Characterizing phospholamban to sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking

Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys²⁷ of PLB was cross-linked to the Ca²⁺ pump at the cytoplasmic extension of M4 (at or near Lys³²⁸) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 Å). Cross-linking was augmented by ATP but abolished by Ca²⁺ or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca²⁺ affinity conformation of the Ca²⁺-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the anti-PLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a and 2D12 stimulation of Ca²⁺-ATPase activity and Ca²⁺ transport. The results suggest that the inhibitory effect of PLB on Ca²⁺-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca²⁺ to the Ca²⁺ pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca²⁺ pump are largely unchanged in failing myocardium.     

Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban

Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca²⁺-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca²⁺. Recent cross-linking studies have suggested that PLB binding and Ca²⁺ binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca²⁺-ATPase, preventing formation of E1, the conformation that binds two Ca²⁺ (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca²⁺ binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca²⁺ pump. Seven SERCA2a mutants with amino acid substitutions at Ca²⁺-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca²⁺ on N30C-PLB cross-linking to Lys³²⁸ of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca²⁺-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca²⁺; however, all were phosphorylatable by P_i to form E2P. Ca²⁺ inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca²⁺-ATPase mutant was observed in the absence of Ca²⁺. Importantly, however, micromolar Ca²⁺ inhibited PLB cross-linking only to mutants retaining a functional Ca²⁺-binding site I. The dynamic equilibrium between Ca²⁺ pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca²⁺-binding site regulating the physical association between PLB and SERCA2a.     

Migalastat HCl Reduces Globotriaosylsphingosine (Lyso-Gb3) in Fabry Transgenic Mice and in the Plasma of Fabry Patients

Fabry disease (FD) results from mutations in the gene (GLA) that encodes the lysosomal enzyme α-galactosidase A (α-Gal A), and involves pathological accumulation of globotriaosylceramide (GL-3) and globotriaosylsphingosine (lyso-Gb₃). Migalastat hydrochloride (GR181413A) is a pharmacological chaperone that selectively binds, stabilizes, and increases cellular levels of α-Gal A. Oral administration of migalastat HCl reduces tissue GL-3 in Fabry transgenic mice, and in urine and kidneys of some FD patients. A liquid chromatography-tandem mass spectrometry method was developed to measure lyso-Gb₃ in mouse tissues and human plasma. Oral administration of migalastat HCl to transgenic mice reduced elevated lyso-Gb₃ levels up to 64%, 59%, and 81% in kidney, heart, and skin, respectively, generally equal to or greater than observed for GL-3. Furthermore, baseline plasma lyso-Gb₃ levels were markedly elevated in six male FD patients enrolled in Phase 2 studies. Oral administration of migalastat HCl (150 mg QOD) reduced urine GL-3 and plasma lyso-Gb₃ in three subjects (range: 15% to 46% within 48 weeks of treatment). In contrast, three showed no reductions in either substrate. These results suggest that measurement of tissue and/or plasma lyso-Gb₃ is feasible and may be warranted in future studies of migalastat HCl or other new potential therapies for FD.     

Prostaglandin E2 receptors are differentially expressed in subpopulations of granulosa cells from primate periovulatory follicles

Prostaglandin E2 (PGE2) mediates many effects of the midcycle luteinizing hormone (LH) surge within the periovulatory follicle. Differential expression of the four PGE2 (EP) receptors may contribute to the specialized functions of each granulosa cell subpopulation. To determine if EP receptors are differentially expressed in granulosa cells, monkeys received gonadotropins to stimulate ovarian follicular development. Periovulatory events were initiated with human chorionic gonadotropin (hCG); granulosa cells and whole ovaries were collected before (0 h) and after (24-36 h) hCG to span the 40-h primate periovulatory interval. EP receptor mRNA and protein levels were quantified in granulosa cell subpopulations. Cumulus cells expressed higher levels of EP2 and EP3 mRNA compared with mural cells 36 h after hCG. Cumulus cell EP2 and EP3 protein levels also increased between 0 and 36 h after hCG. Overall, mural granulosa cells expressed low levels of EP1 protein at 0 h and higher levels 24-36 h after hCG. However, EP1 protein levels were higher in granulosa cells away from the follicle apex compared with apex cells 36 h after hCG. Higher levels of PAI-1 protein were measured in nonapex cells, consistent with a previous study showing EP1-stimulated PAI-1 protein expression in monkey granulosa cells. EP4 protein levels were low in all subpopulations. In summary, cumulus cells likely respond to PGE2 via EP2 and EP3, whereas PGE2 controls rupture of a specific region of the follicle via EP1. Therefore, differential expression of EP receptors may permit each granulosa cell subpopulation to generate a unique response to PGE2 during the process of ovulation.     

P-glycoprotein retains drug-stimulated ATPase activity upon covalent linkage of the two nucleotide binding domains at their C-terminal ends

P-glycoprotein (Pgp), a member of the ABC transporter family, functions as an ATP hydrolysis-driven efflux pump to rid the cell of toxic organic compounds, including a variety of drugs used in anti-cancer chemotherapy. We have recently obtained EM projection images of lipid-bound Pgp without nucleotide and transport substrate that showed the two halves of the transporter separated by a central cavity (Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2002) J. Biol. Chem. 277, 40125-40131). Addition of nucleotide and/or substrate lead to a close association of the two halves of the transporter, thereby closing the central cavity (Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2008) J. Biol. Chem. 283, 5769-5779). Here, we used cysteine-mediated disulfide cross-linking to further delineate the structural rearrangements of the two nucleotide binding domains (NBD1 and NBD2) that take place during catalysis. Cysteines introduced at or near the C-terminal ends of NBD1 and NBD2 allowed for spontaneous disulfide cross-linking under nonreducing conditions. For mutant A627C/S1276C, disulfide formation was with high efficiency and cross-linked Pgp retained 30-68% drug-stimulated ATPase activity compared with reduced or cysteine-less Pgp. Two other cysteine pairs (K615C/S1276C and A627C/K1260C) also formed a disulfide but to a lesser extent, and the cross-linked form of these two mutants had lower drug-stimulated ATPase activity. The data suggest that the C-terminal ends of the two NBDs of Pgp are not required to undergo significant motion with respect to one another during the catalytic cycle.     

