The caveolin-binding motif of the pathogen-related yeast protein Pry1, a member of the CAP protein superfamily,is required for in vivo export of cholesteryl acetate

Proteins belonging to the CAP superfamily are present in all kingdoms of life and have been implicated in different physiological processes. Their molecular mode of action, however, is poorly understood. Saccharomyces cerevisiae expresses three members of this superfamily, pathogen-related yeast (Pry)1, -2, and -3. We have recently shown that Pry function is required for the secretion of cholesteryl acetate and that Pry proteins bind cholesterol and cholesteryl acetate, suggesting that CAP superfamily members may generally act to bind sterols or related small hydrophobic compounds. Here, we analyzed the mode of sterol binding by Pry1. Computational modeling indicates that ligand binding could occur through displacement of a relatively poorly conserved flexible loop, which in some CAP family members displays homology to the caveolin-binding motif. Point mutations within this motif abrogated export of cholesteryl acetate but did not affect binding of cholesterol. Mutations of residues located outside the caveolin-binding motif, or mutations in highly conserved putative catalytic residues had no effect on export of cholesteryl acetate or on lipid binding. These results indicate that the caveolin-binding motif of Pry1, and possibly of other CAP family members, is crucial for selective lipid binding and that lipid binding may occur through displacement of the loop containing this motif.     

Identification of CRISP2 from human sperm as PSP94‐binding protein and generation of CRISP2‐specific anti‐peptide antibodies

Cysteine‐rich secretory proteins (CRISPs) are mainly found in the mammalian male reproductive tract and reported to be involved at different stages of fertilization. CRISPs have been shown to interact with prostate secretory protein of 94 amino acids (PSP94) from diverse sources, and the binding of these evolutionarily conserved proteins across species is proposed to be of functional significance. Of the three mammalian CRISPs, PSP94–CRISP3 interaction is well characterized, and specific binding sites have been identified; whereas, CRISP2 has been shown to interact with PSP94 in vitro. Interestingly, human CRISP3 and CRISP2 proteins are closely related showing 71.4% identity. In this study, we identified CRISP2 as a potential binding protein of PSP94 from human sperm. Further, we generated antisera capable of specifically detecting CRISP2 and not CRISP3. In this direction, specific peptides corresponding to the least conserved ion channel regulatory region were synthesized, and polyclonal antibodies were generated against the peptide in rabbits. The binding characteristics of the anti‐CRISP2 peptide antibody were evaluated using competitive ELISA. Immunoblotting experiments also confirmed that the peptide was able to generate antibodies capable of detecting the mature CRISP2 protein present in human sperm lysate. Furthermore, this anti‐CRISP2 peptide antibody also detected the presence of native CRISP2 on sperm.Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Heligmosomoides polygyrus Venom Allergen-like Protein-4 (HpVAL-4) is a sterol binding protein

Heligmosomoides polygyrus bakeri is a model parasitic hookworm used to study animal and human helminth diseases. During infection, the parasite releases excretory/secretory products that modulate the immune system of the host. The most abundant protein family in excretory/secretory products comprises the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. There are >30 secreted Heligmosomoides polygyrus VAL proteins (HpVALs) and these proteins are characterised by having either one or two 15?kDa CAP (cysteine-rich secretory protein (CRISP)/antigen 5/pathogenesis related-1) domains. The first known HpVAL structure, HpVAL-4, refined to 1.9?Å is reported. HpVAL-4 was produced as a homogeneously glycosylated protein in leaves of Nicotiana benthamiana infiltrated with recombinant plasmids, making this plant expression platform amenable for the production of biological products. The overall topology of HpVAL-4 is a three layered αβα sandwich between a short N-terminal loop and a C-terminal cysteine rich extension. The C-terminal cysteine rich extension has two strands stabilized by two disulfide bonds and superposes well with the previously reported extension from the human hookworm Necator americanus Ancylostoma secreted protein-2 (Na-ASP-2). The N-terminal loop is connected to alpha helix 2 via a disulfide bond previously observed in Na-ASP-2. HpVAL-4 has a central cavity that is more similar to the N-terminal CAP domain of the two CAP Na-ASP-1 from Necator americanus. Unlike Na-ASP-2, mammalian CRISP, and the C-terminal CAP domain of Na-ASP-1, the large central cavity of HpVAL-4 lacks the two histidines required to coordinate divalent cations. HpVAL-4 has both palmitate-binding and sterol-binding cavities and is able to complement the in vivo sterol export phenotype of yeast mutants lacking their endogenous CAP proteins. More studies are required to determine endogenous binding partners of HpVAL-4 and unravel the possible impact of sterol binding on immune-modulatory functions.     

