High Cysteine Content Research Articles

Analytical Methods for Metallothionein Detection

Metallothioneins (MTs) are a group of low molecular mass, cysteine-rich proteins with a variety of functions including involvement in metal homeostasis, free radical scavenging, protection against heavy metal damage, and metabolic regulation via Zn donation. The overexpression of MTs in proliferating cells is turning the attention to the study of MTs as novel promising marker of tumor diseases. Besides, levels of MTs in invertebrates and aquatic vertebrates well correlate with heavy metal pollution of an environment and, thus, serve as bio-environmental marker. It is not surprising that these proteins are of great interest not only for biochemists, molecular biologists clinical chemists, but also for environmental chemists and ecologists. Detection and quantification of MTs, which is needed in all above mentioned areas, is challenging due to the high cysteine content and relatively low molecular mass. This review is giving an overview of the analytical methods used for determination of MTs comprising a broad range of immunochemical, electrochemical as well as spectrometric methods, which have been optimized and even hyphenated with different separation methods to detect MTs. More attention is paid to the structural analysis of these proteins as well as the employment of analytical instruments for environmental, biochemical and clinical purposes.

Analysis of common bean expressed sequence tags identifies sulfur metabolic pathways active in seed and sulfur-rich proteins highly expressed in the absence of phaseolin and major lectins

BackgroundA deficiency in phaseolin and phytohemagglutinin is associated with a near doubling of sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris), particularly cysteine, elevated by 70%, and methionine, elevated by 10%. This mostly takes place at the expense of an abundant non-protein amino acid, S-methyl-cysteine. The deficiency in phaseolin and phytohemagglutinin is mainly compensated by increased levels of the 11S globulin legumin and residual lectins. Legumin, albumin-2, defensin and albumin-1 were previously identified as contributing to the increased sulfur amino acid content in the mutant line, on the basis of similarity to proteins from other legumes.ResultsProfiling of free amino acid in developing seeds of the BAT93 reference genotype revealed a biphasic accumulation of gamma-glutamyl-S-methyl-cysteine, the main soluble form of S-methyl-cysteine, with a lag phase occurring during storage protein accumulation. A collection of 30,147 expressed sequence tags (ESTs) was generated from four developmental stages, corresponding to distinct phases of gamma-glutamyl-S-methyl-cysteine accumulation, and covering the transitions to reserve accumulation and dessication. Analysis of gene ontology categories indicated the occurrence of multiple sulfur metabolic pathways, including all enzymatic activities responsible for sulfate assimilation, de novo cysteine and methionine biosynthesis. Integration of genomic and proteomic data enabled the identification and isolation of cDNAs coding for legumin, albumin-2, defensin D1 and albumin-1A and -B induced in the absence of phaseolin and phytohemagglutinin. Their deduced amino acid sequences have a higher content of cysteine than methionine, providing an explanation for the preferential increase of cysteine in the mutant line.ConclusionThe EST collection provides a foundation to further investigate sulfur metabolism and the differential accumulation of sulfur amino acids in seed of common bean. Identification of sulfur-rich proteins whose levels are elevated in seed lacking phaseolin and phytohemagglutinin and sulfur metabolic genes may assist the improvement of protein quality.

Cloning, expression and purification of the CCN family of proteins in Escherichia coli

The CCN proteins are extracellular matrix associated proteins involved in critical cell activities and several aggressive forms of cancer. The proteins share a modular structure of four discrete domains and 38 conserved cysteine residues. The absence of any structural information of these proteins has resulted in a need for the ability to produce substantial amounts of pure CCN protein. Through bacterial expression and inclusion body based purification, pure recombinant CCN proteins have been produced for use in structural and biochemical experiments.

Refined Crystal Structures of Human Ca 2+/Zn 2+-Binding S100A3 Protein Characterized by Two Disulfide Bridges

S100A3, a member of the EF-hand-type Ca 2+-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn 2+ affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N, N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca 2+/Zn 2+ supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A + C68A) abolished the Ca 2+ binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca 2+ affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A + C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15−1.40 Å resolution shows that SS1 renders the C-terminal classical Ca 2+-binding loop flexible, which are essential for its Ca 2+ binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn 2+ by (Cys) 3His residues that is compatible with SS2 formation in S100A3.

