Trafficking and proteolytic processing of RNF13, a model PA-TM-RING family endosomal membrane ubiquitin ligase.

RING finger protein 13 (RNF13) is a ubiquitously expressed, highly regulated ubiquitin ligase anchored in endosome membranes. A RING domain located in the cytoplasmic half of this type 1 membrane protein mediates ubiquitination in vitro but physiological substrates have not yet been identified. The protein localized in endosomal membranes undergoes extensive proteolysis in a proteasome-dependent manner, but the mRNA level can be increased and the encoded protein stabilized under specific physiological conditions. The cytoplasmic half of RNF13 is released from the membrane by regulatory proteases and therefore has the potential to mediate ubiquitination at distant sites independent of the full-length protein. In response to protein kinase C activation, the full-length protein is stabilized and moves to recycling endosomes and to the inner nuclear membrane, which exposes the RING domain to the nucleoplasm. Thus RNF13 is a ubiquitin ligase that can potentially mediate ubiquitination in endosomes, on the plasma membrane, in the cytoplasm, in the nucleoplasm or on the inner nuclear membrane, with the site(s) regulated by signaling events that modulate protein targeting and proteolysis.

Nuclear targeting of an endosomal E3 ubiquitin ligase.

Ring finger protein 13 (RNF13) is an E3 ubiquitin ligase embedded in endosome membranes. The protein undergoes constitutive post-translational proteolysis, making its detection difficult unless cells are incubated with a proteasome inhibitor to allow biosynthetic forms to accumulate. When cells were treated with phorbol 12-myristate 13-acetate (PMA), RNF13 avoided proteolysis. A similar stabilization was seen on ionomycin treatment of cells. Drug treatment stabilized both the full-length protein and a membrane-embedded C-terminal fragment generated following ectodomain shedding. Immunofluorescence staining revealed that PMA treatment caused the protein to accumulate in recycling endosomes, where it colocalized with transferrin receptor, and on the inner nuclear membrane, where it colocalized with lamin B. Expression of dominant-negative Rab11 inhibited nuclear localization, suggesting RNF13 was targeted to the inner nuclear membrane through recycling endosomes. New protein synthesis was necessary for this targeting. Nuclear localization was confirmed by immunoelectron microscopy and by purification of the inner nuclear membrane. Stress-induced transport of an endosomal protein to the inner nuclear membrane is a novel mechanism for introduction of regulatory proteins to the DNA environment. RNF13, with its ubiquitin ligase-active RING domain, has the potential to turn over key nuclear proteins in response to signals received at the plasma membrane.

The PA-TM-RING protein RING finger protein 13 is an endosomal integral membrane E3 ubiquitin ligase whose RING finger domain is released to the cytoplasm by proteolysis.

PA-TM-RING proteins have an N-terminal protease-associated domain, a structure found in numerous proteases and implicated in protein binding, and C-terminal RING finger and PEST domains. Homologous proteins include GRAIL (gene related to anergy in leukocytes), which controls T-cell anergy, and AtRMR1 (receptor homology region-transmembrane domain-RING-H2 motif protein), a plant protein storage vacuole sorting receptor. Another family member, chicken RING zinc finger (C-RZF), was identified as being upregulated in embryonic chicken brain cells grown in the presence of tenascin-C. Despite algorithm predictions that the cDNA encodes a signal peptide and transmembrane domain, the protein was found in the nucleus. We showed that RING finger protein 13 (RNF13), the murine homolog of C-RZF, is a type I integral membrane protein localized in the endosomal/lysosomal system. By quantitative real-time RT-PCR analysis, we demonstrated that expression of RNF13 is increased in adult relative to embryonic mouse tissues and is upregulated in B35 neuroblastoma cells stimulated to undergo neurite outgrowth. We found that RNF13 is very labile, being subject to extensive proteolysis that releases both the protein-associated domain and the RING domain from the membrane. By analyzing microsomes, we showed that the ectodomain is shed into the lumen of vesicles, whereas the C-terminal half, which possesses the RING finger, is released to the cytoplasm. This C-terminal fragment of RNF13 has the ability to mediate ubiquitination. Proteolytic release of RNF13 from a membrane anchor thus provides unique spatial and temporal regulation that has not been previously described for an endosomal E3 ubiquitin ligase.

