Isolation and characterization of the B-cell marker CD20

The integral membrane protein CD20 has been identified as an important therapeutic target in the treatment of non-Hodgkin's lymphoma (NHL). CD20 binding of many antibodies including the therapeutic antibody, rituximab, has been shown to be critically dependent upon the conformation of a loop structure between the third and fourth helical transmembrane regions. In this work, human and murine CD20 proteins expressed in Escherichia coli are shown to be localized with the cell membrane and are purified in nondenaturing detergent solutions. The purified human and murine CD20 proteins have a substantial helical structure as measured by circular dichroism spectroscopy. Only small changes in the secondary structure are observed following the reduction of CD20, with the addition of SDS, or after heating. The rituximab antibody is shown to bind to purified human CD20 with nanomolar affinity. Rituximab binding is abolished by reduction and alkylation of CD20, with data consistent with the proposed antibody epitope being within the disulfide-bonded loop formed between cysteine residues 167 and 183. Disulfide-bond-dependent antibody binding is partially recovered following reoxidation of reduced CD20. Antibody binding is unaffected by mutations of cysteines proposed to be in the intracellular domain of CD20. The affinities of intact rituximab and its Fab fragment to the isolated and purified CD20 are similar to the observed affinity of rituximab Fab for CD20 on the surface of B cells. However, the intact rituximab antibody shows much higher affinity for CD20 on B cells. This suggests that B cells display CD20 in such a way that allows for marked avidity effects to be observed, perhaps through cross-linking of CD20 monomers into lipid rafts, which limits receptor diffusion in the membrane. Such cross-linking may play a role in partitioning CD20 into lipid rafts and in enhancing antibody-dependent B-cell depletion activities of rituximab and other therapeutic anti-CD20 antibodies.     

CD20 Antibody Primes B Lymphocytes for Type I Interferon Production

CD20 is a B cell surface marker that is expressed in various stages in B lymphocytes and certain lymphomas. Clinical administration of CD20 antibody, such as rituximab, is used widely to treat human B-cell lymphomas and other diseases. However, CD20 antibody failed to treat systemic lupus erythematosus (SLE or lupus). The reason for the failure is currently unknown. Type I interferons (IFN) are a major component for the host innate immunity, and a key pathogenic factor in lupus. We found that CD20 antibody potentiated human B cells for its production of IFNs in vitro. This function was specific to CD20-expressing cells and the potentiation function seems to be instant. In addition, ectopic expression of CD20 in non-B-lymphocytes increased the IFN promoter reporter activities. Because IFNs are a key pathogenic factor in lupus, our data suggest that, in the presence of virus infection, the CD20-antibody-mediated enhancement of IFN production might be related to its failure in lupus treatments. This work may provide new insights for CD20-Ab therapeutic applications.     

Functional characterization of the Drosophila Hmt4-20/Suv4-20 histone methyltransferase

Di- and trimethylation of histone H4 lysine20 (H4K20) are thought to play an important role in controlling gene expression in vertebrates and in Drosophila. By inducing a null mutation in Drosophila Suv4-20, we show that it encodes the histone H4 lysine20 di- and trimethyltransferase. In Suv4-20 mutants, the H4K20 di- and trimethyl marks are strongly reduced or absent, and the monomethyl mark is significantly increased. We find that even with this biochemical function, Suv4-20 is not required for survival and does not control position-effect variegation (PEV).     

