Conservative Management of Urinary Incontinence in Women

Urinary incontinence in women has a high prevalence and causes significant morbidity. Given that urinary incontinence is not generally a progressive disease, conservative therapies play an integral part in the management of these patients. We conducted a nonsystematic review of the literature to identify high-quality studies that evaluated the different components of conservative management of stress urinary incontinence, including behavioral therapy, bladder training, pelvic floor muscle training, lifestyle changes, mechanical devices, vaginal cones, and electrical stimulation. Urinary incontinence can have a severe impact on our healthcare system and patients’ quality of life. There are currently a wide variety of treatment options for these patients, ranging from conservative treatment to surgical treatment. Although further research is required in the area of conservative therapies, nonsurgical treatments are effective and are preferred by some patients.Key words: Urinary incontinence, Women, Conservative managementUrinary incontinence (UI) is a significant cause of decrease in quality of life, especially among women.¹ The prevalence of UI in women is estimated to range from 13% to 46%,^2,3 and studies have shown that incontinence increases with age.⁴ In addition to the significant social impact that UI has on a woman’s quality of life, this condition has a significant financial burden on individual and national healthcare dollars. It has been estimated that the total annual direct and indirect cost for UI in the United States alone is $19.5 billion.⁵UI is defined according to patients’ symptoms. Although definitions vary in the literature, the International Continence Society defines three major subtypes of UI: (1) stress urinary incontinence (SUI) is the complaint of involuntary leakage on effort or exertion, or on sneezing or coughing; (2) urgency urinary incontinence (UUI) is the complaint of involuntary leakage accompanied by or immediately preceded by urgency; and (3) mixed urinary incontinence (MUI) is the complaint of involuntary leakage associated with urgency and also with exertion, effort, sneezing, or coughing.^6,7Although there is a plethora of treatment options, conservative management is the first-line option for most patients with UI. The rationale for conservative treatment is that UI is not necessarily a progressive disease, and that conservative therapies can be effective, well tolerated, and safe. Furthermore, a moderate delay in surgical therapy does not make treatment more difficult or less effective. One of the recommendations of the 1992 Agency for Health Care Policy and Research guideline states that “surgery, except in very specific cases, should be considered only after behavioral and pharmacologic interventions have been tried.”⁸ Similarly, the European Association of Urology guidelines advocate a stepwise approach regarding management of UI, which begins with addressing underlying medical or cognitive issues, progressing to lifestyle modifications, behavioral therapy, and mechanical devices.⁹ In addition, conservative therapies are frequently preferred by many patients. Taking into account the patient’s goals and preferences, it is appropriate to recommend conservative management as an initial approach.     

47,XYY Syndrome and Male Infertility

Men with 47,XYY syndrome present with varying physical attributes and degrees of infertility. A retrospective chart review was performed on a male infertility and genetic anomaly database. Three patients with 47,XYY were found. Each presented with > 2 years of infertility. All were tall with elevated body mass indices. Scrotal findings ranged from normal to atrophic testicles. Semen analyses demonstrated oligospermia and varying endocrine profiles. Because of the diverse phenotype and potential lack of symptoms, identification and diagnosis of men with 47,XYY syndrome may be difficult. We recommend careful screening of 47,XYY patients and referral to primary physicians for long-term follow-up for increased incidence of health-related comorbidities.Key words: Infertility syndromesThe 47,XYY sex chromosome variation is the most common sex chromosome anomaly after Klinefelter syndrome (47,XXY),^1–3 occurring in approximately 1 out of 1000 live male births.^4,5 Parental nondisjunction at meiosis II resulting in an extra Y chromosome produces a 47,XYY karyotype in the affected offspring.^6–8 46,XY/47,XYY mosaics from parental nondisjunction during cell division after postzygotic mitosis can result in addition of the extra Y chromosome in early embryonic development.^6,8Most patients with 47,XYY have a delayed diagnosis, with a median age of 17.1 years at diagnosis, as was shown in a Danish cohort study.⁹ Although most have no phenotypic abnormalities, XYY boys are at greater risk for behavioral problems, mild learning disability, delayed speech and language development, and tall stature.¹⁰ Studies have increasingly reported an association between 47,XYY and fertility problems, noting an increased incidence of chromosomally abnormal spermatozoa in the semen of men with 47,XXY syndrome.^7,11–15 This greater prevalence of hyperhaploid sperm results in an increased risk of passing the extra Y chromosome to offspring.¹⁴ Men with 47,XXY syndrome can have variable sperm counts, ranging from normal to azoospermia.^{3,8,14,16–18}Here we review pertinent findings on physical examination and laboratory evaluation in three men with 47,XXY syndrome diagnosed during infertility evaluation as well as review the available literature on the subject, with special emphasis on male fertility effects.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Impact of the US Preventive Services Task Force Grade D Recommendation: Assessment of Evaluations for Elevated Prostate-specific Antigen and Prostate Biopsies in a Large Urology Group Practice Following Statement Revision