Crystal structure of Brugia malayi venom allergen-like protein-1 (BmVAL-1), a vaccine candidate for lymphatic filariasis

Brugia malayi is a causative agent of lymphatic filariasis, a major tropical disease. The infective L3 parasite stage releases immunomodulatory proteins including the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. BmVAL-1 is a major target of host immunity with >90% of infected B. malayi microfilaraemic cases being seropositive for antibodies to BmVAL-1. This study is part of ongoing efforts to characterize the structures and functions of important B. malayi proteins. Recombinant BmVAL-1 was produced using a plant expression system, crystallized and the structure was solved by molecular replacement and refined to 2.1?Å, revealing the characteristic alpha/beta/alpha sandwich topology of eukaryotic SCP/TAPS proteins. The protein has more than 45% loop regions and these flexible loops connect the helices and strands, which are longer than predicted based on other parasite SCP/TAPS protein structures. The large central cavity of BmVAL-1 is a prototypical CRISP cavity with two histidines required to bind divalent cations. The caveolin-binding motif (CBM) that mediates sterol binding in SCP/TAPS proteins is large and open in BmVAL-1 and is N-glycosylated. N-glycosylation of the CBM does not affect the ability of BmVAL-1 to bind sterol in vitro. BmVAL-1 complements the in vivo sterol export phenotype of yeast mutants lacking their endogenous SCP/TAPS proteins. The in vitro sterol-binding affinity of BmVAL-1 is comparable with Pry1, a yeast sterol transporting SCP/TAPS protein. Sterol binding of BmVAL-1 is dependent on divalent cations. BmVAL-1 also has a large open palmitate-binding cavity, which binds palmitate comparably to tablysin-15, a lipid-binding SCP/TAPS protein. The central cavity, CBM and palmitate-binding cavity of BmVAL-1 are interconnected within the monomer with channels that can serve as pathways for water molecules, cations and small molecules.     

Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

Prostate Secretory Protein of 94 amino acids (PSP94) is one of the major proteins present in the human seminal plasma. Though several functions have been predicted for this protein, its exact role either in sperm function or in prostate pathophysiology has not been clearly defined. Attempts to understand the mechanism of action of PSP94 has led to the search for its probable binding partners. This has resulted in the identification of PSP94 binding proteins in plasma and seminal plasma from human. During the chromatographic separation step of proteins from human seminal plasma by reversed phase HPLC, we had observed that in addition to the main fraction of PSP94, other fractions containing higher molecular weight proteins also showed the presence of detectable amounts of PSP94. This prompted us to hypothesize that PSP94 could be present in the seminal plasma complexed with other protein/s of higher molecular weight. One such fraction containing a major protein of ∼47 kDa, on characterization by mass spectrometric analysis, was identified to be Prostatic Acid Phosphatase (PAP). The ability of PAP present in this fraction to bind to PSP94 was demonstrated by affinity chromatography. Co-immunoprecipitation experiments confirmed the presence of PSP94-PAP complex both in the fraction studied and in the fresh seminal plasma. In silico molecular modeling of the PSP94-PAP complex suggests that β-strands 1 and 6 of PSP94 appear to interact with domain 2 of PAP, while β-strands 7 and 10 with domain 1 of PAP. This is the first report which suggests that PSP94 can bind to PAP and the PAP-bound PSP94 is present in human seminal plasma.     

