The broad phenotypic spectrum of 17α-hydroxylase/17,20-lyase (CYP17A1) deficiency: a case series.

CONTEXT: 17α-Hydroxylase/17,20-lyase deficiency (17OHD) caused by mutations in the CYP17A1 gene is a rare form of congenital adrenal hyperplasia typically characterised by cortisol deficiency, mineralocorticoid excess and sex steroid deficiency. OBJECTIVE: To examine the phenotypic spectrum of 17OHD by clinical and biochemical assessment and corresponding in silico and in vitro functional analysis. DESIGN: Case series. PATIENTS AND RESULTS: We assessed eight patients with 17OHD, including four with extreme 17OHD phenotypes: two siblings presented with failure to thrive in early infancy and two with isolated sex steroid deficiency and normal cortisol reserve. Diagnosis was established by mass spectrometry-based urinary steroid profiling and confirmed by genetic CYP17A1 analysis, revealing homozygous and compound heterozygous sequence variants. We found novel (p.Gly111Val, p.Ala398Glu, p.Ile371Thr) and previously described sequence variants (p.Pro409Leu, p.Arg347His, p.Gly436Arg, p.Phe53/54del, p.Tyr60IlefsLys88X). In vitro functional studies employing an overexpression system in HEK293 cells showed that 17,20-lyase activity was invariably decreased while mutant 17α-hydroxylase activity retained up to 14% of WT activity in the two patients with intact cortisol reserve. A ratio of urinary corticosterone over cortisol metabolites reflective of 17α-hydroxylase activity correlated well with clinical phenotype severity. CONCLUSION: Our findings illustrate the broad phenotypic spectrum of 17OHD. Isolated sex steroid deficiency with normal stimulated cortisol has not been reported before. Attenuation of 17α-hydroxylase activity is readily detected by urinary steroid profiling and predicts phenotype severity. SIGNIFICANCE STATEMENT: Here we report, supported by careful phenotyping, genotyping and functional analysis, a prismatic case series of patients with congenital adrenal hyperplasia due to 17α-hydroxylase (CYP17A1) deficiency (17OHD). These range in severity from the abolition of function, presenting in early infancy, and unusually mild with isolated sex steroid deficiency but normal ACTH-stimulated cortisol in adult patients. These findings will guide improved diagnostic detection of CYP17A1 deficiency.

Dimerization of the Sodium/Iodide Symporter.

Background: The ability of thyroid follicular epithelial cells to accumulate iodide via the sodium/iodide symporter (NIS) is exploited to successfully treat most thyroid cancers, although a subset of patients lose functional NIS activity and become unresponsive to radioiodide therapy, with poor clinical outcome. Our knowledge of NIS regulation remains limited, however. While numerous membrane proteins are functionally regulated via dimerization, there is little definitive evidence of NIS dimerization, and whether this might impact upon radioiodide uptake and treatment success is entirely unknown. We hypothesized that NIS dimerizes and that dimerization is a prerequisite for iodide uptake. Methods: Coimmunoprecipitation, proximity ligation, and Förster resonance energy transfer (FRET) assays were used to assess NIS:NIS interaction. To identify residues involved in dimerization, a homology model of NIS structure was built based on the crystal structure of the dimeric bacterial protein vSGLT. Results: Abundant cellular NIS dimerization was confirmed in vitro via three discrete methodologies. FRET and proximity ligation assays demonstrated that while NIS can exist as a dimer at the plasma membrane (PM), it is also apparent in other cellular compartments. Homology modeling revealed one key potential site of dimeric interaction, with six residues <3Å apart. In particular, NIS residues Y242, T243, and Q471 were identified as critical to dimerization. Individual mutation of residues Y242 and T243 rendered NIS nonfunctional, while abrogation of Q471 did not impact radioiodide uptake. FRET data show that the putative dimerization interface can tolerate the loss of one, but not two, of these three clustered residues. Conclusions: We show for the first time that NIS dimerizes in vitro, and we identify the key residues via which this happens. We hypothesize that dimerization of NIS is critical to its trafficking to the PM and may therefore represent a new mechanism that would need to be considered in overcoming therapeutic failure in patients with thyroid cancer.

Melting Down Protein Stability: PAPS Synthase 2 in Patients and in a Cellular Environment.

