Protein glycosylation mutants of procyclic Trypanosoma brucei: defects in the asparagine-glycosylation pathway

We employed a genetic approach to study protein glycosylation in the procyclic form of the parasite Trypanosoma brucei. Two different mutant parasites, ConA 1-1 and ConA 4-1, were isolated from mutagenized cultures by selecting cells which resisted killing or agglutination by concanavalin A. Both mutant cells show reduced concanavalin A binding. However, the mutants have different phenotypes, as indicated by the fact that ConA 1-1 binds to wheat germ agglutinin but ConA 4-1 and wild type do not. A blot probed with concanavalin A revealed that many proteins in both mutants lost the ability to bind this lectin, and the blots resembled one of wild type membrane proteins treated with PNGase F. This finding suggested that the mutants had altered asparagine- linked glycosylation. This conclusion was confirmed by studies on a flagellar protein (Fla1) and procyclic acidic repetitive protein (PARP). Structural analysis indicated that the N- glycan of wild type PARP is exclusively Man5GlcNAc2 whereas that in both mutants is predominantly a hybrid type with a terminal N- acetyllactosamine. The occupancy of the PARP glycosylation site in ConA 4-1 was much lower than that in ConA 1-1. These mutants will be useful for studying trypanosome glycosylation mechanisms and function.      

Statistical discovery of site inter-dependencies in sub-molecular hierarchical protein structuring

Background

Much progress has been made in understanding the 3D structure of proteins using methods such as NMR and X-ray crystallography. The resulting 3D structures are extremely informative, but do not always reveal which sites and residues within the structure are of special importance. Recently, there are indications that multiple-residue, sub-domain structural relationships within the larger 3D consensus structure of a protein can be inferred from the analysis of the multiple sequence alignment data of a protein family. These intra-dependent clusters of associated sites are used to indicate hierarchical inter-residue relationships within the 3D structure. To reveal the patterns of associations among individual amino acids or sub-domain components within the structure, we apply a k-modes attribute (aligned site) clustering algorithm to the ubiquitin and transthyretin families in order to discover associations among groups of sites within the multiple sequence alignment. We then observe what these associations imply within the 3D structure of these two protein families.

Results

The k-modes site clustering algorithm we developed maximizes the intra-group interdependencies based on a normalized mutual information measure. The clusters formed correspond to sub-structural components or binding and interface locations. Applying this data-directed method to the ubiquitin and transthyretin protein family multiple sequence alignments as a test bed, we located numerous interesting associations of interdependent sites. These clusters were then arranged into cluster tree diagrams which revealed four structural sub-domains within the single domain structure of ubiquitin and a single large sub-domain within transthyretin associated with the interface among transthyretin monomers. In addition, several clusters of mutually interdependent sites were discovered for each protein family, each of which appear to play an important role in the molecular structure and/or function.

Conclusions

Our results demonstrate that the method we present here using a k- modes site clustering algorithm based on interdependency evaluation among sites obtained from a sequence alignment of homologous proteins can provide significant insights into the complex, hierarchical inter-residue structural relationships within the 3D structure of a protein family.

     

Assessing the Pulsatility of Luteinizing Hormone in Female Vervet Monkeys (Chlorocebus aethiops sabaeus)

Specific alterations in the pulsatility of luteinizing hormone (LH) are linked to obesity-related subfertility in ovulatory women. Vervet monkeys (Chlorocebus aethiops sabaeus) are an Old World nonhuman primate that develops obesity and has a menstrual cycle similar to humans. We evaluated follicular-phase LH pulses in 12 adult normal-weight female vervets. Serum was collected every 10 min for 4 h by using a tether device in conscious, freely moving monkeys on menstrual cycle days 2 through 5. Serum estradiol was collected daily during the follicular phase to identify the luteal–follicular transition. For comparison, we used data from 12 ovulatory normal-weight women who had undergone frequent blood sampling of early-follicular LH. LH pulse frequency was similar, with 2.8 ± 0.7 LH pulses during 4 h in vervets compared with 2.3 ± 0.7 LH pulses during 4 h in women. The LH pulse mass (percentage change in the pulse peak over the preceding nadir) was 123.2% ± 27.4% in vervets and 60.9% ± 14.9% in humans. The first day of low serum estradiol after the follicular-phase peak was denoted as the day of the luteal–follicular transition. Luteectomy was performed on luteal days 7 through 9, and corpora lutea were confirmed by histology. We demonstrate that follicular LH patterns in vervets are similar to those in humans and that the luteal phase is easily identified by monitoring daily serum estradiol. These findings demonstrate that vervet monkeys are a suitable animal model for evaluating LH pulse dynamics longitudinally in studies of diet-induced obesity.Abbreviations: CL, corpus luteum; LH, luteinizing hormoneNonhuman primates have been used in biomedical research for decades and have enabled advancements in many areas, including HIV–AIDS, Alzheimer disease, diabetes, asthma, and endometriosis.²³ Neuroendocrine research in menstruating nonhuman primates, such as rhesus and cynomolgus macaques, have provided valuable information regarding the hypothalamic– pituitary–ovarian axis, including modulating factors of pulsatile gonadotropin-releasing hormone secretion and the negative and positive feedback mechanisms of sex steroids.^20,25,33Normal reproductive physiology in women involves highly coordinated communication between the hypothalamus, pituitary gland, and the end organ of female reproduction, the ovary. These processes are governed by the magnitude and frequency of secretory outbursts (pulses) of gonadotropin-releasing hormone from the hypothalamus. The activity of gonadotropin-releasing hormone results in a pulsatile mode of secretion of follicle-stimulating hormone and luteinizing hormone (LH) from the anterior pituitary. In females, follicle-stimulating hormone drives ovarian follicle growth during the follicular phase of the menstrual cycle. The midcycle LH surge results in ovulation and the subsequent formation of a corpus luteum (CL). Secretion of estradiol, produced by the developing follicles, progressively increases over the course of the follicular (proliferative) phase of the menstrual cycle and peaks prior to ovulation. Progesterone, secreted by the CL, is the dominant sex steroid during the luteal (secretory) phase.¹² Both estradiol and progesterone exert tightly regulated negative feedback on the hypothalamus and pituitary and affect gonadotropin release. Alterations in this intricate system can result in anovulation or infertility.Obesity is a growing worldwide hazard that has many adverse health outcomes, including subfertility. Endocrine alterations associated with obesity include relative hypogonadotropic hypogonadism^29,34 and selective impairment of LH pulse amplitude.¹⁴ Progesterone metabolite excretion in morbidly obese women is reduced by 70% compared with that in normal-weight women,²⁹ and pulsatile LH amplitude is suppressed by half in frequent blood-sampling studies.¹⁴ However, despite the recent advances in understanding the endocrine pathophysiology of obesity-related subfertility,¹⁵ its molecular mechanisms are poorly understood.Animal models for obesity-related subfertility are needed for mechanistic studies but are currently unavailable. The hormonal control of the menstrual cycle has been extensively studied in rhesus and cynomolgus macaques and is similar to that of humans.^12,22,26 These nonhuman primates have also been shown to develop obesity and resultant metabolic disturbances.¹ However, demand for rhesus and cynomolgus macaques is high, and the NIH has espoused the need to identify other species of nonhuman primate that are suitable for research.⁶Vervet monkeys (Chlorocebus aethiops sabaeus) are a small, Old World nonhuman primate with an ovarian cycle similar to that in humans; therefore vervets may be an appropriate alternative species in which to do neuroendocrine research. LH pulsatility in this species has not been assessed comprehensively. Our objective in the current study was to characterize the follicular LH pulse pattern in vervet monkeys, to establish the feasibility of using this model in future studies to assess the effect of body mass on pituitary function.     