Crystal Structure of Prostate Secretory Protein PSP94 Shows an Edge-to-Edge Association of two Monomers to Form a Homodimer

Several recent genome-wide association studies have linked the human MSMB gene, encoding prostate secretory protein of 94 residues (PSP94), with prostate cancer susceptibility. PSP94 is one of the most abundant proteins from prostatic secretions and a primary constituent of human semen. PSP94 suppresses tumor growth and metastasis, and its expression gradually decreases during progression of the prostate cancer. It is a rapidly evolving protein with homologues present in several species with 10 conserved cysteine residues. PSP94 homologues show high-affinity binding with different proteins from the cysteine-rich secretory protein family, some of which have been shown to be ion channel blockers. Here, we report the crystal structure of human PSP94 at 2.3 Å resolution. The structure shows that the amino and the carboxyl ends of the polypeptide chain are held in close proximity facing each other. A strong hydrogen bond between these ends, which are located respectively on the first and the last β-strands, leads to formation of an almost straight edge in PSP94 structure. Crystal structure shows that these edges from two PSP94 monomers associate in antiparallel fashion, leading to formation of a dimer. Our studies further show that dimers dissociate into monomers at acidic pH, possibly through distortion of the straight edge. Further, based on several observations, we propose that PSP94 binds to cysteine-rich secretory proteins and immunoglobulin G through the same edge, which is involved in the formation of PSP94 dimeric interface.     

Serum bound forms of PSP94 (prostate secretory protein of 94 amino acids) in prostate cancer patients

PSP94 (prostate secretory protein of 94 amino acids) was regarded as a possible prostate cancer marker, however, it has been controversial. All prior studies were designed to test the free form in serum using antibodies to PSP94. Results presented here demonstrate that PSP94 exists in prostate cancer patients in two forms, free and bound, and that the majority is present as serum bound complexes. This result was demonstrated by using both native and SDS-PAGE analyses of serum proteins from prostate cancer patients. Chromatographic separation of serum total proteins by a molecular sieve column generated two peaks (peak I and II), which were reactive with rabbit antiserum to human PSP94 in Western blot experiments. Peak I was eluted before the IgG fraction at a molecular weight larger than 150 kDa, and peak II appeared after serum albumin ( approximately 67 kDa) was eluted. By using a biotinylated PSP94 as an indicator of the free form of PSP94, we demonstrate that peak I contains serum PSP94-bound complexes and peak II is likely the free form of serum PSP94. Since the molecular weight of serum PSP94-bound complexes is close to IgG during molecular sieve separation, serum PSP94 complexes were further purified through two rounds of protein A column separation, followed by DEAE-ion exchange column chromatography. In vitro dissociation tests of the purified PSP94-bound complexes showed that the binding of serum PSP94-complexes is probably via disulfide bonds and is chemically stable. The results presented here indicate that serum PSP94-bound complexes must be considered in evaluating the clinical utility of PSP94 as a prostate cancer marker.     

Serum bound forms of PSP94 (prostate secretory protein of 94 amino acids) in prostate cancer patients

PSP94 (prostate secretory protein of 94 amino acids) was regarded as a possible prostate cancer marker, however, it has been controversial. All prior studies were designed to test the free form in serum using antibodies to PSP94. Results presented here demonstrate that PSP94 exists in prostate cancer patients in two forms, free and bound, and that the majority is present as serum bound complexes. This result was demonstrated by using both native and SDS‐PAGE analyses of serum proteins from prostate cancer patients. Chromatographic separation of serum total proteins by a molecular sieve column generated two peaks (peak I and II), which were reactive with rabbit antiserum to human PSP94 in Western blot experiments. Peak I was eluted before the IgG fraction at a molecular weight larger than 150 kDa, and peak II appeared after serum albumin (∼67 kDa) was eluted. By using a biotinylated PSP94 as an indicator of the free form of PSP94, we demonstrate that peak I contains serum PSP94‐bound complexes and peak II is likely the free form of serum PSP94. Since the molecular weight of serum PSP94‐bound complexes is close to IgG during molecular sieve separation, serum PSP94 complexes were further purified through two rounds of protein A column separation, followed by DEAE‐ion exchange column chromatography. In vitro dissociation tests of the purified PSP94‐bound complexes showed that the binding of serum PSP94‐complexes is probably via disulfide bonds and is chemically stable. The results presented here indicate that serum PSP94‐bound complexes must be considered in evaluating the clinical utility of PSP94 as a prostate cancer marker. J. Cell. Biochem. 76:71–83, 1999. © 1999 Wiley‐Liss, Inc.     