Within the crowded and complex environment of the cell, a protein experiences stabilizing excluded-volume effects and destabilizing quinary interactions with other proteins. Which of these prevail, needs to be determined on a case-by-case basis. PAPS synthases are dimeric and bifunctional enzymes, providing activated sulfate in the form of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for sulfation reactions. The human PAPS synthases PAPSS1 and PAPSS2 differ significantly in their protein stability as PAPSS2 is a naturally fragile protein. PAPS synthases bind a series of nucleotide ligands and some of them markedly stabilize these proteins. PAPS synthases are of biomedical relevance as destabilizing point mutations give rise to several pathologies. Genetic defects in PAPSS2 have been linked to bone and cartilage malformations as well as a steroid sulfation defect. All this makes PAPS synthases ideal to study protein unfolding, ligand binding, and the stabilizing and destabilizing factors in their cellular environment. This review provides an overview on current concepts of protein folding and stability and links this with our current understanding of the different disease mechanisms of PAPSS2-related pathologies with perspectives for future research and application.

Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.

The high-energy sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), generated by human PAPS synthase isoforms PAPSS1 and PAPSS2, is required for all human sulfation pathways. Sulfotransferase SULT2A1 uses PAPS for sulfation of the androgen precursor dehydroepiandrosterone (DHEA), thereby reducing downstream activation of DHEA to active androgens. Human PAPSS2 mutations manifest with undetectable DHEA sulfate, androgen excess, and metabolic disease, suggesting that ubiquitous PAPSS1 cannot compensate for deficient PAPSS2 in supporting DHEA sulfation. In knockdown studies in human adrenocortical NCI-H295R1 cells, we found that PAPSS2, but not PAPSS1, is required for efficient DHEA sulfation. Specific APS kinase activity, the rate-limiting step in PAPS biosynthesis, did not differ between PAPSS1 and PAPSS2. Co-expression of cytoplasmic SULT2A1 with a cytoplasmic PAPSS2 variant supported DHEA sulfation more efficiently than co-expression with nuclear PAPSS2 or nuclear/cytosolic PAPSS1. Proximity ligation assays revealed protein-protein interactions between SULT2A1 and PAPSS2 and, to a lesser extent, PAPSS1. Molecular docking studies showed a putative binding site for SULT2A1 within the PAPSS2 APS kinase domain. Energy-dependent scoring of docking solutions identified the interaction as specific for the PAPSS2 and SULT2A1 isoforms. These findings elucidate the mechanistic basis for the selective requirement for PAPSS2 in human DHEA sulfation.

Sensing and signaling of oxidative stress in chloroplasts by inactivation of the SAL1 phosphoadenosine phosphatase.

Intracellular signaling during oxidative stress is complex, with organelle-to-nucleus retrograde communication pathways ill-defined or incomplete. Here we identify the 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase SAL1 as a previously unidentified and conserved oxidative stress sensor in plant chloroplasts. Arabidopsis thaliana SAL1 (AtSAL1) senses changes in photosynthetic redox poise, hydrogen peroxide, and superoxide concentrations in chloroplasts via redox regulatory mechanisms. AtSAL1 phosphatase activity is suppressed by dimerization, intramolecular disulfide formation, and glutathionylation, allowing accumulation of its substrate, PAP, a chloroplast stress retrograde signal that regulates expression of plastid redox associated nuclear genes (PRANGs). This redox regulation of SAL1 for activation of chloroplast signaling is conserved in the plant kingdom, and the plant protein has evolved enhanced redox sensitivity compared with its yeast ortholog. Our results indicate that in addition to sulfur metabolism, SAL1 orthologs have evolved secondary functions in oxidative stress sensing in the plant kingdom.

The Regulation of Steroid Action by Sulfation and Desulfation.

Steroid sulfation and desulfation are fundamental pathways vital for a functional vertebrate endocrine system. After biosynthesis, hydrophobic steroids are sulfated to expedite circulatory transit. Target cells express transmembrane organic anion-transporting polypeptides that facilitate cellular uptake of sulfated steroids. Once intracellular, sulfatases hydrolyze these steroid sulfate esters to their unconjugated, and usually active, forms. Because most steroids can be sulfated, including cholesterol, pregnenolone, dehydroepiandrosterone, and estrone, understanding the function, tissue distribution, and regulation of sulfation and desulfation processes provides significant insights into normal endocrine function. Not surprisingly, dysregulation of these pathways is associated with numerous pathologies, including steroid-dependent cancers, polycystic ovary syndrome, and X-linked ichthyosis. Here we provide a comprehensive examination of our current knowledge of endocrine-related sulfation and desulfation pathways. We describe the interplay between sulfatases and sulfotransferases, showing how their expression and regulation influences steroid action. Furthermore, we address the role that organic anion-transporting polypeptides play in regulating intracellular steroid concentrations and how their expression patterns influence many pathologies, especially cancer. Finally, the recent advances in pharmacologically targeting steroidogenic pathways will be examined.

