Substitution Of Proline Research Articles

Effect of Proline Mutations on the Monomer Conformations of Amylin

The formation of human islet amyloid polypeptide (hIAPP) is implicated in the loss of pancreatic β-cells in type II diabetes. Rat amylin, which differs from human amylin at six residues, does not lead to formation of amyloid fibrils. Pramlintide is a synthetic analog of human amylin that shares three proline substitutions with rat amylin. Pramlintide has a much smaller propensity to form amyloid aggregates and has been widely prescribed in amylin replacement treatment. It is known that the three prolines attenuate β-sheet formation. However, the detailed effects of these proline substitutions on full-length hIAPP remain poorly understood. In this work, we use molecular simulations and bias-exchange metadynamics to investigate the effect of proline substitutions on the conformation of the hIAPP monomer. Our results demonstrate that hIAPP can adopt various β-sheet conformations, some of which have been reported in experiments. The proline substitutions perturb the formation of long β-sheets and reduce their stability. More importantly, we find that all three proline substitutions of pramlintide are required to inhibit β conformations and stabilize the α-helical conformation. Fewer substitutions do not have a significant inhibiting effect.

A Naturally Variable Residue in the S1 Subsite of M1 Family Aminopeptidases Modulates Catalytic Properties and Promotes Functional Specialization

M1 family metallo-aminopeptidases fulfill a wide range of critical and in some cases medically relevant roles in humans and human pathogens. The specificity of M1-aminopeptidases is dominated by the interaction of the well defined S1 subsite with the side chain of the first (P1) residue of the substrate and can vary widely. Extensive natural variation occurs at one of the residues that contributes to formation of the cylindrical S1 subsite. We investigated whether this natural variation contributes to diversity in S1 subsite specificity. Effects of 11 substitutions of the S1 subsite residue valine 459 in the Plasmodium falciparum aminopeptidase PfA-M1 and of three substitutions of the homologous residue methionine 260 in Escherichia coli aminopeptidase N were characterized. Many of these substitutions altered steady-state kinetic parameters for dipeptide hydrolysis and remodeled S1 subsite specificity. The most dramatic change in specificity resulted from substitution with proline, which collapsed S1 subsite specificity such that only substrates with P1-Arg, -Lys, or -Met were appreciably hydrolyzed. The structure of PfA-M1 V459P revealed that the proline substitution induced a local conformational change in the polypeptide backbone that resulted in a narrowed S1 subsite. The restricted specificity and active site backbone conformation of PfA-M1 V459P mirrored those of endoplasmic reticulum aminopeptidase 2, a human enzyme with proline in the variable S1 subsite position. Our results provide compelling evidence that changes in the variable residue in the S1 subsite of M1-aminopeptidases have facilitated the evolution of new specificities and ultimately novel functions for this important class of enzymes.

A New PAX8 Mutation Causing Congenital Hypothyroidism in Three Generations of a Family Is Associated with Abnormalities in the Urogenital Tract

Although thyroid dysgenesis is the most common cause of congenital hypothyroidism (CH), its molecular basis remains largely elusive. Indeed, in only a minority of cases with thyroid dysgenesis (2%-3%) was it possible to identify an underlying genetic defect. The objective of this study was to screen the PAX8 gene and the PAX2 gene in a family with six cases of CH spanning three generations and presenting urogenital malformations. Herein, we report a case series and in vitro characterization of the PAX8 gene mutation. Investigations were conducted at a tertiary care referral center. The index case was diagnosed to have congenital hypothyroidism at 7 months of age when he presented with severe impairment of suckling, constipation, and poor development. Treatment with levothyroxine corrected the symptoms and was associated with catch-up growth. His progeny, including two sons, one daughter, and two granddaughters, were affected by CH, and three of them received the diagnosis at neonatal screening. Ultrasound demonstrated normally located thyroid glands with reduced volumes. Five of the six affected family members, including the index case, had urogenital malformations, including incomplete horseshoe kidney, undescended testicles, hydrocele, and ureterocele. Strabismus was found in three out of six affected patients. No other somatic malformations were found. Direct sequencing of the PAX8 gene revealed a new heterozygous mutation (c.74C > G) in all affected individuals. This mutation leads to substitution of proline with arginine at codon 25 (P25R). Fluorescence microscopy showed that P25R is normally located in the nucleus. In transient transfection studies, this mutation causes reduced transcriptional activation ability when using a luciferase reporter construct under the control of a thyroglobulin promoter. This diminished transactivation ability is due to loss of DNA binding capability as shown in electrophoresis mobility shift assay. The sequencing analysis of the PAX2 gene was normal. We conclude that this novel PAX8 mutation is responsible for a severe form of dominantly inherited CH. The mutation seems to be associated with abnormalities of the urogenital tract.