A ubiquitin-binding protein, FAAP20, links RNF8-mediated ubiquitination to the Fanconi anemia DNA repair network

The Fanconi anemia (FA) protein network is necessary for repair of DNA interstrand crosslinks (ICLs), but its control mechanism remains unclear. Here we show that the network is regulated by a ubiquitin signaling cascade initiated by RNF8 and its partner, UBC13, and mediated by FAAP20, a component of the FA core complex. FAAP20 preferentially binds the ubiquitin product of RNF8-UBC13, and this ubiquitin-binding activity and RNF8-UBC13 are both required for recruitment of FAAP20 to ICLs. Both RNF8 and FAAP20 are required for recruitment of FA core complex and FANCD2 to ICLs, whereas RNF168 can modulate efficiency of the recruitment. RNF8 and FAAP20 are needed for efficient FANCD2 monoubiquitination, a key step of the FA network; RNF8 and the FA core complex work in the same pathway to promote cellular resistance to ICLs. Thus, the RNF8-FAAP20 ubiquitin cascade is critical for recruiting FA core complex to ICLs and for normal function of the FA network.     

Domain stealing by receptors in a protein transport complex

The mitochondrion is an essential cellular compartment in eukaryotes. The mitochondrial proteins Tom20 and Tom22 are receptors that ensure recognition and binding of proteins imported for mitochondrial biogenesis. Comparison of the sequence for the Tom20 and Tom22 subunits in the yeasts Saccharomyces cerevisiae and Saccharomyces castellii, show a rare case of domain stealing, where in Saccharomyces castellii Tom22 has lost an acidic domain, and Tom20 has gained one. This example of domain stealing is a snapshot of evolution in action and provides excellent evidence that Tom20 and Tom22 are subunits of a single, composite receptor that binds precursor proteins for import into mitochondria.     

A role for the peroxin Pex8p in Pex20p-dependent thiolase import into peroxisomes of the yeast Yarrowia lipolytica

Peroxins are proteins required for peroxisome assembly. The cytosolic peroxin Pex20p binds directly to the beta-oxidation enzyme thiolase and is necessary for its dimerization and peroxisomal targeting. The intraperoxisomal peroxin Pex8p has a role in the import of peroxisomal matrix proteins, including thiolase. We report the results of yeast two-hybrid analyses with various peroxins of the yeast Yarrowia lipolytica and characterize more fully the interaction between Pex8p and Pex20p. Coimmunoprecipitation showed that Pex8p and Pex20p form a complex, while in vitro binding studies demonstrated that the interaction between Pex8p and Pex20p is specific, direct, and autonomous. Pex8p fractionates with peroxisomes in cells of a PEX20 disruption strain, indicating that Pex20p is not necessary for the targeting of Pex8p to peroxisomes. In cells of a PEX8 disruption strain, thiolase is mostly cytosolic, while Pex20p and a small amount of thiolase associate with peroxisomes, suggesting the involvement of Pex8p in the import of thiolase after docking of the Pex20p-thiolase complex to the membrane. In the absence of Pex8p, peroxisomal thiolase and Pex20p are protected from the action of externally added protease. This finding, together with the fact that Pex8p is intraperoxisomal, suggests that Pex20p may accompany thiolase into peroxisomes during import.     

The CDC20 gene product of Saccharomyces cerevisiae, a beta-transducin homolog, is required for a subset of microtubule-dependent cellular processes.

Previous analysis of cdc20 mutants of the yeast Saccharomyces cerevisiae suggests that the CDC20 gene product (Cdc20p) is required for two microtubule-dependent processes, nuclear movements prior to anaphase and chromosome separation. Here we report that cdc20 mutants are defective for a third microtubule-mediated event, nuclear fusion during mating of G1 cells, but appear normal for a fourth microtubule-dependent process, nuclear migration after DNA replication. Therefore, Cdc20p is required for a subset of microtubule-dependent processes and functions at multiple stages in the life cycle. Consistent with this interpretation, we find that cdc20 cells arrested by alpha-factor or at the restrictive temperature accumulate anomalous microtubule structures, as detected by indirect immunofluorescence. The anomalous microtubule staining patterns are due to cdc20 because intragenic revertants that revert the temperature sensitivity have normal microtubule morphologies. cdc20 mutants have a sevenfold increase in the intensity of antitubulin fluorescence in intranuclear spindles compared with spindles from wild-type cells, yet the total amount of tubulin is indistinguishable by Western immunoblot analysis. This result suggests that Cdc20p modulates microtubule structure in wild-type cells either by promoting microtubule disassembly or by altering the surface of the microtubules. Finally, we cloned and sequenced CDC20 and show that it encodes a member of a family of proteins that share homology to the beta subunit of transducin.     