Factor H binding to PspC of Streptococcus pneumoniae increases adherence to human cell lines in vitro and enhances invasion of mouse lungs in vivo.

Pneumococcal surface protein C (PspC) binds to both human secretory immunoglobulin A (sIgA) and complement factor H (FH). FH, a regulator of the alternative pathway of complement, can also mediate adherence of different host cells. Since PspC contributes to adherence and invasion of host cells, we hypothesized that the interaction of PspC with FH may also mediate adherence of pneumococci to human cells. In this study, we investigated FH- and sIgA-mediated pneumococcal adherence to human cell lines in vitro. Adherence assays demonstrated that preincubation of Streptococcus pneumoniae D39 with FH increased adherence to human umbilical vein endothelial cells (HUVEC) 5-fold and to lung epithelial cells (SK-MES-1) 18-fold, relative to that of D39 without FH on the surface. The presence of sIgA enhanced adherence to SK-MES-1 6-fold and to pharyngeal epithelial cells (Detroit 562) 14-fold. Furthermore, sIgA had an additive effect on adherence to HUVEC; specifically, preincubation of D39 with both FH and sIgA led to a 21-fold increase in adherence. Finally, using a mouse model, we examined the significance of the FH-PspC interaction in pneumococcal nasal colonization and lung invasion. Mice intranasally infected with D39 preincubated with FH had increased bacteremia and lung invasion, but they had similar levels of nasopharyngeal colonization compared to that of mice challenged with D39 without FH.

Antigen three-dimensional structure guides the processing and presentation of helper T-cell epitopes.

Antigen three-dimensional structure potentially controls presentation of CD4(+) T-cell epitopes by limiting the access of proteolytic enzymes and MHC class II antigen-presenting proteins. The protease-sensitive mobile loops of Hsp10s from mycobacteria, Escherichia coli, and bacteriophage T4 (T4Hsp10) are associated with adjacent immunodominant helper T-cell epitopes, and a mobile-loop deletion in T4Hsp10 eliminated the protease sensitivity and the associated epitope immunodominance. In the present work, protease-sensitivity and epitope presentation was analyzed in a group of T4Hsp10 variants. Two mobile-loop sequence variants of T4Hsp10 were constructed by replacing different segments of the mobile loop with an irrelevant sequence from hen egg lysozyme. The variant proteins retained native-like structure, and the mobile loops retained protease sensitivity. Mobile-loop deletion and reconstruction affected the presentation of two epitopes according to whether the epitope was protease-independent or protease-dependent. The protease-independent epitope lies within the mobile loop, and the protease-dependent epitope lies in a well-ordered segment on the carboxy-terminal flank of the mobile loop. The results are consistent with a model for processing of the protease-dependent epitope in which an endoproteolytic nick in the mobile-loop unlocks T4Hsp10 three-dimensional structure, and then the epitope becomes available for binding to the MHC protein.

Interaction of clinical isolates of Streptococcus pneumoniae with human complement factor H.

PspC recruits complement factor H (FH) to the pneumococcal surface. While there is differential expression of pspC during infection, detection of PspC on the surface of viable pneumococci is difficult due to variability among PspCs. We analyzed FH binding to detect PspC expression on the surface of pneumococcal isolates from different pathological sources. Using flow cytometry, we investigated FH-binding to 89 low-passage clinical isolates classified by disease manifestation (systemic, mucosal, or carriage). Carriage isolates recruited significantly more FH to their surfaces than either systemic or mucosal isolates, and this binding was independent of capsular serotype.

In vivo binding of complement regulator factor H by Streptococcus pneumoniae.

Pneumococcal surface protein C (PspC) binds to the complement regulatory protein factor H (FH), which inhibits alternative pathway activation. In the present study, using a mouse model of systemic infection and flow-cytometric analyses, we demonstrated an in vivo interaction between FH and pneumococci and showed differential FH binding during bacteremia. Flow-cytometric analyses of pneumococci harvested after intraperitoneal (ip) challenge demonstrated increased binding of FH, compared with that after intravenous (iv) challenge. Real-time polymerase chain reaction analyses of PspC mRNA showed that, relative to pneumococci grown in vitro, those recovered from the blood of mice 24 h after iv challenge exhibited 23-fold higher mRNA levels; however, after ip challenge, PspC mRNA induction was increased 870-fold. A subsequent increase in PspC expression was detected by flow cytometry using a monoclonal antibody against PspC. Furthermore, pneumococci with FH bound to complement before exposure had increased proliferation, compared with pneumococci not pretreated with FH. These results suggest that the interaction between PspC and FH contributes to pneumococcal virulence.