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for the Design of Functional Monomeric Proteins

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.

Proline substitution independently enhances H‐2Db complex stabilization and TCR recognition of melanoma‐associated peptides

The immunogenicity of H-2D(b) (D(b)) restricted epitopes can be significantly increased by substituting peptide position 3 to a proline (p3P). The p3P modification enhances MHC stability without altering the conformation of the modified epitope allowing for T-cell cross-reactivity with the native peptide. The present study reveals how specific interactions between p3P and the highly conserved MHC heavy chain residue Y159 increase the stability of D(b) in complex with an optimized version of the melanoma-associated epitope gp10025-33 . Furthermore, the p3P modification directly increased the affinity of the D(b)/gp10025-33 -specific T-cell receptor (TCR) pMel. Surprisingly, the enhanced TCR binding was independent from the observed increased stability of the optimized D(b)/gp10025-33 complex and from the interactions formed between p3P and Y159, indicating a direct effect of the p3P modification on TCR recognition.

Autosomal-dominant nystagmus, foveal hypoplasia and presenile cataract associated with a novel PAX6 mutation

Autosomal-dominant idiopathic infantile nystagmus has been linked to 6p12 (OMIM 164100), 7p11.2 (OMIM 608345) and 13q31-q33 (OMIM 193003). PAX6 (11p13, OMIM 607108) mutations can also cause autosomal-dominant nystagmus, typically in association with aniridia or iris hypoplasia. We studied a large multigenerational white British family with autosomal-dominant nystagmus, normal irides and presenile cataracts. An SNP-based genome-wide analysis revealed a linkage to a 13.4-MB region on chromosome 11p13 with a maximum lod score of 2.93. A mutation analysis of the entire coding region and splice junctions of the PAX6 gene revealed a novel heterozygous missense mutation (c.227C>G) that segregated with the phenotype and is predicted to result in the amino-acid substitution of proline by arginine at codon 76 p.(P76R). The amino-acid variation p.(P76R) within the paired box domain is likely to destabilise the protein due to steric hindrance as a result of the introduction of a polar and larger amino acid. Eye movement recordings showed a significant intrafamilial variability of horizontal, vertical and torsional nystagmus. High-resolution in vivo imaging of the retina using optical coherence tomography (OCT) revealed features of foveal hypoplasia, including rudimentary foveal pit, incursion of inner retinal layers, short photoreceptor outer segments and optic nerve hypoplasia. Thus, this study presents a family that segregates a PAX6 mutation with nystagmus and foveal hypoplasia in the absence of iris abnormalities. Moreover, it is the first study showing detailed characteristics using eye movement recordings of autosomal-dominant nystagmus in a multigenerational family with a novel PAX6 mutation.

Emerging targets in human lymphoma: targeting the MYD88 mutation

Emerging targets in human lymphoma: targeting the MYD88 mutation James Q Wang,* Yogesh S Jeelall,* Keisuke Horikawa* Department of Immunology, The John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia *All authors contributed equally to this manuscript Abstract: B cell neoplasms co-opt the molecular machinery of normal B cells for their survival. Technological advances in cancer genomics has significantly contributed to uncovering the root cause of aggressive lymphomas, revealing a previously unknown link between TLR signaling and B cell neoplasm. Recurrent oncogenic mutations in MYD88 have been found in 39% of the activated B cell-like subtype of diffuse large B cell lymphoma (ABC DLBCL). Interestingly, 29% of ABC DLBCL have a single amino acid substitution of proline for the leucine at position 265 (L265P), and the exact same variant has also been identified in a number of lymphoid malignancies. The MYD88 L265P variant was recently identified in 90% of Wadenstrom's macroglobulinemia patients. These recent developments warrant the need for novel diagnostic tools as well as targeted therapeutics. In this review, we discuss the physiological functions of MYD88 and focus on its role in B cell lymphomas, evaluating the potential for targeting oncogenic MYD88 in lymphoma. Keywords: MYD88, L265P mutation, lymphoma, targeted therapy