Application of XANES profiles to X-ray spectromicroscopy for biomedical specimens: Part II. Mapping oxidation state of cysteine in human hair

Human hair fibers are primarily composed of keratin protein, characterized by a very high content of cysteine, a sulfur-containing amino acid, which ordinarily forms cystine via a disulfide bond. It is known that some cystine residues are converted to cysteic acid during permanent waving or hair coloring, although details of their distribution and extent are still unclear. In this study, by using difference in XANES profiles of cystine and cysteic acid at the S-K absorption edge, the formation of cysteic acid was confirmed for homogenized samples of permed or bleached hair. Furthermore chemical mapping of cysteic acid was performed on hair-section samples with X-ray contact microscopy. The peripheral region, cuticle, in bleached hair showed the highest content of cysteic acid compared with the other parts, while permed hair showed relatively uniform distribution. This finding suggests that perming and bleaching damage hair by different mechanisms.

A comparative study on interactions of cisplatin and ruthenium arene anticancer complexes with metallothionein using MALDI-TOF-MS

Metallothioneins (MTs) are a group of low molecular weight (6–7 KDa) proteins featured by high cysteine content which allows the proteins to bind to a diverse range of metals. MTs, of which the gene transcription can be induced by a variety of stimuli including hormones, cytokines and metal ions, have important biological functions such as storage, trafficking and homeostasis of metal ions, detoxification of heavy metals, resistance to ionizing radiation, scavenging hydroxyl radical, etc. Importantly, it has been reported that MTs play a role in oncogenesis and cancer prognosis, and are implicated in resistance of cancer cells to anticancer metallodrug cisplatin. In this work, we present a comparative study on interactions between MTs and cisplatin and ruthenium arene anticancer complexes using MALDI-TOF-MS. The results show that cisplatin coordinates to MT-I and MT-II at either pH 3.0 or 7.4, and exhibits a higher affinity to MT-II than to MT-I. The MT–Pt complexes increase significantly in content with decrease in pH values of solutions, indicative that cisplatin competes with zinc for coordination to cysteine residues on MTs and interferes with the binding of zinc to the proteins. While the ruthenium arene anticancer complexes [(η 6-arene)Ru(en)Cl]PF 6 (arene = benzene or biphenyl, en = ethylenediamine) hardly bind to MTs at acidic pH and coordinate to MTs at a much lower level than cisplatin at neutral pH, which may account for the less toxicity and lack of cross-resistance to cisplatin for this class of ruthenium anticancer complexes. With the MT–Pt complexes as model protein complexes, DDT, the widely used unfolding thiol-containing agent in proteomic research was shown to reduce the coordination of platinum to MTs significantly, implying that DDT used as unfolding reagent at high concentration may cause dissociation of bound metallodrug and should be applied with care.

In silico prediction of potential metallothioneins and metallohistins in actinobacteria

Metallothioneins and metallohistins are short peptides with a high cysteine and/or histidine content able to coordinate metals intracellularly, thereby increasing the tolerance against elevated concentrations of metals. Because of their features, they can be detected by in silico prediction from proteomes annotated from sequenced genomes. Here, we analyzed 73 sequenced actinobacterial genomes for peptides (≤ 100 amino acids) with a high content of cysteine and histidine (≥ 15%) and identified 103 putative metallothioneins and metallohistins. For 45 of these peptides, we found similarities to metal binding protein domains, including zinc fingers, heavy metal transporters or eukaryotic metallothioneins, which can serve as proof-of-principle in underscoring a potential function as metal binding peptides. An evolutionary origin from metal containing domains of enzymes is discussed and metallohistins not containing cysteine are described for the first time for bacteria.