Interplay of JH, 20E and biogenic amines under normal and stress conditions and its effect on reproduction

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) are well known to play a gonadotropic role in adult insects. In Drosophila the mechanism of reciprocal regulation of JH and 20E is shown to be responsible for their proper balance. Dopamine is a mediator in this JH and 20E interplay. A proper balance between JH and 20E is crucial for the normal progress of oogenesis. An imbalance of gonadotropins leads to reproductive defects: a rise in JH titre leads to oviposition arrest, a rise in 20E level, to the degradation of vitellogenic oocytes. Upon a change in the level of one of the gonadotropins, the balance is restored owing to the relative change in the titre of the other.     

Steroid hormone inactivation is required during the juvenile-adult transition in Drosophila

Steroid hormones are systemic signaling molecules that regulate juvenile-adult transitions in both insects and mammals. In insects, pulses of the steroid hormone 20-hydroxyecdysone (20E) are generated by increased biosynthesis followed by inactivation/clearance. Although mechanisms that control 20E synthesis have received considerable recent attention, the physiological significance of 20E inactivation remains largely unknown. We show that the cytochrome P450 Cyp18a1 lowers 20E titer during the Drosophila prepupal to pupal transition. Furthermore, this reduction of 20E levels is a prerequisite to induce βFTZ-F1, a key factor in the genetic hierarchy that controls early metamorphosis. Resupplying βFTZ-F1 rescues Cyp18a1-deficient prepupae. Because Cyp18a1 is 20E-inducible, it appears that the increased production of steroid is responsible for its eventual decline, thereby generating the regulatory pulse required for proper temporal progression of metamorphosis. The coupling of hormone clearance to βFTZ-F1 expression suggests a general mechanism by which transient signaling drives unidirectional progression through a multistep process.     

Thermodynamic analysis of monoclonal antibody binding to duplex DNA.

A technique based on fluorescence polarization (anisotropy) was used to measure the binding of antibodies to DNA under a variety of conditions. Fluorescein-labeled duplexes of 20 bp in length were employed as the standard because they are stable even at low ionic strength yet sufficiently short so that both arms of an IgG cannot bind to the same duplex. IgG Jel 274 binds duplexes in preference to single-stranded DNA; in 80 mM NaCl Kobs for (dG)20.(dC)20 is 4.1x10(7) M-1 compared with 6.4x10(5) M-1 for d(A5C10A5). There is little sequence specificity, but the interaction is very dependent on ionic strength. From plots of log Kobs against log[Na+] it was deduced that five or six ion pairs are involved in complex formation. At low ionic strength,Kobs is independent of temperature and complex formation is entropy driven with DeltaH degrees obs and DeltaC degrees p,obs both zero. In contrast, in 80 mM NaCl DeltaC degrees p,obs is -630 and -580 cal mol-1K-1 for [d(TG)]10.[d(CA)]10 and (dG)20.(dC)20 respectively. IgG Jel 241 also binds more tightly to duplexes than single-stranded DNA, but sequence preferences were apparent. The values for Kobs to [d(AT)]20 and [d(GC)]20 are 2.7x10(8) and 1.3x10(8) M-1 respectively compared with 5.7x10(6) M-1 for both (dA)20. (dT)20 and (dG)20.(dC)20. As with Jel 274, the binding of Jel 241 is very dependent on ionic strength and four or five ionic bonds are involved in complex formation with all the duplex DNAs which were tested. DeltaC degrees p,obs for Jel 241 binding to [d(AT)]20 was negative (-87 cal mol-1K-1) in 80 mM NaCl but was zero at high ionic strength (130 mM NaCl). Therefore, for duplex-specific DNA binding antibodies DeltaC degrees p,obs is dependent on [Na+] and a large negative value does not correlate with sequence-specific interactions.     

