Peptidyl-prolyl Cis-trans Isomerase Domain Research Articles

FK506 BINDING PROTEIN 8 (FKBP8) INTERACTIONS WITH MYOSIN LIGHT CHAIN KINASE 1 ARE REQUIRED FOR TNF-INDUCED TIGHT JUNCTION BARRIER LOSS

Abstract Background Tumor necrosis factor (TNF) regulates intestinal epithelial tight junction permeability by activating myosin light chain kinase 1 (MLCK1) expression and enzymatic activity. MLCK1 recruitment to the apical perijunctional actomyosin ring (PAMR) is, however, required for barrier regulation; Divertin, a small molecule drug that blocks this recruitment, prevents barrier loss and attenuates both acute and chronic experimental diarrheal disease. We therefore hypothesized that MLCK1 recruitment to the PAMR requires interactions with as yet unidentified chaperone protein(s). Objective To identify binding partners and define the mechanisms by which they activate MLCK1 recruitment to the PAMR. Results We performed a yeast-2-hybrid (Y2H) screen using the MLCK1 domains required for PAMR recruitment as bait. FKBP8, which encodes a peptidyl-prolyl cis-trans isomerase (PPI), was recovered, and direct binding to the MLCK1 domains (Kd=~5mM) was confirmed using microscale thermophoresis (MST). This binding interaction required the FK506-binding PPI domain and was specifically inhibited by FK506 (tacrolimus). Immunofluorescent microscopy demonstrated partial colocalization of MLCK1 and FKBP8 within intestinal epithelial monolayers; TNF caused both to concentrate around the PAMR. To further characterize this interaction, we performed proximity ligation assays (PLA) and found that TNF increased interaction between MLCK1 and FKBP8 > 2-fold. FK506 prevented TNF-induced increases in PLA signal. FK506 was also able to reverse TNF-induced increases in myosin light chain (MLC) phosphorylation and tight junction permeability. In Caco-2 monolayers, FKBP8 knockout blocked TNF-induced MLCK1 recruitment, MLC phosphorylation, and tight junction barrier loss; all of which were restored by FKBP8 re-expression. In mice, MLC phosphorylation and intestinal barrier loss triggered by acute, anti-CD3-induced, T cell activation were blocked by luminal FK506. Importantly, this local FK506 treatment did not prevent anti-CD3-induced increases in mucosal TNF, IL-1b, and IFNg. Immunostains of biopsies from IBD patients documented increased PAMR MLC phosphorylation, MLCK1 recruitment, FKBP8 recruitment, and MLCK1-FKBP8 PLA signal relative to control subjects. Conclusions FKBP8 is a chaperone protein required for TNF-induced MLCK1 recruitment and barrier loss. This requires direct interaction between MLCK1 and the PPI domain of FKBP8. FK506 binding to the PPI domain displaces MLCK1 thereby preventing recruitment to the PAMR and barrier loss. These activities are separate from the immunosuppressive effects of FK506. We speculate that molecular blockade of the FKBP8-MLCK1 interaction may be a novel approach to barrier restoration and therapy of diseases associated with intestinal barrier dysfunction. Support NIH (DK068271, DK061931) and the NNSF of China (81800464, 82070548).

Purification and Preliminary X-Ray Crystallographic Analysis of the Peptidyl-Prolyl cis–trans Isomerase Alr5059 from Anabaena sp. PCC 7120

Peptidyl-prolyl cis–trans isomerases (PPIases) have a wide range of functions in all cells. They are typically classified as cyclophilin, the FK506-binding protein-like or parvulins. Most PPIases are two-domain enzymes. While the peptidyl-prolyl cis–trans isomerase domain is common to all PPIlases, different proteins differ in the function of the second domain. Plant PPIases of the cyclophilin family (Cyp38 in A. thaliana) contain a second domain at the N-terminus, but its function is still not known. They are localized in the thylakoid lumen and are involved in the assembly of photosystem II. The protein is conserved among plants and cyanobacteria with high similarity in the PPIase domain, but with some divergence in the N-terminal region of the protein. This prompted protein crystallization to analyze whether a unique feature of plant proteins originates from a cyanobacterial ancestor. We expressed the Alr5059 protein from Anabaena sp. PCC 7120 in E. coli and crystallized protein by the sitting drop method. Single crystals of the Alr5059 protein appeared after 5–7 days. The best crystal gave a diffraction pattern to a resolution of 1.1 A. The crystal belongs to the monoclinic space group P1211 with unit cell parameters a = 41.2 A, b = 103.2 A, c = 44.2 A and β = 114.7° and contains one molecule per asymmetric unit.