On October 7, 2011, the United States Preventive Services Task Force (USPSTF) released their evidence statement and grade D recommendation against prostate-specific antigen (PSA)-based prostate cancer screening. Using a time series design, we assessed the effect of this recommendation upon evaluations for elevated PSA levels and prostate biopsies in our large urology group practice. We found that, despite a 24.1% increase in total visits, the 32 urologists in our practice completed 16.4% fewer evaluations for elevated PSA levels (317 fewer evaluations per month; P = .017) and 21.4% fewer prostate biopsies (42 fewer biopsies per month; P = .001) in the 2 years following the USPSTF grade D recommendation.Key words: Prostate-specific antigen, Prostate cancer screening, Prostate biopsies, United StatesProstate cancer is the most common noncutaneous malignancy in American men. In the United States in 2015, approximately 220,800 men will be diagnosed with prostate cancer and 27,540 men will die from the disease.¹In 1986, the US Food and Drug Administration approved prostatespecific antigen (PSA) testing for monitoring disease progression in men previously diagnosed with prostate cancer.² In 1991, Catalona and colleagues³ published their findings that, when coupled with digital rectal examination and ultrasound, serum PSA measurement improved the detection of prostate cancer.Aiming to clarify the effect of PSA-based prostate cancer screening upon prostate cancer mortality, two large randomized trials of screening matured in 2009: the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO)⁴ and the European Randomized Study of Screening for Prostate Cancer (ERSPC).⁵ After 7 to 10 years of follow-up, the PLCO trial found no difference in prostate cancer mortality between men randomized to annual PSA testing and digital rectal examination versus usual care.⁴ In the ERSPC trial, PSA-based screening reduced the rate of death from prostate cancer by 20% at 9 years median follow-up. This reduction in prostate cancer mortality was associated with a high risk of over-diagnosis: 1410 men needed screening and 48 additional cases of prostate cancer required treatment to prevent 1 death from prostate cancer.⁵Using these two large studies as their evidence foundation for the benefits of early detection and treatment of prostate cancer, the United States Preventive Services Task Force (USPSTF) determined that the harms of PSA-based prostate cancer screening outweighed the benefits. On October 7, 2011, the USPSTF published their evidence statement and draft recommendation against PSA-based prostate cancer screening. ⁶ Extensive media coverage and national discussion ensued, with many publically disagreeing with the Task Force’s draft recommendation. ^7–9 In May 2012, the USPSTF finalized their grade D recommendation: PSA-based prostate cancer screening should be discouraged.¹⁰Although multiple screening guidelines exist that differ from those of the USPSTF,^11–13 primary care physicians are historically most influenced by the USPSTF recommendations. ¹⁴ In a study of primary care providers from Johns Hopkins Community Physicians, a university-affiliated practice including 26 outpatient sites in 11 Maryland counties, following release of the USPSTF draft recommendation against PSA-based prostate cancer screening, fewer than 50% agreed with the new recommendation, suggesting the change may encounter significant barriers to adoption.¹⁵ Consistent with this observation, various effects of the USPSTF recommendation upon the number of PSA tests performed, ^16–19 evaluations for elevated PSA levels,²⁰ and prostate biopsies completed^20–22 have been reported in the literature since 2012. Based on our clinical observations, we hypothesized that the number of evaluations for elevated PSA levels and number of prostate biopsies performed in our community-based, large urology group practice would decrease significantly following the publication of the USPSTF draft recommendation against prostate cancer screening.     

Binding Modes of Aromatic Ligands to Mammalian Heme Peroxidases with Associated Functional Implications: CRYSTAL STRUCTURES OF LACTOPEROXIDASE COMPLEXES WITH ACETYLSALICYLIC ACID, SALICYLHYDROXAMIC ACID, AND BENZYLHYDROXAMIC ACID*

The binding and structural studies of bovine lactoperoxidase with three aromatic ligands, acetylsalicylic acid (ASA), salicylhydoxamic acid (SHA), and benzylhydroxamic acid (BHA) show that all the three compounds bind to lactoperoxidase at the substrate binding site on the distal heme side. The binding of ASA occurs without perturbing the position of conserved heme water molecule W-1, whereas both SHA and BHA displace it by the hydroxyl group of their hydroxamic acid moieties. The acetyl group carbonyl oxygen atom of ASA forms a hydrogen bond with W-1, which in turn makes three other hydrogen-bonds, one each with heme iron, His-109 N^ϵ2, and Gln-105 N^ϵ2. In contrast, in the complexes of SHA and BHA, the OH group of hydroxamic acid moiety in both complexes interacts with heme iron directly with Fe-OH distances of 3.0 and 3.2Å respectively. The OH is also hydrogen bonded to His-109 N^ϵ2 and Gln-105N^ϵ2. The plane of benzene ring of ASA is inclined at 70.7° from the plane of heme moiety, whereas the aromatic planes of SHA and BHA are nearly parallel to the heme plane with inclinations of 15.7 and 6.2°, respectively. The mode of ASA binding provides the information about the mechanism of action of aromatic substrates, whereas the binding characteristics of SHA and BHA indicate the mode of inhibitor binding.Lactoperoxidase (LPO)⁴ (EC 1.11.1.7) is a member of the family of glycosylated mammalian heme-containing peroxidase enzymes which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO), and thyroid peroxidase. These enzymes also show functional similarities to non-homologous plant and fungal peroxidases because they follow a similar scheme of reaction (1–3), but their modes of ligand binding differ considerably. Furthermore, the association of the prosthetic heme group in mammalian peroxidases is through covalent bonds (4–9), whereas covalent linkages are absent in other peroxidases (10–14). Among the four mammalian peroxidases, the prosthetic heme group is linked through three covalent bonds in MPO, whereas in LPO, EPO, and thyroid peroxidase only two covalent linkages are formed. So far the detailed crystal structures of only two mammalian peroxidases, MPO and LPO, are known (15–20). One of the most striking differences between these two mammalian peroxidases is concerned with the basic structural organization in which MPO exists as a covalently linked dimer, whereas LPO is a monomeric protein. At present the fundamental questions pertaining to mammalian heme peroxidases are (i) what distinguishes between the aromatic ligands that one ligand acts as a substrate, whereas the other ligand works as an inhibitor and (ii) how the substrate and inhibitor specificities differ between two enzymes lactoperoxidase and myeloperoxidase.Lactoperoxidase oxidizes inorganic ions, preferentially thiocyanate (SCN⁻), and to a lesser extent, bromide (Br⁻), whereas in the case of myeloperoxidase the chloride (Cl⁻) ion is a preferred substrate (21, 22). The mammalian peroxidases including LPO are also involved in catalyzing the single electron oxidation of various physiologically important organic aromatic substrates including phenols (23, 24), catecholamines, and catechols (25–27) as well as other experimental model compounds such as aromatic amines (28), polychlorinated biphenyls (29), steroid hormones (30–32), and polycyclic aromatic hydrocarbons (33). However, the mode of binding of aromatic ligands and associated functional implications are not yet clearly understood. Surprisingly, the structural data on the complexes of mammalian peroxidases with aromatic ligands are conspicuously lacking. The only available structural report is on the complex of MPO with salicylhydroxamic acid (SHA) (34). Even in this case, the coordinates of this structure are not available for a detailed analysis. In the case of non-homologous plant peroxidases, a few crystal structures of their complexes with aromatic compounds are available (35–38), but their modes of binding are not very similar to those of mammalian peroxidases because the distal ligand binding sites in mammalian and plant peroxidases differ markedly. In this regard it is pertinent to note that the substrate binding site in peroxidases, in general, is observed at the δ-heme edge of the heme moiety on the distal side; in plant peroxidases an additional ligand binding site has also been observed at γ-heme edge (39–41). Unlike those in mammalian peroxidases where the heme moiety is buried deeply in the protein core, in plant peroxidases it is located close to the surface of the protein. Therefore, to characterize the mode of binding of the aromatic substrates and aromatic inhibitors and also for defining the subsites in the substrate binding site, we have determined the crystal structures of three complexes of bovine lactoperoxidase with aromatic ligands, acetylsalicylic acid (ASA), SHA, and benzylhydroxamic acid (BHA). Acetylsalicylic acid can be oxidized by lactoperoxidase to ASA free radical (42), whereas both salicylhydroxamic acid and benzylhydroxamic acid act as potent inhibitors of mammalian peroxidases (43–47). The determination of binding characteristics of these compounds having different actions has helped in establishing the relationship between the modes of binding and their potential actions as the substrates and inhibitors. To the best of our knowledge, this is the first structural report on the modes of binding of three aromatic ligands, ASA, SHA, and BHA, to LPO as well as the first structural study of the complexes of any mammalian peroxidase with ASA and BHA. These studies have shown that ASA, SHA, and BHA bind to LPO at the substrate binding site on the distal side. The SHA and BHA directly interact with the heme iron, whereas ASA interacts through the heme water molecule, which in turn is hydrogen-bonded to the heme iron. These studies have provided a greater insight into the mechanisms of substrate and inhibitor binding in the two mammalian peroxidases.     