Yeast PalA/AIP1/Alix Homolog Rim20p Associates with a PEST-Like Region and Is Required for Its Proteolytic Cleavage

The Saccharomyces cerevisiae zinc finger protein Rim101p is activated by cleavage of its C-terminal region, which resembles PEST regions that confer susceptibility to proteolysis. Here we report that Rim20p, a member of the broadly conserved PalA/AIP1/Alix family, is required for Rim101p cleavage. Two-hybrid and coimmunoprecipitation assays indicate that Rim20p binds to Rim101p, and a two-hybrid assay shows that the Rim101p PEST-like region is sufficient for Rim20p binding. Rim101p-Rim20p interaction is conserved in Candida albicans, supporting the idea that interaction is functionally significant. Analysis of Rim20p mutant proteins indicates that some of its broadly conserved regions are required for processing of Rim101p and for stability of Rim20p itself but are not required for interaction with Rim101p. A recent genome-wide two-hybrid study (T. Ito, T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, and Y. Sakaki, Proc. Natl. Acad. Sci. USA 98:4569-4574, 2000) indicates that Rim20p interacts with Snf7p and that Snf7p interacts with Rim13p, a cysteine protease required for Rim101p proteolysis. We suggest that Rim20p may serve as part of a scaffold that places Rim101p and Rim13p in close proximity.     

Suv4-20h deficiency results in telomere elongation and derepression of telomere recombination

Mammalian telomeres have heterochromatic features, including trimethylated histone H3 at lysine 9 (H3K9me3) and trimethylated histone H4 at lysine 20 (H4K20me3). In addition, subtelomeric DNA is hypermethylated. The enzymatic activities responsible for these modifications at telomeres are beginning to be characterized. In particular, H4K20me3 at telomeres could be catalyzed by the novel Suv4-20h1 and Suv4-20h2 histone methyltransferases (HMTases). In this study, we demonstrate that the Suv4-20h enzymes are responsible for this histone modification at telomeres. Cells deficient for Suv4-20h2 or for both Suv4-20h1 and Suv4-20h2 show decreased levels of H4K20me3 at telomeres and subtelomeres in the absence of changes in H3K9me3. These epigenetic alterations are accompanied by telomere elongation, indicating a role for Suv4-20h HMTases in telomere length control. Finally, cells lacking either the Suv4-20h or Suv39h HMTases show increased frequencies of telomere recombination in the absence of changes in subtelomeric DNA methylation. These results demonstrate the importance of chromatin architecture in the maintenance of telomere length homeostasis and reveal a novel role for histone lysine methylation in controlling telomere recombination.     

Multiple chaperone-assisted formation of mammalian 20S proteasomes

Protein degradation is essential for maintenance of cellular homeostasis. The majority of proteins are selectively degraded in eukaryotic cells by the ubiquitin-proteasome system. The 26S proteasome selects target proteins that are covalently modified with polyubiquitin chains. The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. The catalytic activities are carried out by the core 20S proteasome. The eukaryotic 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, alpha1-7beta1-7beta1-7alpha1-7. Recent studies have revealed the mechanism responsible for the assembly of such a complex structure. This article recounts the observations that disclosed the biogenesis of 20S proteasomes and discusses the difference in the mechanism of assembly between archael, yeast, and mammalian 20S proteasomes.     