Évaluation de l’intérêt nutritionnel d’isolats de protéines de colza chez le rat et l’Homme : application à la prévention du syndrome métabolique

Plant protein have long been opposed to animal protein because some plant protein present a relative mismatch between their amino acid pattern and human requirements for the renewal of body protein. Nevertheless, beyond its theoretical relevance to match the metabolic demand for protein metabolism, the amino acid pattern of dietary protein may also be of importance in the nutritional prevention of certain metabolic diseases. Of major public health concern is the metabolic syndrome, a cluster of risk factors associating obesity, insulin resistance, endothelial dysfunction, and low-grade inflammation. Two amino acids may be of importance in the pathophysiology of the metabolic syndrome: cysteine, the glutathione precursor, and arginine, the nitric oxide precursor. In this context, rapeseed protein, which is rich in cysteine and a good source of arginine, is an attractive candidate for studying the potential of dietary protein to prevent insulin resistance and related metabolic/ physiological abnormalities in the setting of the metabolic syndrome. After having presented a study demonstrating the high postprandial nitrogen utilization of rapeseed protein in humans, this review report two studies from our group showing that 1) rapeseed protein prevents the onset of insulin resistance induced by a diet rich in saturated fatty acids and sucrose in rats; 2) rapeseed protein alleviates the postprandial vascular endothelial dysfunction and oxidative stress coming after a meal rich in saturated fatty acids and sucrose. The beneficial preventive effects can be accounted for, at least to some extent, by the high cysteine and arginine content of the rapeseed protein. Studies are urgently needed to assess the beneficial effect of this emerging protein source in humans.

Effects of toxic and non‐toxic blue mussel meal on health and product quality of laying hens

Blue mussels (Mytilus edulis) are a potential protein source in poultry feeds because of their high methionine and cysteine content. However, mussels may temporarily be contaminated with the toxin, okadaic acid. The aim of this small scale study was to evaluate the effects on animal health and morphology of the digestive tract of laying hens fed three different diets; a commercial feed and diets containing 15% normal or 15% toxic mussel meal, i.e. a concentration of 198.6 microg toxin per kg feed. Twelve laying hens were divided into six groups and fed the experimental diets for 8 weeks. Animal health, production performance and egg quality were recorded. At sacrifice, tissue samples were prepared for histological evaluation using light microscopy. Okadaic acid did not have any adverse effects on animal health or production parameters and no histological changes indicating disturbances in the digestive tract was observed. Internal egg quality was improved as eggs from hens fed either of the two mussel meal diets had an increased yolk colour. These results show that mussel meal functions as a novel protein source for laying hens and those toxic mussels at this level may be included in the feed without negative effects on parameters evaluated in this study.

Determination of Reduced Cysteine in Oenological Cell Wall Fractions of Saccharomyces cerevisiae

Compounds containing cysteine residues, such as glutathione, can affect the redox potential of must and wine by reduction of o-quinones and hydrogen peroxide. The oenological yeast cell wall fractions contain cysteine residues in their protein structure, and they could affect both oxidative and odor properties of wine. An analytical approach based on the derivatization of cysteinyl residues with p-benzoquinone followed by reversed-phase high-performance liquid chromatography separation was developed to quantify glutathione and free and protein cysteine in 16 Saccharomyces cerevisiae strains and 12 commercial samples of yeast mannoproteins, hulls, and lysates. The chemical modifications induced by the Maillard reaction following the industrial preparation of such fractions were evaluated as well. Lysates showed the highest protein cysteine content and high contents of glutathione and free cysteine. Mannoproteins showed an intense Maillard reaction (furosine >60 mg/100 g protein), and most of the samples were able to bind thiol compounds with a potentially detrimental effect toward the thiol-related odors in wine.

Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific, high-affinity binding to CSA

Pregnancy-associated malaria (PAM) is a serious consequence of sequestration of Plasmodium falciparum-parasitized erythrocytes (PE) in the placenta through adhesion to chondroitin sulfate A (CSA) present on placental proteoglycans. Recent work implicates var2CSA, a member of the PfEMP1 family, as the mediator of placental sequestration and as a key target for PAM vaccine development. Var2CSA is a 350 kDa transmembrane protein, whose extracellular region includes six Duffy-binding-like (DBL) domains. Due to its size and high cysteine content, the full-length var2CSA extracellular region has not hitherto been expressed in heterologous systems, thus limiting investigations to individual recombinant domains. Here we report for the first time the expression of the full-length var2CSA extracellular region (domains DBL1X to DBL6epsilon) from the 3D7 parasite strain using the human embryonic kidney 293 cell line. We show that the recombinant extracellular var2CSA region is correctly folded and that, unlike the individual DBL domains, it binds with high affinity and specificity to CSA (K(D) = 61 nM) and efficiently inhibits PE from binding to CSA. Structural characterization by analytical ultracentrifugation and small-angle x-ray scattering reveals a compact organization of the full-length protein, most likely governed by specific interdomain interactions, rather than an extended structure. Collectively, these data suggest that a high-affinity, CSA-specific binding site is formed by the higher-order structure of the var2CSA extracellular region. These results have important consequences for the development of an effective vaccine and therapeutic inhibitors.

Higher sequence coverage and improved confidence in the identification of cysteine-rich proteins from the wool cuticle using combined chemical and enzymatic digestion

Keratin-associated proteins (KAPs) are important constituents of the wool cuticle, comprised of the endo-, exocuticle and a-layers, which contribute significantly to the fibre's molecular and mechanical characteristics. Relatively little is known about the distribution of specific KAPs across these layers, and correct protein identification of individual KAPs is difficult due to extensive homology and identity among individual KAPs. We here present evidence that, by specifically exploiting the high-cysteine content of KAPs in the wool cuticle, using 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage in combination with tryptic digestion, a larger number of KAPs can be identified than with standard trypsin-only digests. A total of 27 KAPs were identified, six of which could only be identified using NTCB. Furthermore, NTCB-mediated cleavage of cuticle proteins generated unique peptides critical for unambiguous identification of two KAPs, as well as significantly increasing the overall sequence coverage of most identified KAPs. Interestingly, some of the peptides found to be unique to particular KAPs could only be found in either the exo- or endocuticle. We conclude that for the analysis of high sulphur proteomes, specific targeting of cysteine residues using chemical agents such as NTCB can provide critical information for unambiguous protein identification.

The polymorphism of core promoter region on metallothionein 2A-metal binding protein in Turkish population

Metallothioneins are low molecular weight proteins composed of 61-68 amino acid residues, and are characterized by high cysteine content with lack of aromatic amino acids. Because of their rich thiol content, Metallothioneins have crucial biological functions including cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification. In this study, we examined metallothionein 2A isoform in 122 kidney tissues from autopsy cases and 186 blood samples from volunteers and detected A --> G single-nucleotide polymorphism in metallothionein 2A core promoter region of Turkish population. The promoter region of metallothionein 2A was amplified with the PCR technique and the amplified oligonucleotides were digested with BsgI restriction enzyme. The genotype frequencies of the single-nucleotide polymorphism in the societies' metallothionein 2A core promoter region were determined as follows: 87% homozygote typical, 12.3% heterozygote and 0.7% homozygote atypical genotype. The allele frequencies were consistent with Hardy-Weinberg equilibrium (chi(2) = 0.2719; P > 0.05).

The secreted salivary proteome of the pea aphid Acyrthosiphon pisum characterised by mass spectrometry.

Nine proteins secreted in the saliva of the pea aphid Acyrthosiphon pisum were identified by a proteomics approach using GE-LC-MS/MS and LC-MS/MS, with reference to EST and genomic sequence data for A. pisum. Four proteins were identified by their sequences: a homolog of angiotensin-converting enzyme (an M2 metalloprotease), an M1 zinc-dependant metalloprotease, a glucose-methanol-choline (GMC)-oxidoreductase and a homolog to regucalcin (also known as senescence marker protein 30). The other five proteins are not homologous to any previously described sequence and included an abundant salivary protein (represented by ACYPI009881), with a predicted length of 1161 amino acids and high serine, tyrosine and cysteine content. A. pisum feeds on plant phloem sap and the metalloproteases and regucalcin (a putative calcium-binding protein) are predicted determinants of sustained feeding, by inactivation of plant protein defences and inhibition of calcium-mediated occlusion of phloem sieve elements, respectively. The amino acid composition of ACYPI009881 suggests a role in the aphid salivary sheath that protects the aphid mouthparts from plant defences, and the oxidoreductase may promote gelling of the sheath protein or mediate oxidative detoxification of plant allelochemicals. Further salivary proteins are expected to be identified as more sensitive MS technologies are developed.