PAPSS2 deficiency causes androgen excess via impaired DHEA sulfation--in vitro and in vivo studies in a family harboring two novel PAPSS2 mutations.

CONTEXT: PAPSS2 (PAPS synthase 2) provides the universal sulfate donor PAPS (3'-phospho-adenosine-5'-phosphosulfate) to all human sulfotransferases, including SULT2A1, responsible for sulfation of the crucial androgen precursor dehydroepiandrosterone (DHEA). Impaired DHEA sulfation is thought to increase the conversion of DHEA toward active androgens, a proposition supported by the previous report of a girl with inactivating PAPSS2 mutations who presented with low serum DHEA sulfate and androgen excess, clinically manifesting with premature pubarche and early-onset polycystic ovary syndrome. PATIENTS AND METHODS: We investigated a family harboring two novel PAPSS2 mutations, including two compound heterozygous brothers presenting with disproportionate short stature, low serum DHEA sulfate, but normal serum androgens. Patients and parents underwent a DHEA challenge test comprising frequent blood sampling and urine collection before and after 100 mg DHEA orally, with subsequent analysis of DHEA sulfation and androgen metabolism by mass spectrometry. The functional impact of the mutations was investigated in silico and in vitro. RESULTS: We identified a novel PAPSS2 frameshift mutation, c.1371del, p.W462Cfs*3, resulting in complete disruption, and a novel missense mutation, c.809G>A, p.G270D, causing partial disruption of DHEA sulfation. Both patients and their mother, who was heterozygous for p.W462Cfs*3, showed increased 5α-reductase activity at baseline and significantly increased production of active androgens after DHEA intake. The mother had a history of oligomenorrhea and chronic anovulation that required clomiphene for ovulation induction. CONCLUSIONS: We provide direct in vivo evidence for the significant functional impact of mutant PAPSS2 on DHEA sulfation and androgen activation. Heterozygosity for PAPSS2 mutations can be associated with a phenotype resembling polycystic ovary syndrome.

Transient domain interactions enhance the affinity of the mitotic regulator Pin1 toward phosphorylated peptide ligands.

The mitotic regulator Pin1 plays an important role in protein quality control and age-related medical conditions such as Alzheimer disease and Parkinson disease. Although its cellular role has been thoroughly investigated during the past decade, the molecular mechanisms underlying its function remain elusive. We provide evidence for interactions between the two domains of Pin1. Several residues displayed unequivocal peak splits in nuclear magnetic resonance spectra, indicative of two different conformational states in equilibrium. Pareto analysis of paramagnetic relaxation enhancement data demonstrates that the two domains approach each other upon addition of a nonpeptidic ligand. Titration experiments with phosphorylated peptides monitored by fluorescence anisotropy and chemical shift perturbation indicate that domain interactions increase Pin1's affinity toward peptide ligands. We propose this interplay of the domains and ligands to be a general mechanism for a large class of two-domain proteins.

Adenosine-5'-phosphosulfate--a multifaceted modulator of bifunctional 3'-phospho-adenosine-5'-phosphosulfate synthases and related enzymes.

All sulfation reactions rely on active sulfate in the form of 3'-phospho-adenosine-5'-phosphosulfate (PAPS). In fungi, bacteria, and plants, the enzymes responsible for PAPS synthesis, ATP sulfurylase and adenosine-5'-phosphosulfate (APS) kinase, reside on separate polypeptide chains. In metazoans, however, bifunctional PAPS synthases catalyze the consecutive steps of sulfate activation by converting sulfate to PAPS via the intermediate APS. This intricate molecule and the related nucleotides PAPS and 3'-phospho-adenosine-5'-phosphate modulate the function of various enzymes from sulfation pathways, and these effects are summarized in this review. On the ATP sulfurylase domain that initially produces APS from sulfate and ATP, APS acts as a potent product inhibitor, being competitive with both ATP and sulfate. For the APS kinase domain that phosphorylates APS to PAPS, APS is an uncompetitive substrate inhibitor that can bind both at the ATP/ADP-binding site and the PAPS/APS-binding site. For human PAPS synthase 1, the steady-state concentration of APS has been modelled to be 1.6 µM, but this may increase up to 60 µM under conditions of sulfate excess. It is noteworthy that the APS concentration for maximal APS kinase activity is 15 µM. Finally, we recognized APS as a highly specific stabilizer of bifunctional PAPS synthases. APS most likely stabilizes the APS kinase part of these proteins by forming a dead-end enzyme-ADP-APS complex at APS concentrations between 0.5 and 5 µM; at higher concentrations, APS may bind to the catalytic centers of ATP sulfurylase. Based on the assumption that cellular concentrations of APS fluctuate within this range, APS can therefore be regarded as a key modulator of PAPS synthase functions.