酵母甾醇转运蛋白研究进展

甾醇是一类广泛存在于生物体内的环戊烷骈多氢菲衍生物,其不仅是细胞膜的重要组成成分,还具有重要的生理和药理活性。随着合成生物学和代谢工程技术的发展,近些年来应用酵母细胞异源合成甾醇的研究不断深入。但由于甾醇是疏水性大分子,倾向于积累在酵母的膜结构中而引发细胞毒性,一定程度上限制了甾醇产量的进一步提升。因此,揭示酵母中甾醇转运机制,特别是与甾醇转运相关的转运蛋白的工作原理,有助于设计新的策略,解除酵母细胞工厂中的甾醇积累毒性、实现甾醇增产。酵母中甾醇转运主要通过蛋白质介导的非囊泡运输机制来完成,本文归纳了酵母中已报道的5类甾醇转运相关蛋白,即OSBP/ORPs家族蛋白、LAM家族蛋白、NPC样甾醇转运蛋白、ABC转运家族蛋白和CAP超家族蛋白,汇总了这些蛋白对细胞内甾醇梯度分布和稳态维持所起的重要作用。此外,本文还综述了甾醇转运蛋白在酵母细胞工厂中的应用现状。     

富含半胱氨酸的分泌蛋白（CRISPs）在哺乳动物授精过程中的作用

雌雄配子间的结合与融合是哺乳动物授精成功的关键步骤,哺乳动物富含半胱氨酸的分泌蛋白CRISPs家族是一个进化上高度保守的蛋白家族,参与了精卵结合与融合过程,并在其中扮演了多种角色。目前从雄性小鼠生殖道中分离出4个CRISPs家族成员：附睾的CRISP1、睾丸的CRISP2、分布广泛的CRISP3以及与人CRISP1同源的CRISP4,对CRISPs家族蛋白成员的晶体结构分析揭示出CRISP蛋白含有两个功能域,一个是位于N末端的结构保守的CAP结构域,另一个是位于C末端的CRISP蛋白家族特有的CRISP功能域。CAP功能域中含有CAP基序,CRISP功能域由一个短的铰链区和一个离子通道调节区组成,并通过铰链区与CAP结构域相连接。简要回顾了各种CRISP蛋白的发现和特性鉴别过程,希望能从CRISPs的角度对哺乳动物精卵识别、结合与融合的分子机制有更好的了解。     

The conserved C2 phospholipid‐binding domain in Delta contributes to robust Notch signalling

Accurate Notch signalling is critical for development and homeostasis. Fine‐tuning of Notch–ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N‐terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so‐called β1‐2 loop that is involved in phospholipid binding. Mutations in the β1‐2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the β1‐2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss‐of‐function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine‐tuning the balance of trans and cis ligand–receptor interactions.     

A novel protein,sperm head and tail associated protein (SHTAP), interacts with cysteine‐rich secretory protein 2 (CRISP2) during spermatogenesis in the mouse

