The T99K variant of glycosylasparaginase shows a new structural mechanism of the genetic disease aspartylglucosaminuria

Aspartylglucosaminuria (AGU) is an inherited disease caused by mutations in a lysosomal amidase called aspartylglucosaminidase (AGA) or glycosylasparaginase (GA). This disorder results in an accumulation of glycoasparagines in the lysosomes of virtually all cell types, with severe clinical symptoms affecting the central nervous system, skeletal abnormalities, and connective tissue lesions. GA is synthesized as a single‐chain precursor that requires an intramolecular autoprocessing to form a mature amidase. Previously, we showed that a Canadian AGU mutation disrupts this obligatory intramolecular autoprocessing with the enzyme trapped as an inactive precursor. Here, we report biochemical and structural characterization of a model enzyme corresponding to a new American AGU allele, the T99K variant. Unlike other variants with known 3D structures, this T99K model enzyme still has autoprocessing capacity to generate a mature form. However, its amidase activity to digest glycoasparagines remains low, consistent with its association with AGU. We have determined a 1.5‐Å‐resolution structure of this new AGU model enzyme and built an enzyme–substrate complex to provide a structural basis to analyze the negative effects of the T99K point mutation on K_M and k_cat of the amidase. It appears that a “molecular clamp” capable of fixing local disorders at the dimer interface might be able to rescue the deficiency of this new AGU variant.     

Comparative Analysis of Transmembrane Regulators of the Filamentous Growth Mitogen-Activated Protein Kinase Pathway Uncovers Functional and Regulatory Differences

Filamentous growth is a microbial differentiation response that involves the concerted action of multiple signaling pathways. In budding yeast, one pathway that regulates filamentous growth is a Cdc42p-dependent mitogen-activated protein kinase (MAPK) pathway. Several transmembrane (TM) proteins regulate the filamentous growth pathway, including the signaling mucin Msb2p, the tetraspan osmosensor Sho1p, and an adaptor Opy2p. The TM proteins were compared to identify common and unique features. Msb2p, Sho1p, and Opy2p associated by coimmunoprecipitation analysis but showed predominantly different localization patterns. The different localization patterns of the proteins resulted in part from different rates of turnover from the plasma membrane (PM). In particular, Msb2p (and Opy2p) were turned over rapidly compared to Sho1p. Msb2p signaled from the PM, and its turnover was a rate-limiting step in MAPK signaling. Genetic analysis identified unique phenotypes of cells overexpressing the TM proteins. Therefore, each TM regulator of the filamentous growth pathway has its own regulatory pattern and specific function in regulating filamentous growth. This specialization may be important for fine-tuning and potentially diversifying the filamentation response.     

Carbohydrate-induced modulation of cell membrane. VIII. Agglutination with mammalian lectin galectin-1 increases osmofragility and membrane fluidity of trypsinized erythrocytes

Interaction of lectins with cell surface determinants may alter membrane properties. Using trypsinized rabbit erythrocytes as model we tested the capacity of an endogenous lectin in this respect. Galectin-1 is a member of an adhesion/growth-regulatory family known to interact for example with ganglioside GM(1) and also the hydrophobic tail of oncogenic H-Ras. Assays on membrane fluidity and osmofragility detect galectin-1's capacity to increase the parameters. Moreover, it increases susceptibility of erythrocytes to radical damage. These observations indicate the potential of this endogenous lectin to affect membrane properties beyond the immediate interaction with cell surface epitopes.     

Simulated tempering yields insight into the low-resolution Rosetta scoring functions

Rosetta is a structure prediction package that has been employed successfully in numerous protein design and other applications.1 Previous reports have attributed the current limitations of the Rosetta de novo structure prediction algorithm to inadequate sampling, particularly during the low-resolution phase.2-5 Here, we implement the Simulated Tempering (ST) sampling algorithm67 in Rosetta to address this issue. ST is intended to yield canonical sampling by inducing a random walk in temperatures space such that broad sampling is achieved at high temperatures and detailed exploration of local free energy minima is achieved at low temperatures. ST should therefore visit basins in accordance with their free energies rather than their energies and achieve more global sampling than the localized scheme currently implemented in Rosetta. However, we find that ST does not improve structure prediction with Rosetta. To understand why, we carried out a detailed analysis of the low-resolution scoring functions and find that they do not provide a strong bias towards the native state. In addition, we find that both ST and standard Rosetta runs started from the native state are biased away from the native state. Although the low-resolution scoring functions could be improved, we propose that working entirely at full-atom resolution is now possible and may be a better option due to superior native-state discrimination at full-atom resolution. Such an approach will require more attention to the kinetics of convergence, however, as functions capable of native state discrimination are not necessarily capable of rapidly guiding non-native conformations to the native state.     

Assessment and comparison of AFLP and SSR based molecular genetic diversity in Indian isolates of Ascochyta rabiei, a causal agent of Ascochyta blight in chickpea (Cicer arietinum L.)

