Structure and assembly of designed beta-hairpin peptides in crystals as models for beta-sheet aggregation

De novo designed beta-hairpin peptides have generally been recalcitrant to crystallization. The crystal structures of four synthetic peptide beta-hairpins, Boc-Leu-Val-Val-DPro-Gly-Leu-Phe-Val-OMe (1), Boc-Leu-Phe-Val-DPro-Ala-Leu-Phe-Val-OMe (2), Boc-Leu-Val-Val-DPro-Aib-Leu-Val-Val-OMe (3), and Boc-Met-Leu-Phe-Val-DPro-Ala-Leu-Val-Val-Phe-OMe (4), are described. The centrally positioned DPro-Xxx segment promotes prime beta-turn formation, thereby nucleating beta-hairpin structures. In all four peptides well-defined beta-hairpins nucleated by central type II' DPro-Xxx beta-turns have been characterized by X-ray diffraction, providing a view of eight crystallographically independent hairpins. In peptides 1-3 three intramolecular cross-strand hydrogen bonds stabilized the observed beta-hairpin, with some fraying of the structures at the termini. In peptide 4, four intramolecular cross-strand hydrogen bonds stabilized the hairpin. Peptides 1-4 reveal common features of packing of beta-hairpins into crystals. Two-dimensional sheet formation mediated by intermolecular hydrogen bonds formed between antiparallel strands of adjacent molecule is a recurrent theme. The packing of two-dimensional sheets into the crystals is mediated in the third dimension by bridging solvents and interactions of projecting side chains, which are oriented on either face of the sheet. In all cases, solvation of the central DPro-Xxx peptide unit beta-turn is observed. The hairpins formed in the octapeptides are significantly buckled as compared to the larger hairpin in peptide 4, which is much flatter. The crystal structures provide insights into the possible modes of beta-sheet packing in regular crystalline arrays, which may provide a starting point for understanding beta-sandwich and cross-beta-structures in amyloid fibrils.     

Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) interacts with PELP1 and activates MAPK

PELP1 (proline-, glutamic acid-, and leucine-rich protein-1) (also known as the modulator of nongenomic activity of estrogen receptor) plays a role in genomic functions of the estrogen receptor via histone interactions and in nongenomic functions via its influence on the MAPK-Src pathway. However, recent studies have shown that differential compartmentalization of PELP1 could play a crucial role in modulating the status of nongenomic signaling by using molecular mechanisms that remain poorly understood. Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) is an early endosomal protein that plays a role in regulating the trafficking of growth factor-receptor complexes through early endosomes. By using a yeast two-hybrid screen, we identified HRS as a novel PELP1-binding protein providing evidence of a physiologic interaction between HRS and PELP1. The noted HRS-PELP1 interaction was accompanied by inhibition of the basal coactivator function of PELP1 upon estrogen receptor transactivation. HRS was found to sequester PELP1 in the cytoplasm, leading to the activation of MAPK in a manner that is dependent on the epidermal growth factor receptor but independent of the estrogen receptor, Shc, and Src. In addition, stimulation of MAPK and the subsequent activation of its downstream effector pathway, Elk-1, by HRS or PELP1 were found to depend on the presence of endogenous PELP1 or HRS. Furthermore, HRS was overexpressed and correlated well with the cytoplasmic PELP1, increased MAPK, and EGFR status in breast tumors. These findings highlight a novel role of HRS in up-regulating MAPK, presumably involving interaction with PELP1.     

2013 William Allan Award: My Multifactorial Journey

Gonadotropin-releasing hormone (GnRH) neurons originate outside the CNS in the olfactory placode and migrate into the CNS, where they become integral components of the hypothalamic-pituitary-gonadal (HPG) axis. Disruption of this migration results in Kallmann syndrome (KS), which is characterized by anosmia and pubertal failure due to hypogonadotropic hypogonadism. Using candidate-gene screening, autozygosity mapping, and whole-exome sequencing in a cohort of 30 individuals with KS, we searched for genes newly associated with KS. We identified homozygous loss-of-function mutations in FEZF1 in two independent consanguineous families each with two affected siblings. The FEZF1 product is known to enable axons of olfactory receptor neurons (ORNs) to penetrate the CNS basal lamina in mice. Because a subset of axons in these tracks is the migratory pathway for GnRH neurons, in FEZF1 deficiency, GnRH neurons also fail to enter the brain. These results indicate that FEZF1 is required for establishment of the central component of the HPG axis in humans.     