Myosin II Tailpiece Determines Its Paracrystal Structure, Filament Assembly Properties, and Cellular Localization

Non muscle myosin II (NMII) is a major motor protein present in all cell types. The three known vertebrate NMII isoforms share high sequence homology but play different cellular roles. The main difference in sequence resides in the C-terminal non-helical tailpiece (tailpiece). In this study we demonstrate that the tailpiece is crucial for proper filament size, overcoming the intrinsic properties of the coiled-coil rod. Furthermore, we show that the tailpiece by itself determines the NMII filament structure in an isoform-specific manner, thus providing a possible mechanism by which each NMII isoform carries out its unique cellular functions. We further show that the tailpiece determines the cellular localization of NMII-A and NMII-B and is important for NMII-C role in focal adhesion complexes. We mapped NMII-C sites phosphorylated by protein kinase C and casein kinase II and showed that these phosphorylations affect its solubility properties and cellular localization. Thus phosphorylation fine-tunes the tailpiece effects on the coiled-coil rod, enabling dynamic regulation of NMII-C assembly. We thus show that the small tailpiece of NMII is a distinct domain playing a role in isoform-specific filament assembly and cellular functions.Non muscle myosin II (NMII)² is a major motor protein present in all cell types participating in crucial processes, including cytokinesis, surface attachment, and cell movement (1–3). NMII units are hexamers of two long heavy chains with two pairs of light chains attached. NMII heavy chain is composed of a globular head containing the actin binding and force generating ATPase domains, followed by a large coiled-coil rod that terminates with a short non-helical tailpiece (tailpiece). To carry out its cellular functions, NMII assembles into dimers and higher order filaments by interactions of the coiled-coil rod (4). The assembly process is governed by electrostatic interactions between adjacent coiled-coil rods containing alternating charged regions with specific periodicity (5–9) and is enhanced by activation of the motor domain through regulatory light chain phosphorylation (10–12). The charge periodicity also determines the register and orientation of each NMII hexamer in the filament. Additionally the C-terminal region of the coiled-coil rod contains a distinctive positively charged region and the assembly-competence domains that are crucial for proper filament assembly (5–9, 13).Three isoforms of NMII (termed NMII-A, NMII-B, and NMII-C) have been identified in mammals (14–16). Although NMII isoforms share somewhat overlapping roles, each isoform has distinctive tissue distribution and specific functions. NMII-A is important for neural growth cone retraction (17, 18) and is distributed to the front of migrating endothelial cells (19). While NMII-B participates in growth cone advancement (20) and was detected in the retracting tails of migrating endothelial cells (19). Furthermore NMII-A and NMII-B have an opposing effect on motility, since depletion of NMII-A leads to increased motility while NMII-B depletion hinders motility (21, 22). NMII-C plays a role in cytokinesis (23) and has distinct distribution in neuronal cells (24). Furthermore one NMII isoform only partly rescue cells in which siRNA was used to reduce the expression of another isoform (23, 25). This functional diversity is achieved despite a significant amino acid sequence identity between the isoforms (overall 64–80%), and the origin of these differential distributions and functions is not completely understood.Recent studies suggest that the C-terminal portion of NMII-A and NMII-B, particularly the last ∼170 amino acids, is responsible for the differential distribution of these NMII isoforms (26, 27). It was shown that swapping this region between NMII-A and NMII-B resulted in chimeric proteins, which adopted cellular localization according to the C-terminal part (26). This C-terminal ∼170 amino acid coiled-coil region contains the assembly-competence domains and other regions that are critical for filament assembly (5–9, 13) as well as the non-helical tailpiece. As the small tailpiece is also an important regulator of NMII filament assembly (27, 28) capable of changing NMII filament assembly properties; and phosphorylation of NMII tailpiece was shown to interfere with filament assembly (29–33) the tailpiece may be important for allowing NMII to perform its dynamic tasks. Because the coiled-coil regions are highly conserved between NMII isoforms, while the tailpiece is the most divergent, it is therefore a good candidate for mediating NMII isoform-specific functions. However, the exact mechanism by which the tailpiece affects NMII function is not fully understood. Here we show that the tailpiece serves as an isoform-specific control mechanism modulating filament order, assembly, and cellular function.     