RECK Forms Cowbell-shaped Dimers and Inhibits Matrix Metalloproteinase-catalyzed Cleavage of Fibronectin

The membrane-anchored protease regulator RECK plays important roles in mammalian development and tumor suppression. The biochemical bases of these bioactivities, however, remain poorly understood. Here we report on the properties of a recombinant RECK protein expressed in mouse fibroblasts and purified to near homogeneity. Multiple lines of evidence indicate that RECK forms dimers. Single particle reconstruction using transmission electron microscopy revealed a unique cowbell-like shaped RECK dimer. RECK is cleaved by MMP-2 and MMP-7 and competitively inhibits MMP-7-catalyzed cleavage of fibronectin. Forced RECK expression in HT1080 cells, whose endogenous RECK expression is minimal, leads to an increase in the amount of fibronectin associated with the cell. Our data demonstrate the ability of RECK to protect fibronectin from MMP-mediated degradation.

Whey Protein Concentrate as a Protective Agent Against Doxorubicin-Induced Cardiotoxicity in Mice

ABSTRACTWhey proteins are considered as functional food ingredients which have high cysteine content and promote the biosynthesis of glutathione—the primary intracellular antioxidant. Doxorubicin (DOX) is one of the most active antitumor antibiotics in current use. The therapeutic value of DOX, however, is limited by its toxicity. Oxidative stress is one of the underlying mechanisms of DOX toxicity in noncancerous (nontargeted) tissues. We investigated the protective effect of whey protein concentrate against DOX toxicity and oxidative stress. The administration of DOX (20 mg/kg i.p.) to BALB/c mice caused significant decrease of tissue glutathione level in the heart and severe histopathological changes, examined by light and transmission electron microscopy. These biochemical and histological alterations were effectively attenuated on pretreatment with whey protein concentrate. We therefore concluded that the protective action of whey is due to the enhancement of tissue glutathione level which might have important cytoprotective effects on oxidative stress, induced by DOX treatment.

Composição de aminoácidos em gerações precoces de feijão obtidas a partir de cruzamentos controlados com parental de alto teor de cisteína

Os objetivos deste trabalho foram avaliar a composição de aminoácidos em gerações precoces de feijão, obtidas a partir de cruzamentos controlados com genitor de alto teor de cisteína, e identificar plantas com alto teor de cisteína nos grãos. A partir do cruzamento entre 'TPS Nobre' x 'Minuano', foram obtidas as gerações F1, F1 recíproco, F2, F2 recíproco, retrocruzamento 1 e retrocruzamento 2. Os aminoácidos foram determinados por cromatografia líquida de alta performance (HPLC) e os dados obtidos foram submetidos à análise de variância e de variáveis canônicas. Os aminoácidos - cisteína, prolina, tirosina, arginina, isoleucina e histidina - apresentaram variabilidade genética entre os genitores e as gerações obtidas. Os teores de aminoácidos essenciais e não-essenciais observados nos genitores e nas gerações F1 e F1 recíproco foram adequados para uso na alimentação. As demais gerações apresentaram deficiência em aminoácidos sulfurados. As duas primeiras variáveis canônicas explicaram 96,43% da variação total dos genótipos e dois grupos foram formados. Nenhuma planta da geração F2 é passível de seleção pelo programa de melhoramento para o desenvolvimento de populações segregantes de feijão com alto teor de cisteína nos grãos.