Quantifying and modeling birth order effects in autism

Autism is a complex genetic disorder with multiple etiologies whose molecular genetic basis is not fully understood. Although a number of rare mutations and dosage abnormalities are specific to autism, these explain no more than 10% of all cases. The high heritability of autism and low recurrence risk suggests multifactorial inheritance from numerous loci but other factors also intervene to modulate risk. In this study, we examine the effect of birth rank on disease risk which is not expected for purely hereditary genetic models. We analyzed the data from three publicly available autism family collections in the USA for potential birth order effects and studied the statistical properties of three tests to show that adequate power to detect these effects exist. We detect statistically significant, yet varying, patterns of birth order effects across these collections. In multiplex families, we identify V-shaped effects where middle births are at high risk; in simplex families, we demonstrate linear effects where risk increases with each additional birth. Moreover, the birth order effect is gender-dependent in the simplex collection. It is currently unknown whether these patterns arise from ascertainment biases or biological factors. Nevertheless, further investigation of parental age-dependent risks yields patterns similar to those observed and could potentially explain part of the increased risk. A search for genes considering these patterns is likely to increase statistical power and uncover novel molecular etiologies.     

High incidence of deafness from three frequent connexin 26 mutations in an isolated community

In a small village founded by few ancestors, three mutations in GJB2, the gene for connexin 26, are responsible for the high prevalence of deafness. A total of 15% of healthy individuals from a random sample were carriers of either 35Gdel (7.8%), W77R (2.4%), or V37I (4.8%). The three mutations appeared in the village approximately 100-150 years ago. The question of why three distinct mutations of similar age are observed at high frequency within a genetic isolate is discussed.     

Genomic Loss of Tumor Suppressor miRNA-204 Promotes Cancer Cell Migration and Invasion by Activating AKT/mTOR/Rac1 Signaling and Actin Reorganization

Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including brain-derived neurotrophic factor (BDNF), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.     

A common genetic variant in the neurexin superfamily member CNTNAP2 increases familial risk of autism

Autism is a childhood neuropsychiatric disorder that, despite exhibiting high heritability, has largely eluded efforts to identify specific genetic variants underlying its etiology. We performed a two-stage genetic study in which genome-wide linkage and family-based association mapping was followed up by association and replication studies in an independent sample. We identified a common polymorphism in contactin-associated protein-like 2 (CNTNAP2), a member of the neurexin superfamily, that is significantly associated with autism susceptibility. Importantly, the genetic variant displays a parent-of-origin and gender effect recapitulating the inheritance of autism.     

Insights into HLA-restricted T cell responses in a novel mouse model of dengue virus infection point toward new implications for vaccine design

The frequency of dengue virus (DENV) infection has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. To date, a convenient murine system to study human T cell responses to DENV has not been available. Mice transgenic for HLA are widely used to model human immune responses, and it has been shown that mouse-passaged DENV is able to replicate to significant levels in IFN-α/βR(-/-) mice. To cover a wide range of HLA phenotypes, we backcrossed IFN-α/βR(-/-) mice with HLA A*0201, A*0101, A*1101, B*0702, and DRB1*0101-transgenic mice. A DENV proteome-wide screen identified a total of 42 epitopes across all HLA-transgenic IFN-α/βR(-/-) strains tested. In contrast, only eight of these elicited responses in the corresponding IFN-α/βR(+/+) mice. We were able to identify T cell epitopes from 9 out of the 10 DENV proteins. However, the majority of responses were derived from the highly conserved nonstructural proteins NS3 and NS5. The relevance of this model is further demonstrated by the fact that most of the epitopes identified in our murine system are also recognized by PBMC from DENV-exposed human donors, and a dominance of HLA B*0702-restricted responses has been detected in both systems. Our results provide new insights into HLA-restricted T cell responses against DENV, and we describe in this study a novel murine model that allows the investigation of T cell-mediated immune mechanisms relevant to vaccine design.