A cone‐rod dystrophy patient with a homozygous RP1L1 mutation

AbstractPurpose To describe a family with a cone‐rod dystrophy patient who has homozygous mutation of the RP1‐like protein 1 (RP1L1) gene.Methods A family including a cone‐rod dystrophy patient underwent detailed ophthalmic clinical evaluations including high resolution cone photorecepor imaging with adaptive optics fundus camera. Mutation screening of the sequence of RP1L1 gene were performed by DNA sequencing analysis in this family membersResults A patient showed a mild reduction of cone and flicker response in full‐field electroretinogram (ERG). Her ERG also showed slight decrease in the amplitude of rod response. IS/OS junction and COST line in SD‐OCT images are severely disturbed. Response of multifocal ERGs (mfERGs) of the patient was severely reduced. A homozygous RP1L1 mutation (c.3628 T>C) was identified in the patient. The mutation c.3628 T>C in exon 4 resulted in the substitution of proline for serine at amino acid position 1210. This mutation was not reported in SNP database. The serine at position 1210 is well conserved among the RP1L1 family in other species. Four out of five computational assessment tools predicted that this mutation is damaging to the protein function. This mutation was not present in 460 control alleles. Family members with heterozygous S1210P mutation showed normal best‐corrected visual acuity (BCVA), SD‐OCT, mfERGs, and focal macular ERGs. Adaptive Optics (AO) images of the patient showed severe reduction of cone density and irregular cone mosaic despite family members with heterozygous S1210P mutation showed normal cone density and regular cone mosaic.Conclusion We have demonstrated a possibility that autosomal recessive cone‐rod dystrophy may be caused by homozygous RP1L1 mutation.

Modulation of Aggregation Propensity of Aβ38 by Site Specific Multiple Proline Substitution

Proline with its unique geometrical feature generates turn in the peptide backbone. Therefore, aggregation potential of Aβ38 can be modulated by insertion of proline at specific positions. Although site specific proline mutation is reported, effect of multiple proline substitution on aggregation potential is still not demonstrated. Therefore, Aβ38, a single proline substituted variant, V18P-Aβ38, a double proline substituted variant, V18P-I31P-Aβ38 and a triple proline substituted version, V12P-V18P-I31P-Aβ38 were synthesized and their aggregation potential were studied. The proline mutants found to have higher solubility and slower aggregation kinetics. The double mutated version was relatively less aggregation prone than its single mutated version. Such analogues can be useful for designing new β sheet breaker peptides, studying the mechanism of aggregation and structural analyses.

Pinpointing Proline Substitution to be Responsible for the Loss of Amyloidogenesis in IAPP

Human islet amyloid polypeptide (hIAPP) is highly amyloidogenic, whereas its homologs in rodents are non-amyloidogenic. This observed non-amyloidogenecity of rodent IAPP has been attributed to substitutions by proline in a region of IAPP that forms the core of the fibril. By employing molecular dynamics simulation, we have analyzed effects of position-specific proline substitution on amyloidogenesis of the core region of the hIAPP fibril (22-28). We depict that substitution to proline at the 25th position is primarily responsible for the loss of amyloidogenecity of the peptide. In addition, 25th and 26th double mutation to proline and valine has been observed to show significant fibril destabilizing ability. On the contrary, substitution at 28th position to proline has the least ability to destabilize the amyloid fibril. Results obtained from this study are particularly important to design variants of the existing antihyperglycemic drug with minimalistic mutation approach for use in patients with diabetes.

Hb Fulton-Georgia [α20(B1)His→Pro; HBA1: c.62A>C]: A New α-Globin Variant Coinherited with α-Thalassemia-2 (3.7 kb deletion) and Hb SC Disease

We report a novel hemoglobin (Hb) variant that we named Hb Fulton-Georgia, caused by a point mutation in exon 1/codon 20 of the α-globin gene [α20(B1)His→Pro; HBA1: c.62A>C]. This α chain variant was identified in an adult African-American female with Hb SC disease who was also heterozygous for the α-thalassemia-2 (α-thal-2) (3.7 kb deletion or αα/–α3.7). The Hb Fulton-Georgia mutation was located on the intact α1-globin gene not involved by α-thal-2. Molecular models indicated that the α20 residue of Hb Fulton-Georgia was the first amino acid of the B helix, and was not involved in α1/β1 or α1/β2 contacts in Hb S [β6(A3)Glu→Val; HBB: c.20A>T] or Hb C [β6(A3)Glu→Lys; HBB: c.19G>A] tetramers. Furthermore, the histidine→proline substitution at α20 did not disrupt the helical structure. High performance liquid chromatography (HPLC) detected Hb Fulton-Georgia in 16.0% of total Hb, consistent with inheritance on the α1 gene. Coinheritance of Hb Fulton-Georgia, heterozygous α-thal-2 and Hb SC disease was associated with a mild phenotype, consisting of microcytosis and anisocytosis, but no anemia or other hematological abnormality.