Cloning, expression, and characterization of cadmium-induced metallothionein-2 from the earthworms Metaphire posthuma and Polypheretima elongata

In this study we report the sequences of MT-2 cDNA from two species of Megascoleidae earthworms, Metaphire posthuma and Polypheretima elongata, by mRNA differential display after exposure of the organisms to cadmium. Complementary (c)DNA was verified as the MT-2 gene by the characteristics of its predicted translation product, namely a high cysteine content, conserved CXC motifs, and a molecular weight of around 8 kDa. Amino acid sequence alignment revealed a conserved TKCCG in the cloned MT-2 of both megascolecid earthworms instead of the corresponding conserved TQCCG found in lumbricid earthworms. The cDNAs corresponding to the two megascolecid MT-2 genes were expressed, and the MT-2 proteins were purified for biochemical characterization. The binding of Cu 2+ exhibited monophasic kinetics and those of Zn 2+ and Cd 2+ biphasic kinetics. The proteins bound more tightly to Cd 2+ than to Zn 2+ and more tightly still to Cu 2+. Zn-MT and apo-MT were the most effective at scavenging free radicals, followed by Cd-MT. In conclusion, MT-2s from M. posthuma and P. elongata showed unique sequence features compared to those of lumbricid earthworms. These earthworms could be used to evaluate heavy-metal pollution in soil due to the inducible MT-2 by cadmium exposure.

Purification and Characterization of a Novel Protease from the Latex of Pedilanthus tithymaloids

A novel protease was purified to homogeneity from the latex of Pedilanthus tithymaloids by a simple purification procedure involving ammonium sulfate precipitation and cation-exchange chromatography. The molecular weight of the protease was estimated to be approximately 63.1 kDa and the extinction coefficient (epsilon(1%)(280nm)) was 28.4. The enzyme hydrolyzes denatured natural substrates like casein, azoalbumin and azocasein with a high specific activity but little activity towards synthetic substrates. The pH and temperature optima were pH 8.0-9.5 and 65-70 degrees C, respectively. The proteolytic activity of the enzyme was inhibited by different protease-specific inhibitors (e.g., thiol, serine, metallo, etc.) up to a certain extent but not completely by any class of inhibitors. The enzyme was relatively stable towards pH change, temperature, denaturants and organic solvents. The enzyme consists of five disulfide bridges compared to three observed in most plant cysteine proteases. Overall, the striking features of this protease are its high molecular weight, high cysteine content and only partial inhibition of activity by different classes of protease inhibitors contrary to known proteases from other plant sources. The enzyme is named as pedilanthin as per the protease nomenclature.

Juglone Inactivates Cysteine-rich Proteins Required for Progression through Mitosis

The parvulin peptidyl-prolyl isomerase Pin1 catalyzes cis-trans isomerization of p(S/T)-P bonds and might alter conformation and function of client proteins. Since the trans conformation of p(S/T)-P bonds is preferred by protein phosphatase 2A (PP2A), Pin1 may facilitate PP2A-mediated dephosphorylation. Juglone irreversibly inhibits parvulins and is often used to study the function of Pin1 in vivo. The drug prevents dephosphorylation of mitotic phosphoproteins, perhaps because they bind Pin1 and are dephosphorylated by PP2A. We show here however that juglone inhibited post-mitotic dephosphorylation and the exit of mitosis, independent of Pin1. This effect involved covalent modification of sulfhydryl groups in proteins essential for metaphase/anaphase transition. Particularly cytoplasmic proteins with a high cysteine content were vulnerable to the drug. Alkylation of sulfhydryl groups altered the conformation of such proteins, as evidenced by the disappearance of antibody epitopes on tubulin and the mitotic checkpoint component BubR1. The latter activates the anaphase-promoting complex/cyclosome, which degrades regulatory proteins, such as cyclin B1 and securins, and is required for mitotic exit. Indeed, juglone-treated cells failed to assemble a mitotic spindle, which correlated with perturbed microtubule dynamics, loss of immunodetectable tubulin, and formation of tubulin aggregates. Juglone also prevented degradation of cyclin B1, independently of the Mps1-controlled mitotic spindle checkpoint. Since juglone affected cell cycle progression at several levels, more specific drugs need to be developed for studies of Pin1 function in vivo.