A Structural Model of the Pore-Forming Region of the Skeletal Muscle Ryanodine Receptor (RyR1)

Ryanodine receptors (RyRs) are ion channels that regulate muscle contraction by releasing calcium ions from intracellular stores into the cytoplasm. Mutations in skeletal muscle RyR (RyR1) give rise to congenital diseases such as central core disease. The absence of high-resolution structures of RyR1 has limited our understanding of channel function and disease mechanisms at the molecular level. Here, we report a structural model of the pore-forming region of RyR1. Molecular dynamics simulations show high ion binding to putative pore residues D4899, E4900, D4938, and D4945, which are experimentally known to be critical for channel conductance and selectivity. We also observe preferential localization of Ca²⁺ over K⁺ in the selectivity filter of RyR1. Simulations of RyR1-D4899Q mutant show a loss of preference to Ca²⁺ in the selectivity filter as seen experimentally. Electrophysiological experiments on a central core disease mutant, RyR1-G4898R, show constitutively open channels that conduct K⁺ but not Ca²⁺. Our simulations with G4898R likewise show a decrease in the preference of Ca²⁺ over K⁺ in the selectivity filter. Together, the computational and experimental results shed light on ion conductance and selectivity of RyR1 at an atomistic level.     

Role of cadherin-17 in oncogenesis and potential therapeutic implications in hepatocellular carcinoma

Cadherin is an important cell adhesion molecule that plays paramount roles in organ development and the maintenance of tissue integrity. Dysregulation of cadherin expression is often associated with disease pathology including tissue dysplasia, tumor formation, and metastasis. Cadherin-17 (CDH17), belonging to a subclass of 7D-cadherin superfamily, is present in fetal liver and gastrointestinal tract during embryogenesis, but the gene becomes silenced in healthy adult liver and stomach tissues. It functions as a peptide transporter and a cell adhesion molecule to maintain tissue integrity in epithelia. However, recent findings from our group and others have reported aberrant expression of CDH17 in major gastrointestinal malignancies including hepatocellular carcinoma (HCC), stomach and colorectal cancers, and its clinical association with tumor metastasis and advanced tumor stages. Furthermore, alternative splice isoforms and genetic polymorphisms of CDH17 gene have been identified in HCC and linked to an increased risk of HCC. CDH17 is an attractive target for HCC therapy. Targeting CDH17 in HCC can inhibit tumor growth and inactivate Wnt signaling pathway in concomitance with activation of tumor suppressor genes. Further investigation on CDH17-mediated oncogenic signaling and cognate molecular mechanisms would shed light on new targeting therapy on HCC and potentially other gastrointestinal malignancies.     

An integrated approach to uncover drivers of cancer

Systematic characterization of cancer genomes has revealed a staggering number of diverse aberrations that differ among individuals, such that the functional importance and physiological impact of most tumor genetic alterations remain poorly defined. We developed a computational framework that integrates chromosomal copy number and gene expression data for detecting aberrations that promote cancer progression. We demonstrate the utility of this framework using a melanoma data set. Our analysis correctly identified known drivers of melanoma and predicted multiple tumor dependencies. Two dependencies, TBC1D16 and RAB27A, confirmed empirically, suggest that abnormal regulation of protein trafficking contributes to proliferation in melanoma. Together, these results demonstrate the ability of integrative Bayesian approaches to identify candidate drivers with biological, and possibly therapeutic, importance in cancer.     

Realized Fungal Diversity Increases Functional Stability of Leaf Litter Decomposition Under Zinc Stress

Freshwaters include some of the most impaired systems on Earth with high rates of species loss, underscoring the significance of investigating whether ecosystems with fewer species will be able to maintain ecological processes. The environmental context is expected to modulate the effects of declining diversity. We conducted microcosm experiments manipulating fungal inoculum diversity and zinc concentration to test the hypothesis that fungal diversity determines the susceptibility of leaf litter decomposition to Zn stress. Realized fungal diversity was estimated by counting released spores and by measuring species-specific biomasses via denaturing gradient gel electrophoresis. In the absence of Zn, positive diversity effects were found for leaf mass loss and fungal biomass through complementary interactions and due to the presence of key species. The variability of leaf decomposition decreased with increasing species number (portfolio effect), particularly under Zn stress. Results suggest that the effect of species loss on ecosystem stability may be exacerbated at higher stress levels.     

Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors

The proinflammatory cytokine tumor necrosis factor (TNF) binds two distinct plasma membrane receptors, TNFR1 and TNFR2. We have produced different receptor mutants fused with enhanced green fluorescent protein to study their membrane dynamics by fluorescence correlation spectroscopy (FCS). TNFR1 mutants show diffusion constants of approximately 1.2 × 10^− 9 cm²/s and a broad distribution of diffusion times, which is hardly affected by ligand binding. However, cholesterol depletion enhances their diffusion, suggesting a constitutive affinity to cholesterol rich membrane microdomains. In contrast, TNFR2 and mutants thereof diffuse rather fast (D? = 3.1 × 10^− 9 cm²/s) with a marked reduction after 30 min of TNF treatment (D? = 0.9 × 10^− 9 cm²/s). This reduction cannot be explained by the formation of higher ordered receptor clusters, since the fluorescence intensity of TNF treated receptors indicate the presence of a few receptor molecules per complex only. Together, these data point to a topological segregation of the two TNF receptors in different microcompartments of the plasma membrane independent of the cytoplasmic signaling domains of the receptors.     

Modeling membrane shaping by proteins: Focus on EHD2 and N-BAR domains

Cellular membranes are highly dynamic, undergoing both persistent and dynamic shape changes driven by specialized proteins. The observed membrane shaping can be simple deformations of existing shapes or membrane remodeling involving fission or fusion. Here we describe several mechanistic principles by which membrane shaping proteins act. We especially consider models for membrane bending and fission by EHD2 proteins and membrane bending by N-BAR domains. There are major challenges ahead to understand the general principles by which diverse membrane bending proteins act and to understand how some proteins appear to span multiple modes of action from driving curvature to inducing membrane remodeling.     

Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol

The formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient samples and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but causalities remain unclear. We used Saccharomyces cerevisiae to analyze how mitochondrial processes regulate the behavior of aggregation‐prone polyQ protein derived from human huntingtin. Expression of Q97‐GFP rapidly led to insoluble cytosolic aggregates and cell death. Although aggregation impaired mitochondrial respiration only slightly, it considerably interfered with the import of mitochondrial precursor proteins. Mutants in the import component Mia40 were hypersensitive to Q97‐GFP, whereas Mia40 overexpression strongly suppressed the formation of toxic Q97‐GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the post‐translational import of mitochondrial precursor proteins into mitochondria competes with aggregation‐prone cytosolic proteins for chaperones and proteasome capacity. Mia40 regulates this competition as it has a rate‐limiting role in mitochondrial protein import. Therefore, Mia40 is a dynamic regulator in mitochondrial biogenesis that can be exploited to stabilize cytosolic proteostasis.