Regulation of Microtubule Dynamic Instability in Vitro by Differentially Phosphorylated Stathmin

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two α:β tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser¹⁶ or Ser⁶³, and doubly phosphorylated at Ser²⁵ and Ser³⁸, on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser¹⁶ or Ser⁶³ strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser¹⁶ and Ser⁶³ and support the hypothesis that selective targeting by Ser¹⁶-specific or Ser⁶³-specific kinases provides complimentary mechanisms for regulating microtubule function.Stathmin is an 18-kDa ubiquitously expressed microtubule-destabilizing phosphoprotein whose activity is modulated by phosphorylation of its four serine residues, Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³ (1–7). Several classes of kinases have been identified that phosphorylate stathmin, including kinases associated with cell growth and differentiation such as members of the mitogen-activated protein kinase (MAPK)2 family, cAMP-dependent protein kinase (1–5, 8–11), and kinases associated with cell cycle regulation such as cyclin-dependent kinase 1 (3, 12–14). Phosphorylation of stathmin is required for cell cycle progression through mitosis and for proper assembly/function of the mitotic spindle (3, 13–16). Inhibition of stathmin phosphorylation produces strong mitotic phenotypes characterized by disassembly and disorganization of mitotic spindles and abnormal chromosome distributions (3, 13–14).Stathmin is known to destabilize microtubules in two ways. One is by binding to soluble tubulin and forming a stable complex that cannot polymerize into microtubules, consisting of one molecule of stathmin and two molecules of tubulin (T2S complex) (17–24). Addition of stathmin to microtubules in equilibrium with soluble tubulin results in sequestration of the tubulin and a reduction in the level of microtubule polymer (17–18, 22, 25–28). In addition to reducing the amount of assembled polymer, tubulin sequestration by stathmin has been shown to increase the switching frequency at microtubule plus ends from growth to shortening (called the catastrophe frequency) as the microtubules relax to a new steady state (17, 29). The second way is by binding directly to microtubules (27–30). The direct binding of stathmin to microtubules increases the catastrophe frequency at both ends of the microtubules and considerably more strongly at minus ends than at plus ends (27). Consistent with its strong catastrophe-promoting activity at minus ends, stathmin increases the treadmilling rate of steady-state microtubules in vitro (27). These results have led to the suggestion that stathmin might be an important cellular regulator of minus-end microtubule dynamics (27).Phosphorylation of stathmin diminishes its ability to regulate microtubule polymerization (3, 14, 25–26). Phosphorylation of Ser¹⁶ or Ser⁶³ appears to be more critical than phosphorylation of Ser²⁵ and Ser³⁸ for the ability of stathmin to bind to soluble tubulin and to inhibit microtubule assembly in vitro (3, 25). Inhibition of stathmin phosphorylation induces defects in spindle assembly and organization (3, 14) suggesting that not only soluble tubulin-microtubule levels are regulated by phosphorylation of stathmin, but the dynamics of microtubules could also be regulated in a phosphorylation-dependent manner.It is not known how phosphorylation at any of the four serine residues of stathmin affects its ability to regulate microtubule dynamics and, specifically, its ability to increase the catastrophe frequency at plus and minus ends due to its direct interaction with microtubules. Thus, we determined the effects of stathmin individually phosphorylated at either Ser¹⁶ or Ser⁶³ and doubly phosphorylated at both Ser²⁵ and Ser³⁸ on dynamic instability at plus and minus ends in vitro at microtubule polymer steady state and physiological pH (pH 7.2). We find that phosphorylation of Ser¹⁶ strongly reduces the direct catastrophe-promoting activity of stathmin at plus ends and abolishes it at minus ends, whereas phosphorylation of Ser⁶³ abolishes the activity at both ends. The effects of phosphorylation of individual serines correlated well with stathmin''s reduced abilities to form stable T2S complexes, to inhibit microtubule polymerization, and to bind to microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not alter the ability of stathmin to modulate dynamic instability at the microtubule ends, its ability to form a stable T2S complex, or its ability to bind to microtubules. The data further support the hypotheses that phosphorylation of stathmin on either Ser¹⁶ or Ser⁶³ plays a critical role in regulating microtubule polymerization and dynamics in cells.     