Amyotrophic lateral sclerosis-related VAPB P56S mutation differentially affects the function and survival of corticospinal and spinal motor neurons

The substitution of Proline with Serine at residue 56 (P56S) of vesicle-associated membrane protein-associated protein B (VAPB) has been linked to an atypical autosomal dominant form of familial amyotrophic lateral sclerosis 8 (ALS8). To investigate the pathogenic mechanism of P56S VAPB in ALS, we generated transgenic (Tg) mice that heterologously express human wild-type (WT) and P56S VAPB under the control of a pan-neuronal promoter Thy1.2. While WT VAPB Tg mice did not exhibit any overt motor behavioral phenotypes, P56S VAPB Tg mice developed progressive hyperactivities and other motor abnormalities. VAPB protein was accumulated as large punctate in the soma and proximal dendrites of both corticospinal motor neurons (CSMNs) and spinal motor neurons (SMNs) in P56S VAPB Tg mice. Concomitantly, a significant increase of endoplasmic reticulum stress and unfolded protein response and the resulting up-regulation of pro-apoptotic factor CCAAT/enhancer-binding protein homologous protein expression were observed in the CSMNs and SMNs of P56S VAPB Tg mice. However, only a progressive loss of CSMNs but not SMNs was found in P56S VAPB Tg mice. In SMNs, P56S VAPB promoted a rather selective translocation of VAPB protein onto the postsynaptic site of C-boutons that altered the morphology of C-boutons and impaired the spontaneous rhythmic discharges of SMNs. Therefore, these findings provide new pathophysiological mechanisms of P56S VAPB that differentially affect the function and survival of CSMNs and SMNs in ALS8.

YIA3: ADAMTS7 CLEAVAGE AND VASCULAR SMOOTH MUSCLE CELL MIGRATION IS AFFECTED BY A CORONARY ARTERY DISEASE ASSOCIATED VARIANT

BackgroundRecently, genome-wide association studies have revealed an association between single-nucleotide-polymorphisms (SNPs) in a genomic region containing the ADAMTS7 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, 7) gene and...

Serine substitution of proline at codon 151 of TP53 confers gain of function activity leading to anoikis resistance and tumor progression of head and neck cancer cells

Mutation of the TP53 gene occurs in more than half of cases of head and neck squamous cell carcinoma (HNSCC). However, little is known about how specific TP53 mutations affect tumor progression. The objective of this study is to determine the gain of function of mutant p53 with a proline-to-serine substitution at codon 151. Laboratory-based study. A panel of HNSCC cell lines was determined with anoikis assays, and orthotopic mouse experiments were performed. TP53 was sequenced. The shRNA knockdown and overexpression approaches were used for testing mutant p53 functions. The crystal structure of the p53 protein was analyzed using an in silico approach. An anoikis-resistant cell line, Tu138, was found to have a proline-to-serine substitution at codon 151 of TP53, which results in loss of wild-type p53 transcriptional activity. Moreover, the mutant p53 was shown to promote anoikis resistance and soft agar growth. Using an in silico approach based on the crystal structure of wild-type p53 protein, substitution of proline by serine at position 151 would create a cavity in a hydrophobic pocket, the loss of van der Waals contacts, and the thermodynamically unfavorable placement of a polar group, the hydroxyl oxygen atom of the serine, within a hydrophobic region, all of which likely cause a locally altered structure. Our data suggest that mutation at position 151 leads to a structural alteration, which results in significant functional changes in the p53 protein that impact tumor progression.