Circadian phase preference in college students: relationships with psychological functioning and academics

The current study offers a comprehensive assessment of psychosocial functioning and academic performance in relation to circadian phase preference in a US sample of undergraduate college students (N?=?838), aged 17-26 (M?=?19.78, SD?=?1.89). Women had greater morning preference than men, and seniors had greater morning preference than freshmen. Circadian phase preference, fatigue, perceived stress, depression, anxiety, and substance use were assessed cross-sectionally and grade point average (GPA) was assessed prospectively. Evening phase preference was related to higher levels of fatigue, alcohol and caffeine use, and worse academic performance than morning or intermediate phase preferences.     

The in vitro resistance of IgG2 to proteolytic attack concurs with a comparative paucity of autoantibodies against peptide analogs of the IgG2 hinge

The mammalian antibody repertoire comprises immunoglobulin (Ig) molecules of multiple isotypes and subclasses with varying functional properties. Among the four subclasses of the human IgG isotype, we found that IgG2 exhibits a particular resistance to human and bacterial proteases that readily cleave the IgG1 hinge region in vitro. Autoantibodies (IgGs) that recognize points of proteolytic cleavage in the IgG1 hinge are widespread in the healthy human population, suggesting that IgG1 fragmentation and the generation of cryptic antigens for host immune surveillance commonly occur in vivo. We previously reported that autoantibodies to cleaved IgG1s can restore Fc-mediated effector functions that are lost following proteolytic cleavage of the hinge. In contrast, it was not possible to demonstrate an analogous cohort of autoantibodies to IgG2 hinge epitope analogs and there appeared to be no functional component in human serum with the ability to reconstitute Fc effector functions to a cell-bound IgG2 fragment. Thus, the results indicate that among the IgG subclasses, human IgG2 is uniquely resistant to a number of known pathological proteases and that autoimmune recognition to potential cleavage points in the IgG2 hinge appears to be absent in human circulation.Key words: antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, autoantibodies     

Colonization of subsurface microbial observatories deployed in young ocean crust

Oceanic crust comprises the largest hydrogeologic reservoir on Earth, containing fluids in thermodynamic disequilibrium with the basaltic crust. Little is known about microbial ecosystems that inhabit this vast realm and exploit chemically favorable conditions for metabolic activities. Crustal samples recovered from ocean drilling operations are often compromised for microbiological assays, hampering efforts to resolve the extent and functioning of a subsurface biosphere. We report results from the first in situ experimental observatory systems that have been used to study subseafloor life. Experiments deployed for 4 years in young (3.5 Ma) basaltic crust on the eastern flank of the Juan de Fuca Ridge record a dynamic, post-drilling response of crustal microbial ecosystems to changing physical and chemical conditions. Twisted stalks exhibiting a biogenic iron oxyhydroxide signature coated the surface of mineral substrates in the observatories; these are biosignatures indicating colonization by iron oxidizing bacteria during an initial phase of cool, oxic, iron-rich conditions following observatory installation. Following thermal and chemical recovery to warmer, reducing conditions, the in situ microbial structure in the observatory shifted, becoming representative of natural conditions in regional crustal fluids. Firmicutes, metabolic potential of which is unknown but may involve N or S cycling, dominated the post-rebound bacterial community. The archaeal community exhibited an extremely low diversity. Our experiment documented in situ conditions within a natural hydrological system that can pervade over millennia, exemplifying the power of observatory experiments for exploring the subsurface basaltic biosphere, the largest but most poorly understood biotope on Earth.     

Shedding of TRAP by a Rhomboid Protease from the Malaria Sporozoite Surface Is Essential for Gliding Motility and Sporozoite Infectivity

Plasmodium sporozoites, the infective stage of the malaria parasite, move by gliding motility, a unique form of locomotion required for tissue migration and host cell invasion. TRAP, a transmembrane protein with extracellular adhesive domains and a cytoplasmic tail linked to the actomyosin motor, is central to this process. Forward movement is achieved when TRAP, bound to matrix or host cell receptors, is translocated posteriorly. It has been hypothesized that these adhesive interactions must ultimately be disengaged for continuous forward movement to occur. TRAP has a canonical rhomboid-cleavage site within its transmembrane domain and mutations were introduced into this sequence to elucidate the function of TRAP cleavage and determine the nature of the responsible protease. Rhomboid cleavage site mutants were defective in TRAP shedding and displayed slow, staccato motility and reduced infectivity. Moreover, they had a more dramatic reduction in infectivity after intradermal inoculation compared to intravenous inoculation, suggesting that robust gliding is critical for dermal exit. The intermediate phenotype of the rhomboid cleavage site mutants suggested residual, albeit inefficient cleavage by another protease. We therefore generated a mutant in which both the rhomboid-cleavage site and the alternate cleavage site were altered. This mutant was non-motile and non-infectious, demonstrating that TRAP removal from the sporozoite surface functions to break adhesive connections between the parasite and extracellular matrix or host cell receptors, which in turn is essential for motility and invasion.