Conformational Properties of ��-PrP

Prion propagation involves a conformational transition of the cellular form of prion protein (PrP^C) to a disease-specific isomer (PrP^Sc), shifting from a predominantly α-helical conformation to one dominated by β-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91–231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP^Sc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant β-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP^C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a β-rich conformation. This precursor state is almost as compact as the folded PrP^C structure and, as it assembles, only residues 126–227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.Prion diseases, such as Creutzfeldt-Jacob and Gerstmann-Sträussler-Scheinker in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, are fatal neurological disorders associated with the deposition of an abnormally folded form of a host-encoded glycoprotein, prion (PrP)² (1). These diseases may be inherited, arise sporadically, or be acquired through the transmission of an infectious agent (2, 3). The disease-associated form of the protein, termed the scrapie form or PrP^Sc, differs from the normal cellular form (PrP^C) through a conformational change, resulting in a significant increase in the β-sheet content and protease resistance of the protein (3, 4). PrP^C, in contrast, consists of a predominantly α-helical structured domain and an unstructured N-terminal domain, which is capable of binding a number of divalent metals (5–12). A single disulfide bond links two of the main α-helices and forms an integral part of the core of the structured domain (13, 14).According to the protein-only hypothesis (15), the infectious agent is composed of a conformational isomer of PrP (16) that is able to convert other isoforms to the infectious isomer in an autocatalytic manner. Despite numerous studies, little is known about the mechanism of conversion of PrP^C to PrP^Sc. The most coherent and general model proposed thus far is that PrP^C fluctuates between the dominant native state and minor conformations, one or a set of which can self-associate in an ordered manner to produce a stable supramolecular structure composed of misfolded PrP monomers (3, 17). This stable, oligomeric species can then bind to, and stabilize, rare non-native monomer conformations that are structurally complementary. In this manner, new monomeric chains are recruited and the system can propagate.In view of the above model, considerable effort has been devoted to generating and characterizing alternative, possibly PrP^Sc-like, conformations in the hope of identifying common properties or features that facilitate the formation of amyloid oligomers. This has been accomplished either through PrP^Sc-dependent conversion reactions (18–20) or through conversion of PrP^C in the absence of a PrP^Sc template (21–25). The latter approach, using mainly disulfide-oxidized recombinant PrP, has generated a wide range of novel conformations formed under non-physiological conditions where the native state is relatively destabilized. These conformations have ranged from near-native (14, 26, 27), to those that display significant β-sheet content (21, 23, 28–33). The majority of these latter species have shown a high propensity for aggregation, although not all are on-pathway to the formation of amyloid. Many of these non-native states also display some of the characteristics of PrP^Sc, such as increased β-sheet content, protease resistance, and a propensity for oligomerization (28, 29, 31) and some have been claimed to be associated with the disease process (34).One such PrP folding intermediate, termed β-PrP, differs from the majority of studied PrP intermediate states in that it is formed by refolding the PrP molecule from the native α-helical conformation (here termed α-PrP), at acidic pH in a reduced state, with the disulfide bond broken (22, 35). Although no covalent differences between the PrP^C and PrP^Sc have been consistently identified to date, the role of the disulfide bond in prion propagation remains disputed (25, 36–39). β-PrP is rich in β-sheet structure (22, 35), and displays many of the characteristics of a PrP^Sc-like precursor molecule, such as partial resistance to proteinase K digestion, and the ability to form amyloid fibrils in the presence of physiological concentrations of salts (40).The β-PrP species previously characterized, spanning residues 91–231 of PrP, was soluble at low ionic strength buffers and monomeric, according to elution volume on gel filtration (22). NMR analysis showed that it displayed radically different spectra to those of α-PrP, with considerably fewer observable peaks and markedly reduced chemical shift dispersion. Data from circular dichroism experiments showed that fixed side chain (tertiary) interactions were lost, in contrast to the well defined β-sheet secondary structure, and thus in conjunction with the NMR data, indicated that β-PrP possessed a number of characteristics associated with a “molten globule” folding intermediate (22). Such states have been proposed to be important in amyloid and fibril formation (41). Indeed, antibodies raised against β-PrP (e.g. ICSM33) are capable of recognizing native PrP^Sc (but not PrP^C) (42–44). Subsequently, a related study examining the role of the disulfide bond in PrP folding confirmed that a monomeric molten globule-like form of PrP was formed on refolding the disulfide-reduced protein at acidic pH, but reported that, under their conditions, the circular dichroism response interpreted as β-sheet structure was associated with protein oligomerization (45). Indeed, atomic force microscopy on oligomeric full-length β-PrP (residues 23–231) shows small, round particles, showing that it is capable of formation of oligomers without forming fibrils (35). Notably, however, salt-induced oligomeric β-PrP has been shown to be a potent inhibitor of the 26 S proteasome, in a similar manner to PrP^Sc (46). Impairment of the ubiquitin-proteasome system in vivo has been linked to prion neuropathology in prion-infected mice (46).Although the global properties of several PrP intermediate states have been determined (30–32, 35), no information on their conformational properties on a sequence-specific basis has been obtained. Their conformational properties are considered important, as the elucidation of the chain conformation may provide information on the way in which these chains pack in the assembly process, and also potentially provide clues on the mechanism of amyloid assembly and the phenomenon of prion strains. As the conformational fluctuations and heterogeneity of molten globule states give rise to broad NMR spectra that preclude direct observation of their conformational properties by NMR (47–50), here we use denaturant titration experiments to determine the conformational properties of β-PrP, through the population of the unfolded state that is visible by NMR. In addition, we use circular dichroism and analytical ultracentrifugation to examine the global structural properties, and the distribution of multimeric species that are formed from β-PrP.     

A Ca2+ Release-activated Ca2+ (CRAC) Modulatory Domain (CMD) within STIM1 Mediates Fast Ca2+-dependent Inactivation of ORAI1 Channels

STIM1 and ORAI1, the two limiting components in the Ca²⁺ release-activated Ca²⁺ (CRAC) signaling cascade, have been reported to interact upon store depletion, culminating in CRAC current activation. We have recently identified a modulatory domain between amino acids 474 and 485 in the cytosolic part of STIM1 that comprises 7 negatively charged residues. A STIM1 C-terminal fragment lacking this domain exhibits enhanced interaction with ORAI1 and 2–3-fold higher ORAI1/CRAC current densities. Here we focused on the role of this CRAC modulatory domain (CMD) in the fast inactivation of ORAI1/CRAC channels, utilizing the whole-cell patch clamp technique. STIM1 mutants either with C-terminal deletions including CMD or with 7 alanines replacing the negative amino acids within CMD gave rise to ORAI1 currents that displayed significantly reduced or even abolished inactivation when compared with STIM1 mutants with preserved CMD. Consistent results were obtained with cytosolic C-terminal fragments of STIM1, both in ORAI1-expressing HEK 293 cells and in RBL-2H3 mast cells containing endogenous CRAC channels. Inactivation of the latter, however, was much more pronounced than that of ORAI1. The extent of inactivation of ORAI3 channels, which is also considerably more prominent than that of ORAI1, was also substantially reduced by co-expression of STIM1 constructs missing CMD. Regarding the dependence of inactivation on Ca²⁺, a decrease in intracellular Ca²⁺ chelator concentrations promoted ORAI1 current fast inactivation, whereas Ba²⁺ substitution for extracellular Ca²⁺ completely abrogated it. In summary, CMD within the STIM1 cytosolic part provides a negative feedback signal to Ca²⁺ entry by triggering fast Ca²⁺-dependent inactivation of ORAI/CRAC channels.The Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel is one of the best characterized store-operated entry pathways (1–7). Substantial efforts have led to identification of two key components of the CRAC channel machinery: the stromal interaction molecule 1 (STIM1), which is located in the endoplasmic reticulum and acts as a Ca²⁺ sensor (8–10), and ORAI1/CRACM1, the pore-forming subunit of the CRAC channel (11–13). Besides ORAI1, two further homologues named ORAI2 and ORAI3 belong to the ORAI channel family (12, 14).STIM1 senses endoplasmic reticulum store depletion primarily by its luminal EF-hand in its N terminus (8, 15), redistributes close to the plasma membrane, where it forms puncta-like structures, and co-clusters with ORAI1, leading to inward Ca²⁺ currents (12, 16–19). The STIM1 C terminus, located in the cytosol, contains two coiled-coil regions overlapping with an ezrin-radixin-moesin (ERM)-like domain followed by a serine/proline- and a lysine-rich region (2, 8, 20–22). Three recent studies have described the essential ORAI-activating region within the ERM domain, termed SOAR (Stim ORAI-activating region) (23), OASF (ORAI-activating small fragment) (24), and CAD (CRAC-activating domain) (25), including the second coiled coil domain and the following ∼55 amino acids. We and others have provided evidence that store depletion leads to a dynamic coupling of STIM1 to ORAI1 (26–28) that is mediated by a direct interaction of the STIM1 C terminus with ORAI1 C terminus probably involving the putative coiled-coil domain in the latter (27).Furthermore, different groups have proven that the C terminus of STIM1 is sufficient to activate CRAC as well as ORAI1 channels independent of store depletion (22–25, 27, 29). We have identified that OASF-(233–474) or shorter fragments exhibit further enhanced coupling to ORAI1 resulting in 3-fold increased constitutive Ca²⁺ currents. A STIM1 fragment containing an additional cluster of anionic amino acids C-terminal to position 474 displays weaker interaction with ORAI1 as well as reduced Ca²⁺ current comparable with that mediated by wild-type STIM1 C terminus. Hence, we have suggested that these 11 amino acids (474–485) act in a modulatory manner onto ORAI1; however, their detailed mechanistic impact within the STIM1/ORAI1 signaling machinery has remained so far unclear.In this study, we focused on the impact of this negative cluster on fast inactivation of STIM1-mediated ORAI Ca²⁺ currents. Lis et al. (30) have shown that all three ORAI homologues display distinct inactivation profiles, where ORAI2 and ORAI3 show a much more pronounced fast inactivation than ORAI1. Moreover, it has been reported (31) that different expression levels of STIM1 to ORAI1 affect the properties of CRAC current inactivation. Yamashita et al. (32) have demonstrated a linkage between the selectivity filter of ORAI1 and its Ca²⁺-dependent fast inactivation. Here we provide evidence that a cluster of acidic residues within the C terminus of STIM1 is involved in the fast inactivation of ORAI1 and further promotes that of ORAI3 and native CRAC currents.     