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) synthases, naturally fragile enzymes specifically stabilized by nucleotide binding.

Activated sulfate in the form of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is needed for all sulfation reactions in eukaryotes with implications for the build-up of extracellular matrices, retroviral infection, protein modification, and steroid metabolism. In metazoans, PAPS is produced by bifunctional PAPS synthases (PAPSS). A major question in the field is why two human protein isoforms, PAPSS1 and -S2, are required that cannot complement for each other. We provide evidence that these two proteins differ markedly in their stability as observed by unfolding monitored by intrinsic tryptophan fluorescence as well as circular dichroism spectroscopy. At 37 °C, the half-life for unfolding of PAPSS2 is in the range of minutes, whereas PAPSS1 remains structurally intact. In the presence of their natural ligand, the nucleotide adenosine 5'-phosphosulfate (APS), PAPS synthase proteins are stabilized. Invertebrates only possess one PAPS synthase enzyme that we classified as PAPSS2-type by sequence-based machine learning techniques. To test this prediction, we cloned and expressed the PPS-1 protein from the roundworm Caenorhabditis elegans and also subjected this protein to thermal unfolding. With respect to thermal unfolding and the stabilization by APS, PPS-1 behaved like the unstable human PAPSS2 protein suggesting that the less stable protein is evolutionarily older. Finally, APS binding more than doubled the half-life for unfolding of PAPSS2 at physiological temperatures and effectively prevented its aggregation on a time scale of days. We propose that protein stability is a major contributing factor for PAPS availability that has not as yet been considered. Moreover, naturally occurring changes in APS concentrations may be sensed by changes in the conformation of PAPSS2.

Human PAPS synthase isoforms are dynamically regulated enzymes with access to nucleus and cytoplasm.

In higher eukaryotes, PAPS synthases are the only enzymes producing the essential sulphate-donor 3'-phospho-adenosine-5'-phosphosulphate (PAPS). Recently, PAPS synthases have been associated with several genetic diseases and retroviral infection. To improve our understanding of their pathobiological functions, we analysed the intracellular localisation of the two human PAPS synthases, PAPSS1 and PAPSS2. For both enzymes, we observed pronounced heterogeneity in their subcellular localisation. PAPSS1 was predominantly nuclear, whereas PAPSS2 localised mainly within the cytoplasm. Treatment with the nuclear export inhibitor leptomycin B had little effect on their localisation. However, a mutagenesis screen revealed an Arg-Arg motif at the kinase interface exhibiting export activity. Notably, both isoforms contain a conserved N-terminal basic Lys-Lys-Xaa-Lys motif indispensable for their nuclear localisation. This nuclear localisation signal was more efficient in PAPSS1 than in PAPSS2. The activities of the identified localisation signals were confirmed by microinjection studies. Collectively, we describe unusual localisation signals of both PAPS synthase isoforms, mobile enzymes capable of executing their function in the cytoplasm as well as in the nucleus.

Single-domain parvulins constitute a specific marker for recently proposed deep-branching archaeal subgroups.

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes assisting protein folding and protein quality control in organisms of all kingdoms of life. In contrast to the other sub-classes of PPIases, the cyclophilins and the FK-506 binding proteins, little was formerly known about the parvulin type of PPIase in Archaea. Recently, the first solution structure of an archaeal parvulin, the PinA protein from Cenarchaeum symbiosum, was reported. Investigation of occurrence and frequency of PPIase sequences in numerous archaeal genomes now revealed a strong tendency for thermophilic microorganisms to reduce the number of PPIases. Single-domain parvulins were mostly found in the genomes of recently proposed deep-branching archaeal subgroups, the Thaumarchaeota and the ARMANs (archaeal Richmond Mine acidophilic nanoorganisms). Hence, we used the parvulin sequence to reclassify available archaeal metagenomic contigs, thereby, adding new members to these subgroups. A combination of genomic background analysis and phylogenetic approaches of parvulin sequences suggested that the assigned sequences belong to at least two distinct groups of Thaumarchaeota. Finally, machine learning approaches were applied to identify amino acid residues that separate archaeal and bacterial parvulin proteins from each other. When mapped onto the recent PinA solution structure, most of these positions form a cluster at one site of the protein possibly indicating a different functionality of the two groups of parvulin proteins.

