Phenotypes of SERCA and PMCA knockout mice

P-type Ca2+-ATPases of the sarco(endo)plasmic reticulum (SERCAs) and plasma membrane (PMCAs) are responsible for maintaining the Ca2+ gradients across cellular membranes that are required for regulation of Ca2+-mediated signaling and other biological processes. Gene-targeting studies of SERCA isoforms 1, 2, and 3 and PMCA isoforms 1, 2, and 4 have confirmed some of the general functions proposed for these pumps, such as a major role in excitation-contraction coupling for SERCA1 and SERCA2 and housekeeping functions for PMCA1 and SERCA2, but have also revealed some unexpected phenotypes. These include squamous cell cancer and plasticity in the regulation of Ca2+-mediated exocytosis in SERCA2 heterozygous mutant mice, modulation of Ca2+ signaling in SERCA3-deficient mice, deafness and balance disorders in PMCA2 null mice, and male infertility in PMCA4 null mice. These unique phenotypes provide new information about the cellular functions of these pumps, the requirement of their activities for higher order physiological processes, and the pathophysiological consequences of pump dysfunction.     

Thermodynamic stability of domain 2 of epithelial cadherin

Cadherin is a cell adhesion molecule that participates in ordered calcium-dependent self-association interactions both between molecules on the same cell surface (cis-interactions) and on neighboring cell surfaces (trans-interactions). Cadherin is a transmembrane protein that has 3-7 independently folded beta-barrel extracellular domains. Both types of self-association interactions are mediated through the most N-terminal domain (Domain 1). Although the structural nature of the trans-interactions is clear, the nature of the cis-interactions is ambiguous despite several high-resolution structural studies. From earlier studies, it is understood that for the trans-interactions to happen, cis-interactions are mandatory. Hence, our first steps are to study the energetic driving forces for the cis-interactions. We have simplified the approach by first examining participating extracellular domains individually. We report here our initial experiments into the stability of Domain 2 of E-cadherin (ECAD2). ECAD2 appears monomeric, according to results from mass spectrometry and sedimentation equilibrium studies. We report denaturation data from differential scanning calorimetric experiments, and temperature and denaturant-induced unfolding experiments monitored by circular dichroism. These studies give a unified picture of the energetics of ECAD2-folding and stability, for which DeltaG degrees is 6.6 kcal/mol, T(m) is 54 degrees C, DeltaH(m) is 90 kcal/mol, and DeltaC(p) is 1300 cal/Kmol. These parameters are independent of calcium up to 5 mM, indicating that ECAD2 does not bind calcium at physiological calcium levels.     

Structure/function studies of phosphoryl transfer by phosphoenolpyruvate carboxykinase

Phosphoenolpyruvate carboxykinase (PCK) catalyzes the conversion of oxaloacetate (OAA) to PEP and carbon dioxide with the subsequent conversion of nucleoside triphosphate to nucleoside diphosphate (NDP). The 1.9 A resolution structure of Escherichia coli PCK consisted of a 275-residue N-terminal domain and a 265-residue C-terminal domain with the active site located in a cleft between these domains. Each domain has an alpha/beta topology and the overall structure represents a new protein fold. Furthermore, PCK has a unique mononucleotide-binding fold. The 1.8 A resolution structure of the complex of ATP/Mg(2+)/oxalate with PCK revealed a 20 degrees hinge-like rotation of the N- and C-terminal domains, which closed the active site cleft. The ATP was found in the unusual syn conformation as a result of binding to the enzyme. Along with the side chain of Lys254, Mg(2+) neutralizes charges on the P beta and P gamma oxygen atoms of ATP and stabilizes an extended, eclipsed conformation of the P beta and P gamma phosphoryl groups. The sterically strained high-energy conformation likely lowers the free energy of activation for phosphoryl transfer. Additionally, the gamma-phosphoryl group becomes oriented in-line with the appropriate enolate oxygen atom, which strongly supports a direct S(N)2-type displacement of this gamma-phosphoryl group by the enolate anion. In the 2.0 A resolution structure of the complex of PCK/ADP/Mg(2+)/AlF(3), the AlF(3) moiety represents the phosphoryl group being transferred during catalysis. There are three positively charged groups that interact with the fluorine atoms, which are complementary to the three negative charges that would occur for an associative transition state.     

Structure of A beta(25-35) peptide in different environments

The fragment A beta(25-35) of the Alzheimer's amyloid beta-peptide, like its full-length peptide A beta(1-42), has shown neurotoxic activities in cultured cells. The conformational preference of this important peptide is examined here in solution, gel, and film states (obtained with organic and aqueous solvents) by vibrational circular dichroism spectroscopy for the first time. For comparative studies, vibrational absorption and electronic circular dichroism measurements were also carried out under identical conditions. The peptide was found to adopt beta-sheet and beta-turn structures, with their relative proportions changing in different environments.     