Characterization of WDR20: A new regulator of the ERAD machinery

ERAD is an important process of protein quality control that eliminates misfolded or unassembled proteins from ER. Before undergoing proteasome degradation, the misfolded proteins are dislocated from ER membrane into cytosol, which requires the AAA ATPase p97/VCP and its cofactor, the NPL4-UFD1 dimer. Here, we performed a CRISPR-based screen and identify many candidates for ERAD regulation. We further confirmed four proteins, FBOX2, TRIM6, UFL1 and WDR20, are novel regulators for ERAD. Then the molecular mechanism for WDR20 in ERAD is further characterized. Depletion of WDR20 inhibits the degradation of TCRα, a typical ERAD substrate, while WDR20 overexpression reduces TCRα protein level. WDR20 associates with TCRα and central regulators of the ERAD system, p97, GP78 and HRD1. A portion of WDR20 localizes to the ER-containing microsomal membrane. WDR20 expression increases TCRα ubiquitination, and HRD1 E3 ligase is essential for the process. WDR20 seems to serve as an adaptor protein to mediate the interaction between p97 and TCRα. Our study provides novel candidates and reveals an unexpected role of WDR20 in ERAD regulation.     

Significance of the N-terminal domain for the function of chloroplast cpn20 chaperonin

Chaperonins cpn60 and cpn10 are essential proteins involved in cellular protein folding. Plant chloroplasts contain a unique version of the cpn10 co-chaperonin, cpn20, which consists of two homologous cpn10-like domains (N-cpn20 and C-cpn20) that are connected by a short linker region. Although cpn20 seems to function like other single domain cpn10 oligomers, the structure and specific functions of the domains are not understood. We mutated amino acids in the "mobile loop" regions of N-cpn20, C-cpn20 or both: a highly conserved glycine, which was shown to be important for flexibility of the mobile loop, and a leucine residue shown to be involved in binding of co-chaperonin to chaperonin. The mutant proteins were purified and their oligomeric structure validated by gel filtration, native gel electrophoresis, and circular dichroism. Functional assays of protein refolding and inhibition of GroEL ATPase both showed (i) mutation of the conserved glycine reduced the activity of cpn20, whether in N-cpn20 (G32A) or C-cpn20 (G130A). The same mutation in the bacterial cpn10 (GroES G24A) had no effect on activity. (ii) Mutations in the highly conserved leucine of N-cpn20 (L35A) and in the corresponding L27A of GroES resulted in inactive protein. (iii) In contrast, mutant L133A, in which the conserved leucine of C-cpn20 was altered, retained 55% activity. We conclude that the structure of cpn20 is much more sensitive to alterations in the mobile loop than is the structure of GroES. Moreover, only N-cpn20 is necessary for activity of cpn20. However, full and efficient functioning requires both domains.     

Speriolin is a novel spermatogenic cell-specific centrosomal protein associated with the seventh WD motif of Cdc20

The fundamental mechanisms of mitosis are conserved throughout evolution in eukaryotes, including ubiquitin-mediated proteolysis of cell cycle regulators by the anaphase-promoting complex/cyclosome. The spindle checkpoint protein Cdc20 activates the anaphase-promoting complex/cyclosome in a substrate-specific manner. It is present in the cytoplasm and concentrated in the centrosomes throughout the cell cycle, accumulates at the kinetochores in metaphase, and is no longer detected following anaphase. However, it is unknown whether Cdc20 has the same activities and distribution during meiosis in male germ cells. We found that in mice, Cdc20 accumulates in the cytoplasm of pachytene spermatocytes during meiosis I, is distributed throughout spermatocytes undergoing meiotic division, and is present in the cytoplasm of postmeiotic spermatids. Several proteins bind to and regulate the function of Cdc20 during mitosis. We identified speriolin and determined that it is a novel spermatogenic cell-specific Cdc20-binding protein, is present in the cytoplasm, and is concentrated at the centrosomes of spermatocytes and spermatids and that a leucine zipper domain is required to target speriolin to the centrosome. The seven tandem WD motifs of Cdc20 probably fold into a seven-blade beta-propeller structure, and we determined that they are required for speriolin binding and for localization of Cdc20 to the centrosomes and nucleus, suggesting that speriolin might regulate or stabilize the folding of Cdc20 during meiosis in spermatogenic cells.