Crystallographic proof for an extended hydrogen-bonding network in small prolyl isomerases.

Parvulins compose a family of small peptidyl-prolyl isomerases (PPIases) involved in protein folding and protein quality control. A number of amino acids in the catalytic cavity are highly conserved, but their precise role within the catalytic mechanism is unknown. The 0.8 Å crystal structure of the prolyl isomerase domain of parvulin Par14 shows the electron density of hydrogen atoms between the D74, H42, H123, and T118 side chains. This threonine residue has previously not been associated with catalysis, but a corresponding T152A mutant of Pin1 shows a dramatic reduction of catalytic activity without compromising protein stability. The observed catalytic tetrad is strikingly conserved in Pin1- and parvulin-type proteins and hence constitutes a common feature of small peptidyl prolyl isomerases.

Structural basis for the role of the Sir3 AAA+ domain in silencing: interaction with Sir4 and unmethylated histone H3K79.

The silent information regulator 2/3/4 (Sir2/3/4) complex is required for gene silencing at the silent mating-type loci and at telomeres in Saccharomyces cerevisiae. Sir3 is closely related to the origin recognition complex 1 subunit and consists of an N-terminal bromo-adjacent homology (BAH) domain and a C-terminal AAA(+) ATPase-like domain. Here, through a combination of structure biology and exhaustive mutagenesis, we identified unusual, silencing-specific features of the AAA(+) domain of Sir3. Structural analysis of the putative nucleotide-binding pocket in this domain reveals a shallow groove that would preclude nucleotide binding. Mutation of this site has little effect on Sir3 function in vivo. In contrast, several surface regions are shown to be necessary for the Sir3 silencing function. Interestingly, the Sir3 AAA(+) domain is shown here to bind chromatin in vitro in a manner sensitive to histone H3K79 methylation. Moreover, an exposed loop on the surface of this Sir3 domain is found to interact with Sir4. In summary, the unique folding of this conserved Sir3 AAA(+) domain generates novel surface regions that mediate Sir3-Sir4 and Sir3-nucleosome interactions, both being required for the proper assembly of heterochromatin in living cells.

Structure and dynamics of the first archaeal parvulin reveal a new functionally important loop in parvulin-type prolyl isomerases.

Parvulins are a group of peptidyl-prolyl isomerases (PPIases) responsible for important biological processes in all kingdoms of life. The PinA protein from the psychrophilic archaeon Cenarchaeum symbiosum is a parvulin-like PPIase. Due to its striking similarity to the human parvulins Pin1 and Par14, PinA constitutes an interesting subject for structural and functional studies. Here, we present the first high resolution NMR structure of an archaeal parvulin, PinA, based on 1798 conformational restraints. Structure calculation yields an ensemble of 20 convergent low energy structures with a backbone r.m.s.d. value of 0.6 Å within the secondary structure elements. The overall fold of PinA comprises the ß-α(3)-ß-α-ß(2) fold typical for all parvulin structures known so far, but with helix III being a short 3(10)-helix. A detailed comparison of this high resolution structure of the first archaeal PinA protein with bacterial and eukaryotic parvulin PPIase structures reveals an atypically large catalytic binding site. This feature provides an explanation for cold-adapted protein function. Moreover, the residues in and around 3(10)-helix III exhibit strong intramolecular dynamics on a microsecond to millisecond timescale and display structural heterogeneity within the NMR ensemble. A putative peptide ligand was found for PinA by phage display and was used for (1)H-(15)N-HSQC titrations. Again, the flexible region around 3(10)-helix III as well as residues of the peptide binding pocket showed the strongest chemical shift perturbations upon peptide binding. The local flexibility of this region also was modulated by ligand binding. A glycine and two positively charged residues are conserved in most parvulin proteins in this flexible loop region, which may be of general functional importance for parvulin-type PPIases.