Deletion of the Chloride Transporter Slc26a7 Causes Distal Renal Tubular Acidosis and Impairs Gastric Acid Secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl⁻/HCO₃⁻ exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.The collecting duct segment of the distal kidney nephron plays a major role in systemic acid base homeostasis by acid secretion and bicarbonate absorption. The acid secretion occurs via H⁺-ATPase and H-K-ATPase into the lumen and bicarbonate is absorbed via basolateral Cl⁻/HCO₃⁻ exchangers (1–4). The tubules, which are located within the outer medullary region of the kidney collecting duct (OMCD),² have the highest rate of acid secretion among the distal tubule segments and are therefore essential to the maintenance of acid base balance (2).The gastric parietal cell is the site of generation of acid and bicarbonate through the action of cytosolic carbonic anhydrase II (5, 6). The intracellular acid is secreted into the lumen via gastric H-K-ATPase, which works in conjunction with a chloride channel and a K⁺ recycling pathway (7–10). The intracellular bicarbonate is transported to the blood via basolateral Cl⁻/HCO₃⁻ exchangers (11–14).SLC26 (human)/Slc26 (mouse) isoforms are members of a conserved family of anion transporters that display tissue-specific patterns of expression in epithelial cells (15–24). Several SLC26 members can function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9 (25–31). SLC26A7 and SLC26A9 can also function as chloride channels (32–34).SLC26A7/Slc26a7 is predominantly expressed in the kidney and stomach (28, 29). In the kidney, Slc26a7 co-localizes with AE1, a well-known Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of (acid-secreting) A-intercalated cells in OMCD cells (29, 35, 36) (supplemental Fig. 1). In the stomach, Slc26a7 co-localizes with AE2, a major Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of acid secreting parietal cells (28). To address the physiological function of Slc26a7 in the intact mouse, we have generated Slc26a7 ko mice. We report here that Slc26a7 ko mice exhibit distal renal tubular acidosis and impaired gastric acidification in the absence of morphological abnormalities in kidney or stomach.     

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na⁺/nucleoside and H⁺/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H⁺ dependence (all mutants), shift in Na⁺:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na⁺-specific hCNT1, uncoupled Na⁺ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)³ proteins for transport into or out of cells (1–4). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (3–6). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1–3, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na⁺-specific and transport pyrimidine and purine nucleosides, respectively (11–13). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na⁺- but not H⁺-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H⁺-coupled. Na⁺:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H⁺:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na⁺-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na⁺- and H⁺-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX₃NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (20–30). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX₃NEFVA(Y/M/F) motif.     

