shell of the bound Na+ in thrombin, but is oriented in a manner incompatible with Na+ binding in trypsin because of constraints imposed by P225 on the protein backbone. Pro at position 225 is therefore incompatible with Na+ binding and is a direct predictor of the lack of allosteric regulation in serine proteases. To directly test this hypothesis, we have engineered the thrombin mutant Y225P. This mutant has lost the ability to bind Na+ and behaves like the allosteric slow (Na(+)-free) form. The Na(+)-induced allosteric regulation also bears on the molecular evolution of serine proteases. A strong correlation exists between residue 225 and the codon used for the active site S195. Proteases with P225 typically use a TCN codon for S195, whereas proteases with Y225 use an AGY codon. It is proposed that serine proteases evolved from two main lineages: (i) TCN/P225 with a trypsin-like ancestor and (ii) AGY/Y225 with a thrombin-like ancestor. We predict that the Na(+)-induced allosteric regulation of catalytic activity can be introduced in the TCN/P225 lineage using the P225Y replacement.

Identification of three physically and functionally distinct binding sites for C3b in human complement factor H by deletion mutagenesis.

Human complement factor H controls spontaneous activation of complement in plasma and appears to play a role in distinguishing host cells from activators of the alternative pathway of complement. In both mice and humans, the protein is composed of 20 homologous short consensus repeat (SCR) domains. The size of the protein suggests that portions of the structure outside the known C3b binding site (SCR 1-4) possess a significant biological role. We have expressed the full-length cDNA of factor H in the baculovirus system and have shown the recombinant protein to be fully active. Mutants of this full-length protein have now been prepared, purified, and examined for cofactor activity and binding to C3b and heparin. The results demonstrate (i) that factor H has at least three sites that bind C3b, (ii) that one of these sites is located in SCR domains 1-4, as has been shown by others, (iii) that a second site exists in the domain 6-10 region, (iv) that a third site resides in the SCR 16-20 region, and (v) that two heparin binding sites exist in factor H, one near SCR 13 and another in the SCR 6-10 region. Functional assays demonstrated that only the first C3b site located in SCR 1-4 expresses factor I cofactor activity. Mutant proteins lacking any one of the three C3b binding sites exhibited 6- to 8-fold reductions in affinity for C3b on sheep erythrocytes, indicating that all three sites contribute to the control of complement activation on erythrocytes. The identification of multiple functionally distinct sites on factor H clarifies many of the heretofore unexplainable behaviors of this protein, including the heterogeneous binding of factor H to surface-bound C3b, the effects of trypsin cleavage, and the differential control of complement activation on activators and nonactivators of the alternative pathway of complement.