Background information. CRISP2 (cysteine‐rich secretory protein 2) is a sperm acrosome and tail protein with the ability to regulate Ca²⁺ flow through ryanodine receptors. Based on these properties, CRISP2 has a potential role in fertilization through the regulation of ion signalling in the acrosome reaction and sperm motility. The purpose of the present study was to determine the expression, subcellular localization and the role in spermatogenesis of a novel CRISP2‐binding partner, which we have designated SHTAP (sperm head and tail associated protein). Results. Using yeast two‐hybrid screens of an adult testis expression library, we identified SHTAP as a novel mouse CRISP2‐binding partner. Sequence analysis of all Shtap cDNA clones revealed that the mouse Shtap gene is embedded within a gene encoding the unrelated protein NSUN4 (NOL1/NOP2/Sun domain family member 4). Five orthologues of the Shtap gene have been annotated in public databases. SHTAP and its orthologues showed no significant sequence similarity to any known protein or functional motifs, including NSUN4. Using an SHTAP antiserum, multiple SHTAP isoforms (～20–87 kDa) were detected in the testis, sperm, and various somatic tissues. Interestingly, only the ～26 kDa isoform of SHTAP was able to interact with CRISP2. Furthermore, yeast two‐hybrid assays showed that both the CAP (CRISP/antigen 5/pathogenesis related‐1) and CRISP domains of CRISP2 were required for maximal binding to SHTAP. SHTAP protein was localized to the peri‐acrosomal region of round spermatids, and the head and tail of the elongated spermatids and sperm tail where it co‐localized with CRISP2. During sperm capacitation, SHTAP and the SHTAP—CRISP2 complex appeared to be redistributed within the head. Conclusions. The present study is the first report of the identification, annotation and expression analysis of the mouse Shtap gene. The redistribution observed during sperm capacitation raises the possibility that SHTAP and the SHTAP—CRISP2 complex play a role in the attainment of sperm functional competence.     

RNA‐binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral activity

The type I interferon (IFN‐I, IFN‐α/β)‐mediated immune response is the first line of host defense against invading viruses. IFN‐α/β binds to IFN‐α/β receptors (IFNARs) and triggers the expression of IFN‐stimulated genes (ISGs). Thus, stabilization of IFNARs is important for prolonging antiviral activity. Here, we report the induction of an RNA‐binding motif‐containing protein, RBM47, upon viral infection or interferon stimulation. Using multiple virus infection models, we demonstrate that RBM47 has broad‐spectrum antiviral activity in vitro and in vivo. RBM47 has no noticeable impact on IFN production, but significantly activates the IFN‐stimulated response element (ISRE) and enhances the expression of interferon‐stimulated genes (ISGs). Mechanistically, RBM47 binds to the 3''UTR of IFNAR1 mRNA, increases mRNA stability, and retards the degradation of IFNAR1. In summary, this study suggests that RBM47 is an interferon‐inducible RNA‐binding protein that plays an essential role in enhancing host IFN downstream signaling.     

Self-activating G protein α subunits engage seven-transmembrane regulator of G protein signaling (RGS) proteins and a Rho guanine nucleotide exchange factor effector in the amoeba Naegleria fowleri

The free-living amoeba Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis and is highly resistant to current therapies, resulting in mortality rates >97%. As many therapeutics target G protein–centered signal transduction pathways, further understanding the functional significance of G protein signaling within N. fowleri should aid future drug discovery against this pathogen. Here, we report that the N. fowleri genome encodes numerous transcribed G protein signaling components, including G protein–coupled receptors, heterotrimeric G protein subunits, regulator of G protein signaling (RGS) proteins, and candidate Gα effector proteins. We found N. fowleri Gα subunits have diverse nucleotide cycling kinetics; Nf Gα5 and Gα7 exhibit more rapid nucleotide exchange than GTP hydrolysis (i.e., “self-activating” behavior). A crystal structure of Nf Gα7 highlights the stability of its nucleotide-free state, consistent with its rapid nucleotide exchange. Variations in the phosphate binding loop also contribute to nucleotide cycling differences among Gα subunits. Similar to plant G protein signaling pathways, N. fowleri Gα subunits selectively engage members of a large seven-transmembrane RGS protein family, resulting in acceleration of GTP hydrolysis. We show Nf Gα2 and Gα3 directly interact with a candidate Gα effector protein, RGS-RhoGEF, similar to mammalian Gα_12/13 signaling pathways. We demonstrate Nf Gα2 and Gα3 each engage RGS-RhoGEF through a canonical Gα/RGS domain interface, suggesting a shared evolutionary origin with G protein signaling in the enteric pathogen Entamoeba histolytica. These findings further illuminate the evolution of G protein signaling and identify potential targets of pharmacological manipulation in N. fowleri.     