Hepcidin Revisited, Disulfide Connectivity, Dynamics, and Structure

Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. All techniques confirmed the following disulfide bond connectivity: Cys¹–Cys⁸, Cys³–Cys⁶, Cys²–Cys⁴, and Cys⁵–Cys⁷. NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.Regulation of iron levels is critical to the survival of species that live in an oxygen-rich environment (1). In mammals, iron homeostasis is principally regulated by hepcidin, a 25-residue peptide hormone containing a complex network of four disulfide bonds. Hepcidin was discovered by three groups investigating either novel anti-microbial peptides or iron regulation (2–4), and subsequent genetic evidence has shown that mutation of the hepcidin gene can lead to systemic iron overload or hemochromatosis (5). Similarly, mutations in upstream control proteins HFE and hemojuvelin or mutation of the gene for ferroportin, the hepcidin receptor, cause forms of hemochromatosis of varying clinical severity (6–9). Genetic studies in mice have confirmed these relationships, identifying the hepcidin pathway as a critical component in the control of iron metabolism (10–12). Dysfunction of the hepcidin pathway and the resulting iron imbalance may play a role in multiple diseases such as anemia of inflammation (13), atherosclerosis (14), and neurodegenerative disorders (15). In anemia of inflammation, suppression of hepcidin constituted a successful treatment, suggesting that it may be an appropriate therapeutic target in the treatment of disease.³The human hepcidin gene encodes an 84-residue prepropeptide that contains a 24-residue N-terminal signal peptide that is subsequently cleaved to produce pro-hepcidin. Pro-hepcidin is then processed to produce a mature 25-amino acid hepcidin that is detectable in both blood and urine. Mass spectrometry and chemical analysis have revealed that all eight cysteines in hepcidin are involved in disulfide bonds (3) suggesting a highly constrained structure containing a precise disulfide bonding pattern.The NMR solution structure of hepcidin first reported by Hunter et al. (16) revealed a compact fold with β-sheet and β-hairpin loop elements. From structure calculations and dynamic signatures in NMR spectra, the authors inferred a disulfide connectivity of Cys¹–Cys⁸, Cys²–Cys⁷, Cys³–Cys⁶,⁴ and a rare vicinal disulfide bond at Cys⁴–Cys⁵. A later study of bass hepcidin (17) determined essentially the same fold and confirmed the same disulfide connectivity. Both studies, however, were based on incomplete NMR data because the resonances from two adjacent cysteines, Cys-13 and Cys-14 of hepcidin, were not detected, presumably due to exchange broadening.Here we demonstrate a new pattern of disulfide connectivity obtained independently from chemical and spectroscopic analysis. In addition, we present the first complete solution NMR structure of hepcidin and x-ray structure of the peptide in complex with an anti-hepcidin Fab. NMR data obtained at different temperatures reveal that hepcidin exhibits significant conformational dynamics in solution, a problem that likely occluded previous NMR studies. Data presented here show that these dynamics can be almost completely resolved by temperature variation, yielding two distinct structures of hepcidin, one at 325 K and one at 253 K in supercooled water. In addition to inferring disulfide bonds from structure calculations, we present an argument based on probabilistic interpretation of NMR data, which unequivocally establishes the same connectivity as obtained from chemical analysis.Because of the complexity of the disulfide network, hepcidin production is prone to misfolding artifacts. We demonstrate this through biophysical and biological activity characterization of hepcidin samples obtained from different sources. This information is essential for establishing accurate standards for quantitation of hepcidin levels in humans. In our experience, the highest quality material appeared to be critical for the structural studies presented here.     

The Ternary Structure of the Double-headed Arrowhead Protease Inhibitor API-A Complexed with Two Trypsins Reveals a Novel Reactive Site Conformation

The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a ternary complex at 2.48 Å resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 Å apart. The overall fold of API-A belongs to the β-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The two P1 residues were unambiguously assigned as Leu⁸⁷ and Lys¹⁴⁵, and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met⁸³ to P5′ Ala⁹², adopts a novel conformation with the Leu⁸⁷ completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting of residues P5 Cys¹⁴¹ to P5′ Glu¹⁵⁰, binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering of the reactive sites.Protease inhibitors (PIs)⁴ are ubiquitously distributed in all organisms, including plants, animals, and microorganisms (1). They play vital roles in regulating their corresponding proteases, which are involved in many biological processes such as protein digestion, cell signal transmission, inflammation, apoptosis, blood coagulation, and embryogenesis (2). The clinical applications of PIs are widespread, and there is great interest in developing more potent therapeutic PIs for treating human diseases related to cancer (3), pancreatitis (4), thrombosis (5), and AIDS (6). To this end, the soybean Kunitz-type serine proteases inhibitors have been extensively studied (1, 7–11). The inhibitors of this family generally contain 170–200 residues and have two disulfide bonds. Most members have only one reactive site located in the region of residues 60–70 (7, 10, 12–14). However, a few members possess two reactive sites that simultaneously bind two protease molecules and are thus termed double-headed inhibitors (15–18). All of these inhibitors are classified into family I3 of peptidase inhibitors (19). Most members are further grouped into subfamily I3A. However, the double-headed arrowhead PIs API-A and -B are grouped in subfamily I3B because of their very low sequence similarity to other members (19). In contrast to other double-headed PIs such as the Bowman-Birk and ovomucoid inhibitors, which have two identical reactive sites that have evolved by domain shuffling and gene duplication (1, 20–25), both API-A and -B have two distinct reactive sites.API-A and -B were first purified from the tubers of Sagittaria sagittifolia (Linn) in 1979 (26). Both consist of 179 residues with three disulfide bonds and can inhibit a variety of serine proteases, including trypsin, chymotrypsin, and porcine tissue kallikrein (17, 26–28). Although the sequence identity of API-A and -B is as high as 91%, their inhibitory specificities differ. The former can bind one molecule of trypsin and one molecule of chymotrypsin, whereas the latter can simultaneously bind two molecules of trypsin (26). The two P1 residues of the reactive sites of API-A and -B were first predicted to be Lys⁴⁴ and Arg⁷⁶ based on their surrounding sequences, which are similar to those of the reactive sites of bovine pancreas trypsin inhibitor and soybean Kunitz trypsin inhibitor (29). However, their identities were later revised to Arg⁷⁶ and Leu⁸⁷ (for API-A) or Lys⁸⁷ (for API-B) based on results from sited-directed mutagenesis studies (30).To clarify these controversies, we solved the crystal structure of API-A in complex with two molecules of bovine trypsin. To the best of our knowledge, this is the first report on the three-dimensional structure of the double-headed Kunitz-type trypsin inhibitor in complex with two molecules of protease. On the basis of this structure, the two P1 residues have now been identified as Leu⁸⁷ and Lys¹⁴⁵ for reactive site 1 (RS1) and 2 (RS2), respectively. The results were further confirmed by site-directed mutagenesis. It was earlier shown that the first P1 residue Leu⁸⁷ interacts preferentially with chymotrypsin (30). In our structure, Leu⁸⁷ is snugly embedded in the S1 pocket of trypsin, as a consequence of the broad interface contributed by the surrounding residues. Comprehensive analyses of the two reactive site interfaces have provided functional insights into the novel inhibitory patterns of this unique double-headed protease inhibitor.     