Ascochyta blight (AB), caused by Ascochyta rabiei (Pass.) Labr. (anamorph), is the most damaging disease of chickpea (Cicer arietinum L.) and is a serious biotic stress constraint for chickpea production. To understand the molecular diversity in A. rabiei populations of India, a total of 64 isolates collected from AB-infected chickpea plants from different agroclimatic regions in the North Western Plain Zone (NWPZ) of India were analyzed with 11 AFLP (amplified fragment length polymorphism) and 20 SSR (simple sequence repeat) markers. A total of 9 polymorphic AFLP primer pairs provided a total of 317 fragments, of which 130 were polymorphic and showed an average PIC value 0.28. Of the SSR markers, 12 showed polymorphism and provided a total of 29 alleles with an average PIC value 0.35. To the best of our knowledge, this is the first report on a comparison of AFLP and SSR diversity estimates in A. rabiei populations. The dendrogram developed based on AFLP and SSR data separately, as well as on the combined marker dataset, grouped the majority of AB isolates as per geographic regions. Model based population structure analysis revealed four distinct populations with varying levels of ancestral admixtures among 64 isolates studied. Interestingly, several AFLP primer combinations and SSR markers showed the locus/allele specific to AB isolates of certain regions, e.g., Hisar, Sriganganagar, Gurdaspur, and Sundarnagar. Genetic variability present in AB isolates of the NWPZ of India suggests the continuous monitoring of changes in A. rabiei population to anticipate the breakdown of AB resistance in chickpea cultivars grown in India.     

Interspecific interactions of the critically endangered Forest Owlet (Athene blewitti)

The Forest Owlet (Athene blewitti) is critically endangered and at extremely high risk of extinction owing to its restricted distribution. An expedition was organized to determine the density of the Forest Owlet in the Melghat Tiger Reserve in February 2004 where they had been observed sporadically in the previous 5 years. We hoped to identify as many individuals as possible and to observe interspecific interactions in order to understand the social framework in which the species survives. A total of 43.4 km of jungle roads was checked; we confirmed the presence of three of the 13 previously reported individuals, and found 11 previously undetected owlets. Owlets were found in areas with several interconnected forest clearings which allowed the owlets to forage in them. In all cases where the Forest Owlet occurred, a village or agricultural fields of the indigenous people (Adivasis) was within a 0.5-km radius. It appears that Forest Owlets preferred to establish feeding territories in areas disturbed by anthropogenic activity such as clearing dead trees and undergrowth for fire, trampling undergrowth while searching for firewood, burning areas around the agricultural plots, or driving herds of cattle through the area. All of these activities appear to optimize the habitat for the sit-and-wait foraging Forest Owlet, facilitating detection and tracking of prey in open areas with sparse and short undergrowth, allowing a better all-round view due to a lower density of trees.     

Plasma membrane calcium pump activity in rat pancreatic islets

The aim of this study was to quantify the glucose modulation of the plasma membrane calcium pump (PMCA) function in rat pancreatic islets. Ca²⁺-ATPase activity and levels of phosphorylated PMCA intermediates both transiently declined to a minimum in response to stimulation by glucose. Strictly dependent on Ca²⁺ concentration, this inhibitory effect was fully expressed at physiological concentrations of the cation (less than 0.5 μM), then progressively diminished at higher concentrations. These results, together with those previously reported on the effects of insulin secretagogues and blockers on the activity, expression and cellular distribution of the PMCA, support the concept that the PMCA plays a key role in the regulation of Ca²⁺ signaling and insulin secretion in pancreatic islets.     

Agrobacterium-mediated Transfer of Arginine Decarboxylase and Ornithine Decarboxylase Genes to Datura innoxia Enhances Shoot Regeneration and Hyoscyamine Biosynthesis

The genes (adc and odc) for two enzymes, arginine decarboxylase and ornithine decarboxylase involved in polyamine biosynthesis, were introduced into anther-derived calli of Datura innoxia through Agrobacterium tumefaciens. The transformed calli exhibited increased regeneration frequency as compared to control. Transgenic lines showed higher polyamine levels, mainly in the putrescine titre, and such lines also yielded a high level of the alkaloid, hyoscyamine. The results suggest that polyamines can modulate in vitro morphogenesis and polyamine biosynthetic pathway can be exploited for enhancement of polyamine-derived alkaloids of pharmaceutical importance.     

Biochemical and hormonal composition of follicular cysts in water buffalo (Bubalus bubalis)

The objective of this study was to examine the follicular fluid biochemical and hormonal changes associated with ovarian follicular cysts in buffalo. Follicular fluid was aspirated from eight cysts and eight preovulatory follicles, and subjected to biochemical and hormonal analyses. Cysts were characterized by a greater (P<0.01) concentration of nitric oxide and lesser concentrations of ascorbic acid and glucose than that of preovulatory follicles (P<0.01 and P<0.05, respectively). Furthermore, follicular cysts had greater concentrations of progesterone (P<0.001), triiodothyronine (T(3)) and cortisol (P<0.05) and lesser concentrations of insulin (P<0.001) than preovulatory follicles. The results indicate follicular cysts in buffalo have an altered biochemical and hormonal composition. The alterations include increases in nitric oxide, progesterone, cortisol and T(3) concentrations with a concurrent reduction in ascorbic acid, insulin and glucose concentrations. The study suggests that greater progesterone concentrations possibly inhibit the onset of LH surge resulting in formation of follicular cysts in buffalo. In addition, it implies the plausible role of intra-ovarian regulators such as nitric oxide, ascorbic acid and insulin in development of the condition.