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (I) immuno-dominant and immuno-recessive area

PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v. either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immno-dominant N-terminus and an immuno-recessive C-terminus. J. Cell. Biochem. 65:172–185. © 1997 Wiley-Liss, Inc.     

Designer proteins that competitively inhibit Gαq by targeting its effector site

During signal transduction, the G protein, Gα_q, binds and activates phospholipase C-β isozymes. Several diseases have been shown to manifest upon constitutively activating mutation of Gα_q, such as uveal melanoma. Therefore, methods are needed to directly inhibit Gα_q. Previously, we demonstrated that a peptide derived from a helix-turn-helix (HTH) region of PLC-β3 (residues 852–878) binds Gα_q with low micromolar affinity and inhibits Gα_q by competing with full-length PLC-β isozymes for binding. Since the HTH peptide is unstructured in the absence of Gα_q, we hypothesized that embedding the HTH in a folded protein might stabilize the binding-competent conformation and further improve the potency of inhibition. Using the molecular modeling software Rosetta, we searched the Protein Data Bank for proteins with similar HTH structures near their surface. The candidate proteins were computationally docked against Gα_q, and their surfaces were redesigned to stabilize this interaction. We then used yeast surface display to affinity mature the designs. The most potent design bound Gα_q/i with high affinity in vitro (K_D = 18 nM) and inhibited activation of PLC-β isozymes in HEK293 cells. We anticipate that our genetically encoded inhibitor will help interrogate the role of Gα_q in healthy and disease model systems. Our work demonstrates that grafting interaction motifs into folded proteins is a powerful approach for generating inhibitors of protein–protein interactions.     

Phosphorylation regulates arginine-rich RNA-binding protein solubility and oligomerization

Posttranslational modifications (PTMs) such as phosphorylation of RNA-binding proteins (RBPs) regulate several critical steps in RNA metabolism, including spliceosome assembly, alternative splicing, and mRNA export. Notably, serine-/arginine- (SR)-rich RBPs are densely phosphorylated compared with the remainder of the proteome. Previously, we showed that dephosphorylation of the splicing factor SRSF2 regulated increased interactions with similar arginine-rich RBPs U1-70K and LUC7L3. However, the large-scale functional and structural impact of these modifications on RBPs remains unclear. In this work, we dephosphorylated nuclear extracts using phosphatase in vitro and analyzed equal amounts of detergent-soluble and -insoluble fractions by mass-spectrometry-based proteomics. Correlation network analysis resolved 27 distinct modules of differentially soluble nucleoplasm proteins. We found classes of arginine-rich RBPs that decrease in solubility following dephosphorylation and enrich the insoluble pelleted fraction, including the SR protein family and the SR-like LUC7L RBP family. Importantly, increased insolubility was not observed across broad classes of RBPs. We determined that phosphorylation regulated SRSF2 structure, as dephosphorylated SRSF2 formed high-molecular-weight oligomeric species in vitro. Reciprocally, phosphorylation of SRSF2 by serine/arginine protein kinase 2 (SRPK2) in vitro decreased high-molecular-weight SRSF2 species formation. Furthermore, upon pharmacological inhibition of SRPKs in mammalian cells, we observed SRSF2 cytoplasmic mislocalization and increased formation of cytoplasmic granules as well as cytoplasmic tubular structures that associated with microtubules by immunocytochemical staining. Collectively, these findings demonstrate that phosphorylation may be a critical modification that prevents arginine-rich RBP insolubility and oligomerization.     