From Whole-body Sections Down to Cellular Level,Multiscale Imaging of Phospholipids by MALDI Mass Spectrometry

Significant progress in instrumentation and sample preparation approaches have recently expanded the potential of MALDI imaging mass spectrometry to the analysis of phospholipids and other endogenous metabolites naturally occurring in tissue specimens. Here we explore some of the requirements necessary for the successful analysis and imaging of phospholipids from thin tissue sections of various dimensions by MALDI time-of-flight mass spectrometry. We address methodology issues relative to the imaging of whole-body sections such as those cut from model laboratory animals, sections of intermediate dimensions typically prepared from individual organs, as well as the requirements for imaging areas of interests from these sections at a cellular scale spatial resolution. We also review existing limitations of MALDI imaging MS technology relative to compound identification. Finally, we conclude with a perspective on important issues relative to data exploitation and management that need to be solved to maximize biological understanding of the tissue specimen investigated.Since its introduction in the late 90s (1), MALDI imaging mass spectrometry (MS) technology has witnessed a phenomenal expansion. Initially introduced for the mapping of intact proteins from fresh frozen tissue sections (2), imaging MS is now routinely applied to a wide range of different compounds including peptides, proteins, lipids, metabolites, and xenobiotics (3–7). Numerous compound-specific sample preparation protocols and analytical strategies have been developed. These include tissue sectioning and handling (8–14), automated matrix deposition approaches and data acquisition strategies (15–21), and the emergence of in situ tissue chemistries (22–25). Originally performed on sections cut from fresh frozen tissue specimens, methodologies incorporating an in situ enzymatic digestion step prior to matrix application have been optimized to access the proteome locked in formalin-fixed paraffin-embedded tissue biopsies (25–29). The possibility to use tissues preserved using non-cross-linking approaches has also been demonstrated (30–32). These methodologies are of high importance for the study of numerous diseases because they potentially allow the retrospective analysis for biomarker validation and discovery of the millions of tissue biopsies currently stored worldwide in tissue banks and repositories.In the past decade, instrumentation for imaging MS has also greatly evolved. Whereas the first MS images were collected with time-of-flight instruments (TOF) capable of repetition rates of a few hertz, modern systems are today capable of acquiring data in the kilohertz range and above with improved sensitivity, mass resolving power, and accuracy, significantly reducing acquisition time and improving image quality (33, 34). Beyond time-of-flight analyzers, other MALDI-based instruments have been used such as ion traps (35–37), Qq TOF instruments (38–40), and trap-TOF (16, 41). Ion mobility technology has also been used in conjunction with imaging MS (42–44). More recently, MALDI FT/ICR and Orbitrap mass spectrometers have been demonstrated to be extremely valuable instruments for the performance of imaging MS at very high mass resolving power (45–47). These non-TOF-based systems have proven to be extremely powerful for the imaging of lower molecular weight compounds such as lipids, drugs, and metabolites. Home-built instrumentation and analytical approaches to probe tissues at higher spatial resolution (1–10 μm) have also been described (48–50). In parallel to instrumentation developments, automated data acquisition, image visualization, and processing software packages have now also been developed by most manufacturers.To date, a wide range of biological systems have been studied using imaging MS as a primary methodology. Of strong interest are the organization and identification of the molecular composition of diseased tissues in direct correlation with the underlying histology and how it differs from healthy tissues. Such an approach has been used for the study of cancers (51–54), neurologic disorders (55–57), and other diseases (58, 59). The clinical potential of the imaging MS technology is enormous (7, 60, 61). Results give insights into the onset and progression of diseases, identify novel sets of disease-specific markers, and can provide a molecular confirmation of diagnosis as well as aide in outcome prediction (62–64). Imaging MS has also been extensively used to study the development, functioning, and aging of different organs such as the kidney, prostate, epididymis, and eye lens (65–70). Beyond the study of isolated tissues or organs, whole-body sections from several model animals such as leeches, mice, and rats have been investigated (71–74). For these analyses, specialized instrumentation and protocols are necessary for tissue sectioning and handling (72, 73). Whole-body imaging MS opens the door to the study of the localization and accumulation of administered pharmaceuticals and their known metabolites at the level of entire organisms as well as the monitoring of their efficacy or toxicity as a function of time or dose (72, 73, 75, 76).There is considerable interest in determining the identification and localization of small biomolecules such as lipids in tissues because they are involved in many essential biological functions including cell signaling, energy storage, and membrane structure and function. Defects in lipid metabolism play a role in many diseases such as muscular dystrophy and cardiovascular disease. Phospholipids in tissues have been intensively studied by several groups (37, 40, 77–83). In this respect, for optimal recovery of signal, several variables such as the choice of matrix for both imaging and fragmentation, solvent system, and instrument polarity have been investigated (20, 84). Particularly, the use of lithium cation adducts to facilitate phospholipid identification by tandem MS directly from tissue has also been reported (85). Of significant interest is the recent emergence of two new solvent-free matrix deposition approaches that perform exceptionally well for phospholipid imaging analyses. The first approach, described by Hankin et al. (86), consists in depositing the matrix on the sections through a sublimation process. The described sublimation system consists of sublimation glassware, a heated sand or oil bath (100–200 °C), and a primary vacuum pump (∼5 × 10⁻² torr). Within a few minutes of initiating the sublimation process, an exceptionally homogeneous film of matrix forms on the section. The thickness of the matrix may be controlled by regulating pressure, temperature, and sublimation time. The second approach, described by Puolitaival et al.(87), uses a fine mesh sieve (≤20 μm) to filter finely ground matrix on the tissue sections. Agitation of the sieve results in passage of the matrix through the mesh and the deposition of a fairly homogeneous layer of submicrometer matrix crystals of the surface of the sections. The matrix density on the sections is controlled by direct observation using a standard light microscope. This matrix deposition approach was also found to be ideal to image certain drug compounds (88, 89). Both strategies allow very rapid production of homogeneous matrix coatings on tissue sections with a fairly inexpensive setup. Signal recovery was found to be comparable with those obtained by conventional spray deposition. With the appropriate size sublimation device or sieve, larger sections with dimensions of several centimeters such as those cut from mouse or rat whole bodies can also be rapidly and homogeneously coated.Here we present several examples of MALDI imaging MS of phospholipids from tissue sections using TOF mass spectrometers over a wide range of dimensions from whole-body sections (several centimeters), to individual organs (several millimeters), down to high spatial resolution imaging of selected tissue areas (hundreds of micrometers) at 10-μm lateral resolution and below. For all of these dimension ranges, technological considerations and practical aspects are discussed. In light of the imaging MS results, we also address issues faced for compound identification by tandem MS analysis performed directly on the sections. Finally, we discuss under “Perspective” our vision of the future of the field as well as the technological improvements and analytical tools that need to be improved upon and developed.     

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.