The conformation of the activation peptide of protein C is influenced by Ca2+ and Na+ binding

Previous studies have suggested that the conformation of the activation peptide of protein C is influenced by the binding of Ca(2+). To provide direct evidence for the linkage between Ca(2+) binding and the conformation of the activation peptide, we have constructed a protein C mutant in the gamma-carboxyglutamic acid-domainless form in which the P1 Arg(169) of the activation peptide is replaced with the fluorescence reporter Trp. Upon binding of Ca(2+), the intrinsic fluorescence of the mutant decreases approximately 30%, as opposed to only 5% for the wild-type, indicating that Trp(169) is directly influenced by the divalent cation. The K(d) of Ca(2+) binding for the mutant protein C was impaired approximately 4-fold compared with wild-type. Interestingly, the conformation of the activation peptide was also found to be sensitive to the binding of Na(+), and the affinity for Na(+) binding increased approximately 5-fold in the presence of Ca(2+). These findings suggest that Ca(2+) changes the conformation of the activation peptide of protein C and that protein C is also capable of binding Na(+), although with a weaker affinity compared with the mature protease. The mutant protein C can no longer be activated by thrombin but remarkably it can be activated efficiently by chymotrypsin and by the thrombin mutant D189S. Activation of the mutant protein C by chymotrypsin proceeds at a rate comparable to the activation of wild-type protein C by the thrombin-thrombomodulin complex.     

BTBD1 and BTBD2 colocalize to cytoplasmic bodies with the RBCC/tripartite motif protein,TRIM5delta

We previously identified BTBD1 and BTBD2 as novel topoisomerase I-interacting proteins that share 80% amino acid identity. Here we report the characterization of their subcellular localization. In a number of mouse and human cells, BTBD1 and BTBD2 (BTBD1/2) colocalized to punctate or elongated cytoplasmic bodies (< 5 microm long and several per cell) that were larger and more elongated in cancer cell lines than in fibroblasts and myoblasts. A search for potential colocalizing proteins identified TRIM family members that localize to morphologically similar cytoplasmic bodies, which were then tested for colocalization with BTBD1/2. TRIM5delta, expressed as a GFP fusion, colocalized with BTBD1/2 immunostaining and appeared to serve as a scaffold for the assembly of endogenous BTBD1/2 proteins. TRIM family members contain a RING domain, B-box(es), and coiled-coil regions, which have a characteristic order and spacing (RBCC domain). RING-dependent ubiquitin ligase activity and multimerization via the coiled-coil region may be defining properties of the RBCC/TRIM protein family. We found that TRIM5delta with a deleted coiled-coil region or a mutated RING domain failed to colocalize with BTBD1/2. Additionally, TRIM5delta ubiquitylated itself in a RING finger- and UbcH5B-dependent manner. BTBD1/2 each contain a PHR-similarity region, repeated twice on the putative ubiquitin ligases PAM, highwire and RPM-1, which also contain a RING and B-box. Thus, four protein modules found on each of these putative ubiquitin ligases, a RING, a B-box and two PHR repeats, are present on BTBD1/2 and TRIM5delta that are colocalized to cytoplasmic bodies.     

Double-stranded RNA interference of a rice PI/GLO paralog, OsMADS2, uncovers its second-whorl-specific function in floral organ patterning

Unlike many eudicot species, grasses have duplicated PI/GLO-like genes. Functional analysis of one of the rice PI/GLO paralogs, OsMADS2, is reported here. Our data demonstrate its essential role in lodicule development and implicate the second PI/GLO paralog, OsMADS4, to suffice for stamen specification. We provide the first evidence for differential contributions of grass PI/GLO paralogs in patterning second- and third-whorl floral organs.     

Cloning and expression of the gene for a novel protein from Mycobacterium smegmatis with functional similarity to eukaryotic calmodulin

A calmodulin-like protein (CAMLP) from Mycobacterium smegmatis was purified to homogeneity and partially sequenced; these data were used to produce a full-length clone, whose DNA sequence contained a 55-amino-acid open reading frame. M. smegmatis CAMLP, expressed in Escherichia coli, exhibited properties characteristic of eukaryotic calmodulin: calcium-dependent stimulation of eukaryotic phosphodiesterase, which was inhibited by the calmodulin antagonist trifluoperazine, and reaction with anti-bovine brain calmodulin antibodies. Consistent with the presence of nine acidic amino acids (16%) in M. smegmatis CAMLP, there is one putative calcium-binding domain in this CAMLP, compared to four such domains for eukaryotic calmodulin, reflecting the smaller molecular size (approximately 6 kDa) of M. smegmatis CAMLP. Ultracentrifugation and mass spectral studies excluded the possibility that calcium promotes oligomerization of purified M. smegmatis CAMLP.     

Food utilization efficiency in fifth instar larvae of Antheraea mylitta (Lepidoptera: Saturniidae) infected with Nosema sp. and its effect on reproductive potential and silk production

Antheraea mylitta, a sericigenous insect of economical importance is often infected with an intracellular parasite of the genus Nosema. This pathogen is known to cause fatal pebrine disease and is considered as an important factor that strongly influences the development of the host. Larvae developed from the eggs laid by a female infected with Nosema sp. showed extended development period. The increment in the larval weight declined significantly in infected larvae in comparison to uninfected ones. Food consumption, digestion, relative consumption rate (RCR), efficiency of conversion of ingested food (ECI), efficiency of conversion of digested food (ECD), and relative growth rate (RGR) values declined significantly, but at the same time a significant increase in approximate digestibility (AD) was also observed. Silk production declined in infected larvae. Silk gland weight and shell weight also significantly declined following infection over uninfected larvae. The reproductive potential in adults declined significantly (P<0.001) with decrease in ovary weight (31.6%), fecundity (54.1%), and fertility (34.9%). Egg chorionation was also affected in adults, which developed from infected larvae. The maternal infection level in one generation (10.4 x 10(6) spores/female) decreased significantly in the next generation (8.0 x 10(6) spores/female).