Dual roles of PspC, a surface protein of Streptococcus pneumoniae, in binding human secretory IgA and factor H.

Streptococcus pneumoniae, also known as the pneumococcus, contains several surface proteins that along with the polysaccharide capsule function in antiphagocytic activities and evasion of the host immune system. These pneumococcal proteins interact with the host immune system in various ways and possess a wide range of biological activities that suggests that they may be involved at different stages of pneumococcal infection. PspC, also known as CbpA and SpsA, is one of several pneumococcal surface proteins that binds host proteins, including factor H (FH) and secretory IgA (sIgA) via the secretory component. Previous work by our laboratory has demonstrated that PspC on the surface of live pneumococcal cells binds FH. This paper provides evidence that FH activity is maintained in the presence of PspC and that the PspC binding site is located in the short consensus repeat 6-10 region of FH. We also report for the first time that although both FH and sIgA binding has been localized to the alpha-helical domain of PspC, the binding of FH to PspC is not inhibited by sIgA. ELISA, surface plasmon resonance, and flow cytometry indicate that the two host proteins do not compete for binding with PspC and likely do not share the same binding sites. We confirmed by Western analysis that the binding sites are separate using recombinant PspC proteins. These PspC variants bind FH yet fail to bind sIgA. Thus, we conclude that FH and sIgA can bind concurrently to the alpha-helical region of PspC.

Structural basis for helper T-cell and antibody epitope immunodominance in bacteriophage T4 Hsp10. Role of disordered loops.

Antigen three-dimensional structure potentially limits the access of endoproteolytic processing enzymes to cleavage sites and of class II major histocompatibility antigen-presenting proteins to helper T-cell epitopes. Helper T-cell epitopes in bacteriophage T4 Hsp10 have been mapped by restimulation of splenocytes from CBA/J and C57BL/6J mice immunized in conjunction with mutant (R192G) heat-labile enterotoxin from Escherichia coli. Promiscuously immunogenic sequences were associated with unstable loops in the three-dimensional structure of T4 Hsp10. The immunodominant sequence lies on the N-terminal flank of the 22-residue mobile loop, which is sensitive to proteolysis in divergent Hsp10s. Several mobile loop deletions that inhibited proteolysis in vitro caused global changes in the helper T-cell epitope map. A mobile loop deletion that strongly stabilized the protein dramatically reduced the immunogenicity of the flanking immunodominant helper T-cell epitope, although the protein retained good overall immunogenicity. Antisera against the mobile loop deletion variants exhibited increased cross-reactivity, most especially the antisera against the strongly stabilized variant. The results support the hypothesis that unstable loops promote the presentation of flanking epitopes and suggest that loop deletion could be a general strategy to increase the breadth and strength of an immune response.

Proteolytic sensitivity and helper T-cell epitope immunodominance associated with the mobile loop in Hsp10s.

Antigen three-dimensional structure potentially limits antigen processing and presentation to helper T-cell epitopes. The association of helper T-cell epitopes with the mobile loop in Hsp10s from mycobacteria and bacteriophage T4 suggests that the mobile loop facilitates proteolytic processing and presentation of adjacent sequences. Sites of initial proteolytic cleavage were mapped in divergent Hsp10s after treatment with a variety of proteases including cathepsin S. Each protease preferentially cleaved the Hsp10s in the mobile loop. Flexibility in the 22-residue mobile loop most probably allows it to conform to protease active sites. Three variants of the bacteriophage T4 Hsp10 were constructed with deletions in the mobile loop to test the hypothesis that shorter loops would be less sensitive to proteolysis. The two largest deletions effectively inhibited proteolysis by several proteases. Circular dichroism spectra and chemical cross-linking of the deletion variants indicate that the secondary and quaternary structures of the variants are native-like, and all three variants were more thermostable than the wild-type Hsp10. Local structural flexibility appears to be a general requirement for proteolytic sensitivity, and thus, it could be an important factor in antigen processing and helper T-cell epitope immunogenicity.