Plasma plant sterols serve as poor markers of cholesterol absorption in man

The validation of the use of plasma plant sterols as a marker of cholesterol absorption is frail. Nevertheless, plant sterol concentrations are routinely used to describe treatment-induced changes in cholesterol absorption. Their use has also been advocated as a clinical tool to tailor cholesterol-lowering therapy. Prior to wider implementation, however, the validity of plant sterols as absorption markers needs solid evaluation. Therefore, we compared plasma plant sterol concentrations to gold-standard stable isotope-determined cholesterol absorption. Plasma campesterol/TC concentrations (camp/TC) were measured in a population of 175 mildly hypercholesterolemic individuals (age: 59.7 ± 5.6 years; BMI: 25.5 ± 2.9kg/m²; LDL-C: 4.01 ± 0.56 mmol/l). We compared cholesterol absorption according to the plasma dual-isotope method in subjects with the highest camp/TC concentrations (N = 41, camp/TC: 2.14 ± 0.68 μg/mg) and the lowest camp/TC concentrations (N = 39, camp/TC: 0.97 ± 0.22 μg/mg). Fractional cholesterol absorption did not differ between the groups (24 ± 12% versus 25 ± 16%, P = 0.60), nor was it associated with plasma camp/TC concentrations in the total population of 80 individuals (β = 0.13; P = 0.30, adjusted for BMI and plasma triglycerides). Our findings do not support a relation between plasma plant sterol concentrations and true cholesterol absorption and, therefore, do not favor the use of these sterols as markers of cholesterol absorption. This bears direct consequences for the interpretation of earlier studies, as well as for future studies targeting intestinal regulation of cholesterol metabolism.     

Transportin 1 is a major nuclear import receptor of the nitric oxide synthase interacting protein

The nitric oxide synthase interacting protein (NOSIP), an E3-ubiquitin ligase, is involved in various processes like neuronal development, craniofacial development, granulopoiesis, mitogenic signaling, apoptosis, and cell proliferation. The best-characterized function of NOSIP is the regulation of endothelial nitric oxide synthase activity by translocating the membrane-bound enzyme to the cytoskeleton, specifically in the G2 phase of the cell cycle. For this, NOSIP itself has to be translocated from its prominent localization, the nucleus, to the cytoplasm. Nuclear import of NOSIP was suggested to be mediated by the canonical transport receptors importin α/β. Recently, we found NOSIP in a proteomic screen as a potential importin 13 cargo. Here, we describe the nuclear shuttling characteristics of NOSIP in living cells and in vitro and show that it does not interact directly with importin α. Instead, it formed stable complexes with several importins (−β, −7, −β/7, −13, and transportin 1) and was also imported into the nucleus in digitonin-permeabilized cells by these factors. In living HeLa cells, transportin 1 seems to be the major nuclear import receptor for NOSIP. A detailed analysis of the NOSIP-transportin 1 interaction revealed a high affinity and an unusual binding mode, involving the N-terminal half of transportin 1. In contrast to nuclear import, nuclear export of NOSIP seems to occur mostly by passive diffusion. Thus, our results uncover additional layers in the larger process of endothelial nitric oxide synthase regulation.     

Measuring Affinities of Fission Yeast Spindle Pole Body Proteins in Live Cells across the Cell Cycle

Characterizing protein-protein interactions is essential for understanding molecular mechanisms, although reproducing cellular conditions in vitro is challenging and some proteins are difficult to purify. We developed a method to measure binding to cellular structures using fission yeast cells as reaction vessels. We varied the concentrations of Sid2p and Mob1p (proteins of the septation initiation network) and measured their binding to spindle pole bodies (SPBs), the centrosome equivalent of yeast. From our measurements we infer that Sid2p and Mob1p both exist as monomeric, heterodimeric, and homodimeric species throughout the cell cycle. During interphase these species have widely different affinities for their common receptor Cdc11p on the SPB. The data support a model with a subset of Cdc11p binding the heterodimeric species with a K_d < 0.1 μM when Sid2p binds Mob1p-Cdc11p and K_d in the micromolar range when Mob1p binds Sid2p-Cdc11p. During mitosis an additional species presumed to be the phosphorylated Sid2p−Mob1p heterodimer binds SPBs with a lower affinity. Homodimers of Sid2p or Mob1p bind to the rest of Cdc11p at SPBs with lower affinity: K_ds > 10 μM during interphase and somewhat stronger during mitosis. These measurements allowed us to account for the fluctuations in Sid2p binding to SPBs throughout the cell cycle.