Cyclophilin AtROC1S58F confers Arabidopsis cold tolerance by modulating jasmonic acid signaling and antioxidant metabolism

Cyclophilins (CYPs), a class of proteins with a conserved peptidyl-prolyl cis-trans isomerase domain, are widely involved in the regulation of plant growth and development, as well as in the response to abiotic stresses including cold. In our previous study, we identified an Arabidopsis gain-of-function mutant ROC1S58F with enhanced cold-tolerance and enhanced expression of jasmonic acid (JA) and oxidative stress responsive genes. Here, we show the underlying molecular mechanisms for the improved cold tolerance observed in the ROC1S58F mutant. Compared to the WT, the ROC1S58F mutant showed an increased survival rates and a reduced level of electrolyte leakage and endogenous JA content under the freezing treatment. Correspondingly, the JA biosynthesis genes (AtAOC1 and AtOPR3) and signaling genes (AtJAZ5, AtJAZ10 and AtMYB15) are down-regulated in the ROC1S58F mutant compared with the WT. Moreover, both the transcripts and activities of the ROS-scavenging enzymes (SOD/POD/MDHAR) increased in cold-stressed ROC1S58F mutant, which might mitigate the ROS-induced oxidative stress and contribute to the mutant freezing tolerance. Taken together, our findings indicate that AtROC1S58F confers Arabidopsis freezing tolerance by modulating JA signaling and antioxidant metabolism jointly. This research thus provides a molecular mechanism for AtROC1S58F-conferred freezing resistance in Arabidopsis and offers guidance for crop breeding towards an improved cold tolerance.

NMR assignments of the peptidyl-prolyl cis-trans isomerase domain of trigger factor from E. coli.

Trigger factor (TF) is a highly conserved multi-domain molecular chaperone in bacteria. It binds via its ribosome binding domain (RBD) to the ribosomal tunnel exit and facilitates co-translational folding of a broad range of protein substrates primarily through interactions with the substrate binding domain (SBD) adjacent to the RBD. Within the SBD, a peptidyl-prolyl cis-trans isomerase (PPIase) domain is inserted leading to an unusual domain insertion, which may provide stabilizing effect to the highly plastic SBD. Here we report the near complete NMR assignments of TF PPIase providing the basis for subsequent structural and folding in the context of the chaperone activity of TF.

Kuskokwim Syndrome, a Recessive Congenital Contracture Disorder, Extends the Phenotype ofFKBP10 Mutations

Recessive mutations in FKBP10 at 17q21.2, encoding FKBP65, cause both osteogenesis imperfecta (OI) and Bruck syndrome (OI plus congenital contractures). Contractures are a variable manifestation of null/missense FKBP10 mutations. Kuskokwim syndrome (KS) is an autosomal recessive congenital contracture disorder found among Yup'ik Eskimos. Linkage mapping of KS to chromosome 17q21, together with contractures as a feature of FKBP10 mutations, made FKBP10 a candidate gene. We identified a homozygous three-nucleotide deletion in FKBP10 (c.877_879delTAC) in multiple Kuskokwim pedigrees; 3% of regional controls are carriers. The mutation deletes the highly conserved p.Tyr293 residue in FKBP65's third peptidyl-prolyl cis-trans isomerase domain. FKBP10 transcripts are normal, but mutant FKBP65 is destabilized to a residual 5%. Collagen synthesized by KS fibroblasts has substantially decreased hydroxylation of the telopeptide lysine crucial for collagen cross-linking, with 2%-10% hydroxylation in probands versus 60% in controls. Matrix deposited by KS fibroblasts has marked reduction in maturely cross-linked collagen. KS collagen is disorganized in matrix, and fibrils formed in vitro had subtle loosening of monomer packing. Our results imply that FKBP10 mutations affect collagen indirectly, by ablating FKBP65 support for collagen telopeptide hydroxylation by lysyl hydroxylase 2, thus decreasing collagen cross-links in tendon and bone matrix. FKBP10 mutations may also underlie other arthrogryposis syndromes.