A potential transcriptional regulator is out-of-frame translated from the metallothionein 2A messenger RNA.

In a number of yeast two-hybrid screens, we have found clones that contained parts of the human metallothionein 2A (MT2A) nucleotide sequence. All of these clones were out-of-frame relative to the MT2A coding sequence and activated the yeast reporters in the presence of the Gal4 DNA binding domain but irrespective of the bait protein. Reporter gene activation was abolished when activation domain and MT2A coding sequences were brought in-frame. In light of these findings, we evaluated all recently reported interactions with metallothioneins because our out-of-frame proline-rich protein might have been the actual interaction partner in some of these studies.

A heterodimer of human 3'-phospho-adenosine-5'-phosphosulphate (PAPS) synthases is a new sulphate activating complex.

3'-Phospho-adenosine-5'-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Förster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that--in spite of its low quantum yield--is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5'-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.

The solution structure of pGolemi, a high affinity Mena EVH1 binding miniature protein, suggests explanations for paralog-specific binding to Ena/VASP homology (EVH) 1 domains.

Ena/VASP homology 1 (EVH1) domains are polyproline binding domains that are present in a wide range of adaptor proteins, among them Ena/VASP proteins involved in actin remodeling and axonal guidance. The interaction of ActA, a transmembrane protein from the food-borne pathogen Listeria monocytogenes, with EVH1 domains has been shown to be crucial for recruitment of the host's actin skeleton and, as a consequence, for the infectivity of this bacterium. We present the structure of a synthetic high-affinity Mena EVH1 ligand, pGolemi, capable of paralog-specific binding, solved by NMR spectroscopy. This peptide shares the common pancreatic peptide fold with its scaffold, avian pancreatic peptide, but shows pivotal differences in the amino-terminus. The interplay of spatial fixation and flexibility appears to be the reason for its high affinity towards Mena EVH1. Combined with earlier investigations, our structural data shed light on the specificity determinants of pGolemi and the importance of additional binding epitopes around the residues Thr74 and Phe32 on EVH1 domains regulating paralog specificity. Our results are expected to facilitate the design of other high-affinity, paralog-specific EVH1 domain ligands, and serve as a fundament for the investigation of the molecular mode of action of EVH1 domains.

Small family with key contacts: par14 and par17 parvulin proteins, relatives of pin1, now emerge in biomedical research.

The parvulin-type peptidyl-prolyl cis/trans isomerase Pin1 is subject of intense biochemical and clinical research as it seems to be involved in the pathogenesis of certain cancers and protein folding illnesses like Alzheimer's and Parkinson's disease. In addition to Pin1, the human genome only contains a single other parvulin locus encoding two protein species-Par14 and Par17. Much less is known about these enzymes although their sequences are highly conserved in all metazoans. Parvulin has been proposed to function as Pin1 complementing enzyme in cell cycle regulation and in chromatin remodelling. Pharmaceutical modulation of Par14 might therefore have benefits for certain types of cancer. Moreover, the Par17 protein that has been shown to be confined to anthropoid primate species only might provide a deeper understanding for human-specific brain development. This review aims at stimulating further research on Par14 and Par17 that are overlooked drug targets in the shadow of an overwhelming plethora of Pin1 literature by summarising all current knowledge on these parvulin proteins.

Human TPST1 transmembrane domain triggers enzyme dimerisation and localisation to the Golgi compartment.

TPST1 is a human tyrosylprotein sulfotransferase that uses 3'phosphoadenosine-5'phosphosulfate (PAPS) to transfer the sulfate moiety to proteins predominantly designated for secretion. To achieve a general understanding of the cellular role of human tyrosine-directed sulfotransferases, we investigated targeting, structure and posttranslational modification of TPST1. Golgi localisation of the enzyme in COS-7 and HeLa cells was visualised by fluorescence imaging techniques. PNGase treatment and mutational studies determined that TPST1 bears N-linked glycosyl residues exclusively at position Asn60 and Asn262. By alanine mutation of these asparagine residues, we could determine that the N-linked oligosaccharides do not have an influence on Golgi retention of TPST1. In concert with N and C-terminal flanking residues, the transmembrane domain of TPST1 was determined to act in targeting and retention of the enzyme to the trans-Golgi compartment. This domain exhibits a pronounced secondary structure in a lipid environment. Further in vivo FRET studies using the transmembrane domain suggest that the human tyrosylprotein sulfotransferase may be functional as homodimer/oligomer in the trans-Golgi compartment.