Japanese patients with Fabry disease predominantly showing cardiac and neurological manifestation with novel missense mutation: R220P

BackgroundFabry disease, an X-linked lysosomal sphingolipid storage disorder caused by mutation of the α-galactosidase A (GLA) gene, results in systemic organ damage. However, the age of onset of clinical manifestations and course of the disease are variable even within the same family. ObjectiveIn this study, we evaluated the clinical phenotype and the molecular lesions associated with the GLA gene in a Japanese family with Fabry disease that predominantly showed cardiac and neurological manifestations. MethodsA genetic analysis of the GLA gene using conventional genomic sequencing was performed in all seven members of this family, including four hemizygous males and three heterozygous females. Endomyocardial biopsy was performed in two patients with severe left ventricular (LV) hypertrophy. ResultsA novel missense mutation was identified at codon 220 in exon 5, thus resulting in an arginine to proline substitution (R220P) in all seven family members. The three adult hemizygous males had LV hypertrophy and developed neurological manifestations in their 50s. One of the adult hemizygotes developed complete atrioventricular block. On the other hand, we could not find any organ damage in a young hemizygous male or the three heterozygous females. ConclusionWe identified a novel missense mutation in a Japanese family with Fabry disease showing cardiac and neurological manifestations. In patients with Fabry disease, advanced organ damage in the heart and brain can be life-threatening, even if renal failure is lacking.

A Proline Scan Approach to Investigate the Activation Gate of hERG Channels

In Shaker channels, the activation gate is formed at the bundle crossing by the convergence of the inner S6 helices near a conserved proline-valine-proline (PVP) motif, which introduces a kink in the helices that allows for electromechanical coupling with voltage sensor motions via the S4-S5 linker. Human ether-a-go-go related gene (hERG) channels lack the PVP motif and the location of the intracellular pore gate and how it is coupled to S4 movement is less clear. Here, we performed a proline scan of the inner S6 helix, from I655 to Y667, to determine the position of the gate. The rationale was that proline-induced S6 disruption would impede gate function when a proline was engineered above, but not below, the native gate region. We discovered that proximal substitutions (I655P-Q664P) impeded gate closure trapping channels in the open state, while distal substitutions (R665P, L666P and Y667P) preserved wild type-like gating. That proline substitutions below Q664 preserved channel gating, while residues above disrupted gate function, strongly suggests that the position of the intracellular gate is formed at Q664. These data are consistent with previous homology model-based predictions1. Interestingly, in V659P channels the gate was trapped open, but upon strong hyperpolarization channels slowly activated into a distinct voltage-dependent open state, reminiscent of the well-studied hERG mutation, D540K. The presence of voltage-dependent gating in this mutant suggests that the trapped open phenotype is due to uncoupling of gate closure from voltage sensor gating, rather than an immobilization of sensor movement. Moreover, the activation of V659P channels upon hyperpolarization suggests a ‘down' configuration of the voltage sensor that is distinct from that occupied at −80 mV and that leads to hyperpolarization-activated pore opening, as in HCN channels.1 Wynia-Smith et. al., J. Gen. Physiol. 132:507-520, 2008.

Effect of Prolines on the Intrinsic Stiffness of Islet Amyloid Polypeptide Variants

Human islet amyloid polypeptide (hIAPP) is an intrinsically disordered hormone that is co-secreted with insulin in the beta-cells of the pancreas. The aggregation of hIAPP into insoluble amyloid fibrils is believed to play a causal role in type 2 diabetes. The rodent variant (rIAPP), differing by 6 of 37 amino acids, does not aggregate into amyloid fibrils and inhibits hIAPP amyloid formation. Both these properties have been attributed to rIAPP's three proline mutations (A25P, S28P and S29P). Single proline mutants of hIAPP have also been shown to kinetically inhibit hIAPP fibril formation. Because of their intrinsic dihedral angle preferences, prolines are expected to affect monomer conformational sampling. However, the specific effect of proline substitutions on IAPP structure and dynamics has not yet been explored. Detecting such properties is challenging due to the low molecular weight, fast reconfiguration times, and very low solubility of IAPP peptides. High resolution, time resolved techniques are needed.We use a nanosecond laser spectroscopy technique to measure end-to-end contact formation rates in IAPP mutants. Using this technique, Vaiana et al. showed that rIAPP populates more extended conformations, characterized by larger end-to-end distances, compared to hIAPP. Here we study the effects of proline substitutions in IAPP and characterize them in terms of intrinsic chain stiffness. We find that the three proline mutations alone do not explain rIAPP's increased chain stiffness. Interestingly, early experiments by Green et al. show that mutating the non-proline residues in rIAPP renders it capable of forming amyloids. Together these results suggest that rIAPP's unique chain stiffness is a determinant for its non-amyloidogenic properties. We discuss the reasons for this peptide's unique chain stiffness and the implications of our findings on the effect of prolines in IDPs.