Solution structure of the parvulin-type PPIase domain of Staphylococcus aureus PrsA – Implications for the catalytic mechanism of parvulins

BackgroundStaphylococcus aureus is a Gram-positive pathogenic bacterium causing many kinds of infections from mild respiratory tract infections to life-threatening states as sepsis. Recent emergence of S. aureus strains resistant to numerous antibiotics has created a need for new antimicrobial agents and novel drug targets. S. aureus PrsA is a membrane associated extra-cytoplasmic lipoprotein which contains a parvulin-type peptidyl-prolyl cis-trans isomerase domain. PrsA is known to act as an essential folding factor for secreted proteins in Gram-positive bacteria and thus it is a potential target for antimicrobial drugs against S. aureus.ResultsWe have solved a high-resolution solution structure of the parvulin-type peptidyl-prolyl cis-trans isomerase domain of S. aureus PrsA (PrsA-PPIase). The results of substrate peptide titrations pinpoint the active site and demonstrate the substrate preference of the enzyme. With detailed NMR spectroscopic investigation of the orientation and tautomeric state of the active site histidines we are able to give further insight into the structure of the catalytic site. NMR relaxation analysis gives information on the dynamic behaviour of PrsA-PPIase.ConclusionDetailed structural description of the S. aureus PrsA-PPIase lays the foundation for structure-based design of enzyme inhibitors. The structure resembles hPin1-type parvulins both structurally and regarding substrate preference. Even though a wealth of structural data is available on parvulins, the catalytic mechanism has yet to be resolved. The structure of S. aureus PrsA-PPIase and our findings on the role of the conserved active site histidines help in designing further experiments to solve the detailed catalytic mechanism.

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.

The Arabidopsis cyclophilin gene family.

Database searching has allowed the identification of a number of previously unreported single and multidomain isoform members of the Arabidopsis cyclophilin gene family. In addition to the cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, the latter contain a variety of other domains with characterized functions. Transcriptional analysis showed they are expressed throughout the plant, and different isoforms are present in all parts of the cell including the cytosol, nucleus, mitochondria, secretory pathway, and chloroplast. The abundance and diversity of cyclophilin isoforms suggests that, like their animal counterparts, plant cyclophilins are likely to be important proteins involved in a wide variety of cellular processes. As well as fulfilling the basic role of protein folding, they may also play important roles in mRNA processing, protein degradation, and signal transduction and thus may be crucial during both development and stress responsiveness.

Cloning, sequencing, and nucleolar targeting of the basal-body-binding nucleolar protein BN46/51.

BN46/51 is an acidic protein found in the granular component of the nucleolus of the amebo-flagellate Naegleria gruberi. When Naegleria amebae differentiate into swimming flagellates, BN46/51 is found associated with the basal body complex at the base of the flagella. In order to determine the factors responsible for targeting BN46/51 to a specific subnucleolar region, cDNAs coding for both subunits were isolated and sequenced. Two clones, JG4.1 and JG12.1 representing the 46 kDa and 51 kDa subunits, respectively, were investigated in detail. JG12.1 encoded a polypeptide of 263 amino acids with a predicted size of 30.1 kDa that co-migrated with the 51 kDa subunit of BN46/51 when expressed in yeast. JG4.1 encoded a polypeptide of 249 amino acids with a predicted size of 28.8 kDa that co-migrated with the 46 kDa subunit of BN46/51. JG4.1 was identical to JG12.1 except for the addition of an aspartic acid between positions 94 and 95 of the JG12.1 sequence and the absence of 45 amino acids beginning at position 113. The predicted amino acid sequences were not closely related to any previously reported. However, the sequences did have 26-31% identity to a group of FKPBs (FK506 binding proteins) but lacked the peptidyl-prolyl cis-trans isomerase domain of the FKBPs. Both subunits contained two KKE and three KKX repeats found in other nucleolar proteins and in some microtubule binding proteins. Using 'Far Western' blots of nucleolar proteins, BN46/51 bound to polypeptides of 44 kDa and 74 kDa. The 44 kDa component was identified as the Naegleria homologue of fibrillarin. BN46/51 bound specifically to the nucleoli of fixed mammalian cells, cells which lack a BN46/51 related polypeptide. When the JG4.1 and JG12.1 cDNAs were expressed in yeast, each subunit was independently targeted to the yeast nucleolus. We conclude that BN46/51 represents a unique nucleolar protein that can form specific complexes with fibrillarin and other nucleolar proteins. We suggest that the association of BN46/51 with the MTOC of basal bodies may reflect its role in connecting the nucleolus with the MTOC activity for the mitotic spindle. This would provide a mechanism for nucleolar segregation during the closed mitosis of Naegleria amebae.