Sequential activation of inflammatory signaling pathways during graft-versus-host disease (GVHD): early role for STAT1 and STAT3

The aim of this study was to delineate the temporal and spatial sequence of STAT1 and STAT3 activation during development of GVHD following fully Major Histocompatibility Complex (MHC)-mismatched allogeneic Bone Marrow Transplantation (BMT). Activation of inflammatory signaling pathways in GVHD target organs was assessed by western blotting, phospho-flow cytometry and electromobility shift assays (EMSA). Development of GVHD was associated with significant expansion of phospho[p]-STAT1 and p-STAT3 expressing CD4⁺ T cells and CD8⁺ T cells. GVHD-specific STAT3/STAT1 activation preceded activation of Nuclear Factor-κB (NF-κB) and Mitogen Activated Protein Kinase (MAPK) and was associated with subsequent induction of STAT1 or STAT3-dependent inflammatory gene-expression programs (e.g. expression of IRF-1, SOCS1, IL-17). Our studies may help to establish a functional hierarchy of the signaling events leading to the development of GVHD and could be helpful in designing new molecularly targeted treatment approaches for GVHD.     

Intra-tumor genetic heterogeneity and alternative driver genetic alterations in breast cancers with heterogeneous HER2 gene amplification

BackgroundHER2 is overexpressed and amplified in approximately 15% of invasive breast cancers, and is the molecular target and predictive marker of response to anti-HER2 agents. In a subset of these cases, heterogeneous distribution of HER2 gene amplification can be found, which creates clinically challenging scenarios. Currently, breast cancers with HER2 amplification/overexpression in just over 10% of cancer cells are considered HER2-positive for clinical purposes; however, it is unclear as to whether the HER2-negative components of such tumors would be driven by distinct genetic alterations. Here we sought to characterize the pathologic and genetic features of the HER2-positive and HER2-negative components of breast cancers with heterogeneous HER2 gene amplification and to define the repertoire of potential driver genetic alterations in the HER2-negative components of these cases.ResultsWe separately analyzed the HER2-negative and HER2-positive components of 12 HER2 heterogeneous breast cancers using gene copy number profiling and massively parallel sequencing, and identified potential driver genetic alterations restricted to the HER2-negative cells in each case. In vitro experiments provided functional evidence to suggest that BRF2 and DSN1 overexpression/amplification, and the HER2 I767M mutation may be alterations that compensate for the lack of HER2 amplification in the HER2-negative components of HER2 heterogeneous breast cancers.ConclusionsOur results indicate that even driver genetic alterations, such as HER2 gene amplification, can be heterogeneously distributed within a cancer, and that the HER2-negative components are likely driven by genetic alterations not present in the HER2-positive components, including BRF2 and DSN1 amplification and HER2 somatic mutations.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0657-6) contains supplementary material, which is available to authorized users.     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]     

Integrating spatially resolved three-dimensional MALDI IMS with in vivo magnetic resonance imaging

We have developed a method for integrating three dimensional-volume reconstructions of spatially resolved matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) ion images of whole mouse heads with high-resolution images from other modalities in an animal-specific manner. This approach enabled us to analyze proteomic profiles from MALDI IMS data with corresponding in vivo data provided by magnetic resonance imaging.