Hepatitis B escape mutants in Scottish blood donors

Hepatitis B virus (HBV) remains as the viral infection with the highest risk of transmission by transfusion. This risk is associated with window period donations, occult HBV infection (OBI) and the emergence of escape mutants, which render blood donations false negative for hepatitis B surface antigen (HBsAg) serological testing. A retrospective study was conducted to gain insights into the molecular epidemiology of HBV escape mutants in Scottish blood donors. The criterion for selection was HBV positivity either by serology or nucleic acid testing (NAT). HBsAg detection was compared across several commercial immunoassays. The full length S gene from plasma samples was PCR amplified, cloned and expressed in HepG2 cells. Eight samples showed HBsAg discordant results, while 5 OBI samples were found. Four escape mutants, containing missense mutations in the S gene, are described here. These mutations impaired HBsAg detection both from HBV infected plasma samples and from recombinant proteins derived from its infected donors. Phylogenetic analysis showed that most of the mutants were clustered in the genotype D and were closely related to strains from Asia and the Middle East. We report here a proline substitution, outside the major hydrophilic region, that impaired HBsAg detection in vivo and in vitro, warning about the risk for the emergence of vaccine escape mutants with mutations outside the major neutralisation site.

Severe Growth Deficiency is Associated with STAT5b Mutations that Disrupt Protein Folding and Activity

The first genetic defect in human signal transducer and activator of transcription (STAT)5b was identified in an individual with profound short stature and GH insensitivity, immune dysfunction, and severe pulmonary disease, and was caused by an alanine to proline substitution (A630P) within the Src homology-2 (SH2) domain. STAT5b(A630P) was found to be an inactive transcription factor based on its aberrant folding, diminished solubility, and propensity for aggregation triggered by its misfolded SH2 domain. Here we have characterized the second human STAT5b amino acid substitution mutation in an individual with similar pathophysiological features. This single nucleotide transition, predicted to change phenyalanine 646 to serine (F646S), also maps to the SH2 domain. Like STAT5b(A630P), STAT5b(F646S) is prone to aggregation, as evidenced by its detection in the insoluble fraction of cell extracts, the presence of dimers and higher-order oligomers in the soluble fraction, and formation of insoluble cytoplasmic inclusion bodies in cells. Unlike STAT5b(A630P), which showed minimal GH-induced tyrosine phosphorylation and no transcriptional activity, STAT5b(F646S) became tyrosine phosphorylated after GH treatment and could function as a GH-activated transcription factor, although to a substantially lesser extent than STAT5b(WT). Biochemical characterization demonstrated that the isolated SH2 domain containing the F646S substitution closely resembled the wild-type SH2 domain in secondary structure, but exhibited reduced thermodynamic stability and altered tertiary structure that were intermediate between STAT5b(A630P) and STAT5b(WT). Homology-based structural modeling suggests that the F646S mutation disrupts key hydrophobic interactions and may also distort the phosphopeptide-binding face of the SH2 domain, explaining both the reduced thermodynamic stability and impaired biological activity.

Mutational analysis of the FST gene in Chinese women with idiopathic premature ovarian failure

Objective Recent animal studies have suggested that loss of follistatin (FST) may result in premature cessation of ovarian function. Our objective was to investigate whether mutations in the FST coding region are present in Chinese women with idiopathic premature ovarian failure (POF).Method The matched case-control study took place in the First Affiliated Hospital of Nanjing Medical University with 80 idiopathic POF patients and 80 matched controls. There were no interventions. The entire FST coding region was analyzed by direct sequencing in all subjects.Results Three FST gene variants were identified, namely c.270C> G, c.598G> C and c.953–184A> T (rs722910). The synonymous variant (c.270C> G) in exon 2 was present in the heterozygous state in a single POF patient. The novel c.598G> C missense mutation, located in exon 4 and resulting in an alanine to proline substitution at amino acid 200, was detected in a single healthy control. There was no difference in genotype distribution and allele frequency of the known single nucleotide polymorphism rs722910 between POF patients and controls.Conclusion Although we did not find any evidence that it is a disease-causing gene, our study is the first to evaluate the role of the FST gene in Chinese women with idiopathic POF.