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Products of oxidative damage to lipids include 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), both of which are cytotoxic electrophiles. ONE reacts more rapidly with nucleophilic amino acid side chains, resulting in covalent protein adducts, including residue-residue cross-links. Previously, we demonstrated that peptidylprolyl cis/trans isomerase A1 (Pin1) was highly susceptible to adduction by HNE and that the catalytic cysteine (Cys113) was the preferential site of modification. Here, we show that ONE also preferentially adducts Pin1 at the catalytic Cys but results in a profoundly different modification. Results from experiments using purified Pin1 incubated with ONE revealed the principal product to be a Cys-Lys pyrrole-containing cross-link between the side chains of Cys113 and Lys117. In vitro competition assays between HNE and ONE demonstrate that ONE reacts more rapidly than HNE with Cys113. Exposure of RKO cells to alkynyl-ONE (aONE) followed by copper-mediated click chemistry and streptavidin purification revealed that Pin1 is also modified by ONE in cells. Analysis of the Pin1 crystal structure reveals that Cys113 and Lys117 are oriented toward each other in the active site, facilitating formation of an ONE cross-link.
BACKGROUND - Protein folding is crucial for proteins' specific functions and is facilitated by various types of enzymes and molecular chaperones. The peptidyl prolyl cis/trans isomerases (PPIase) are one of these families of enzymes. They ubiquitously exist inside the cell and there are eight PPIases in the rough endoplasmic reticulum (rER), a compartment where the folding of most secreted proteins occurs.
SCOPE OF REVIEW - We review the functional and structural aspects of individual rER resident PPIases. Furthermore, we specifically discuss the role of these PPIases during collagen biosynthesis, since collagen is the most abundant protein in humans, is synthesized in the rER, and contains a proportionally high number of proline residues.
MAJOR CONCLUSIONS - The rER resident PPIases recognize different sets of substrates and facilitate their folding. Although they are clearly catalysts for protein folding, they also have more broad and multifaceted functions. We propose that PPIases coordinate collagen biosynthesis in the rER.
GENERAL SIGNIFICANCE - This review expands our understanding of collagen biosynthesis by explaining the influence of novel indirect mechanisms of regulating folding and this is also explored for PPIases. We also suggest future directions of research to obtain a better understanding of collagen biosynthesis and functions of PPIases in the rER. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.
Copyright © 2015 Elsevier B.V. All rights reserved.
The FK506-binding protein (FKBP) family consists of proteins with a variety of protein-protein interaction domains and versatile cellular functions. It is assumed that all members are peptidyl-prolyl cis-trans isomerases with the enzymatic function attributed to the FKBP domain. Six members of this family localize to the mammalian endoplasmic reticulum (ER). Four of them, FKBP22 (encoded by the FKBP14 gene), FKBP23 (FKBP7), FKBP60 (FKBP9), and FKBP65 (FKBP10), are unique among all FKBPs as they contain the EF-hand motifs. Little is known about the biological roles of these proteins, but emerging genetics studies are attracting great interest to the ER resident FKBPs, as mutations in genes encoding FKBP10 and FKBP14 were shown to cause a variety of matrix disorders. Although the structural organization of the FKBP-type domain as well as of the EF-hand motif has been known for a while, it is difficult to conclude how these structures are combined and how it affects the protein functionality. We have determined a unique 1.9 Å resolution crystal structure for human FKBP22, which can serve as a prototype for other EF hand-containing FKBPs. The EF-hand motifs of two FKBP22 molecules form a dimeric complex with an elongated and predominantly hydrophobic cavity that can potentially be occupied by an aliphatic ligand. The FKBP-type domains are separated by a cleft and their putative active sites can catalyze isomerazation of two bonds within a polypeptide chain in extended conformation. These structural results are of prime interest for understanding biological functions of ER resident FKBPs containing EF-hand motifs.
© 2013 The Protein Society.
Prostatic adenocarcinoma with aberrant diffuse expression of p63 (p63-PCa) is a recently described variant of prostatic adenocarcinoma. The aim of this study was to investigate the clinical and pathologic features of p63-PCa at radical prostatectomy (RP). We reviewed 21 cases of p63-PCa diagnosed on needle biopsy at subsequent RP. Immunohistochemical analysis for PIN4 and Ki-67 was performed in all RP cases. p63-PCa showed a distinctive morphology consisting of atrophic, poorly formed glands, with multilayered and often spindled nuclei. Gleason grading was 3+3=6 in 28.5%, 3+5=8 in 38%, 3+4=7 in 14.3%, and 4+3=7, 5+3=8, and 5+4=9 in 9.5%. Usual-type acinar carcinoma coexisted in 85.7% with only p63-PCa present in the remaining cases. The usual-type carcinoma was Gleason grade 3+2=5 in 4.7%, 3+3=6 in 57%, 3+4=7 in 19%, and 4+3=7 in 4.3%. Overall, p63-PCa represented 65% of the total cancer volume (median 80%). The tumor was organ-confined in 16 cases (76.2%). In the remaining 5 cases, 2 had p63-PCa extending to the margin in areas of intraprostatic incisions, 2 had usual-type acinar adenocarcinoma extending to the margin and extraprostatic tissue, respectively, and 1 had p63-PCa with an unusual cribriform morphology involving the bladder neck. Ki-67 was low, <5% in all cases of p63-PCa, with similar expression in the coexisting acinar-type carcinoma. In summary, it is recommended that these tumors not be assigned a Gleason score and their favorable findings at RP be noted.
Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)7 with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)7-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.
Oxidation of membrane phospholipids is associated with inflammation, neurodegenerative disease, and cancer. Oxyradical damage to phospholipids results in the production of reactive aldehydes that adduct proteins and modulate their function. 4-Hydroxynonenal (HNE), a common product of oxidative damage to lipids, adducts proteins at exposed Cys, His, or Lys residues. Here, we demonstrate that peptidyl-prolyl cis/trans-isomerase A1 (Pin1), an enzyme that catalyzes the conversion of the peptide bond of pSer/pThr-Pro moieties in signaling proteins from cis to trans, is highly susceptible to HNE modification. Incubation of purified Pin1 with HNE followed by MALDI-TOF/TOF mass spectrometry resulted in detection of Michael adducts at the active site residues His-157 and Cys-113. Time and concentration dependencies indicate that Cys-113 is the primary site of HNE modification. Pin1 was adducted in MDA-MB-231 breast cancer cells treated with 8-alkynyl-HNE as judged by click chemistry conjugation with biotin followed by streptavidin-based pulldown and Western blotting with anti-Pin1 antibody. Furthermore, orbitrap MS data support the adduction of Cys-113 in the Pin1 active site upon HNE treatment of MDA-MB-231 cells. siRNA knockdown of Pin1 in MDA-MB-231 cells partially protected the cells from HNE-induced toxicity. Recent studies indicate that Pin1 is an important molecular target for the chemopreventive effects of green tea polyphenols. The present study establishes that it is also a target for electrophilic modification by products of lipid peroxidation.
The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.
Green tea and its major polyphenolic flavonoid, epigallocatechin gallate (EGCG), have been credited with cancer chemopreventive activity for many years; the mechanism for this activity, however, has remained obscure. Now, as reported in this issue of the journal (beginning on page 1366), Urusova and colleagues showed direct binding of EGCG to the peptidyl prolyl cis/trans isomerase Pin1, which inhibited Pin1 enzymatic activity. They showed that Pin1 expression is required for EGCG effects on cell growth, c-Jun activation, and transcription regulation mediated by NF-κB and activator protein-1. The data provide a glimpse of the mechanism of action of EGCG and set a new bar for the future study of natural products with chemopreventive activity.
Pin1 specifically recognizes and catalyzes the cis-trans isomerization of phosphorylated-Ser/Thr-Pro bonds, which modulate the stability, localization, and function of numerous Pin1 targets involved in tumor progression. However, the role of Pin1 in cancer remains enigmatic as the gene is located on chromosome 19p13.2, which is a region subject to loss of heterozygosity in several tumors. Since Pin1 protein is frequently under-expressed in kidney cancer, we have explored its role in human clear cell renal cell carcinoma (ccRCC). Here we show evidence for PIN1 gene deletion and mRNA under-expression as a mechanism of Pin1 reduction in ccRCC tumors. We demonstrate that restoration of Pin1 in cell lines found to be deficient in Pin1 protein expression can attenuate the growth of ccRCC cells in soft agar and a xenograft tumor model. Moreover, this ability of Pin1 to negatively influence tumor growth in ccRCC cells may be dependent on the presence of functional p53, which is infrequently mutated in ccRCC. These observations suggest Pin1 may have a mild tumor suppressive role in ccRCC.
Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
The molecular machinery governing glutamatergic-GABAergic neuronal subtype specification is unclear. Here we describe a cerebellar mutant, cerebelless, which lacks the entire cerebellar cortex in adults. The primary defect of the mutant brains was a specific inhibition of GABAergic neuron production from the cerebellar ventricular zone (VZ), resulting in secondary and complete loss of external germinal layer, pontine, and olivary nuclei during development. We identified the responsible gene, Ptf1a, whose expression was lost in the cerebellar VZ but was maintained in the pancreas in cerebelless. Lineage tracing revealed that two types of neural precursors exist in the cerebellar VZ: Ptf1a-expressing and -nonexpressing precursors, which generate GABAergic and glutamatergic neurons, respectively. Introduction of Ptf1a into glutamatergic neuron precursors in the dorsal telencephalon generated GABAergic neurons with representative morphological and migratory features. Our results suggest that Ptf1a is involved in driving neural precursors to differentiate into GABAergic neurons in the cerebellum.