The Connecdenn DENN domain: a GEF for Rab35 mediating cargo-specific exit from early endosomes.

The DENN domain is an evolutionarily ancient protein module. Mutations in the DENN domain cause developmental defects in plants and human diseases, yet the function of this common module is unknown. We now demonstrate that the connecdenn/DENND1A DENN domain functions as a guanine nucleotide exchange factor (GEF) for Rab35 to regulate endosomal membrane trafficking. Loss of Rab35 activity causes an enlargement of early endosomes and inhibits MHC class I recycling. Moreover, it prevents early endosomal recruitment of EHD1, a common component of tubules involved in endosomal cargo recycling. Our data reveal an enzymatic activity for a DENN domain and demonstrate that distinct Rab GTPases can recruit a common protein machinery to various sites within the endosomal network to establish cargo-selective recycling pathways.

Interplay between Rab35 and Arf6 controls cargo recycling to coordinate cell adhesion and migration.

Cells inversely adjust the plasma membrane levels of integrins and cadherins during cell migration and cell-cell adhesion but the regulatory mechanisms that coordinate these trafficking events remain unknown. Here, we demonstrate that the small GTPase Rab35 maintains cadherins at the cell surface to promote cell-cell adhesion. Simultaneously, Rab35 supresses the activity of the GTPase Arf6 to downregulate an Arf6-dependent recycling pathway for ß1-integrin and EGF receptors, resulting in inhibition of cell migration and attenuation of signaling downstream of these receptors. Importantly, the phenotypes of decreased cell adhesion and increased cell migration observed following Rab35 knock down are consistent with the epithelial-mesenchymal transition, a feature of invasive cancer cells, and we show that Rab35 expression is suppressed in a subset of cancers characterized by Arf6 hyperactivity. Our data thus identify a key molecular mechanism that efficiently coordinates the inverse intracellular sorting and cell surface levels of cadherin and integrin receptors for cell migration and differentiation.

Phenome-wide association study identifies new clinical phenotypes associated with Staphylococcus aureus infections.

BACKGROUND: Phenome-Wide Association study (PheWAS) is a powerful tool designed to systematically screen clinical observations derived from medical records (phenotypes) for association with a variable of interest. Despite their usefulness, no systematic screening of phenotypes associated with Staphylococcus aureus infections (SAIs) has been done leaving potential novel risk factors or complications undiscovered. METHOD AND COHORTS: We tailored the PheWAS approach into a two-stage screening procedure to identify novel phenotypes correlating with SAIs. The first stage screened for co-occurrence of SAIs with other phenotypes within medical records. In the second stage, significant findings were examined for the correlations between their age of onset with that of SAIs. The PheWAS was implemented using the medical records of 754,401 patients from the Marshfield Clinic Health System. Any novel associations discovered were subsequently validated using datasets from TriNetX and All of Us, encompassing 109,884,571 and 118,538 patients respectively. RESULTS: Forty-one phenotypes met the significance criteria of a p-value < 3.64e-5 and odds ratios of > 5. Out of these, we classified 23 associations either as risk factors or as complications of SAIs. Three novel associations were discovered and classified either as a risk (long-term use of aspirin) or complications (iron deficiency anemia and anemia of chronic disease). All novel associations were replicated in the TriNetX cohort. In the All of Us cohort, anemia of chronic disease was replicated according to our significance criteria. CONCLUSIONS: The PheWAS of SAIs expands our understanding of SAIs interacting phenotypes. Additionally, the novel two-stage PheWAS approach developed in this study can be applied to examine other disease-disease interactions of interest. Due to the possibility of bias inherent in observational data, the findings of this study require further investigation.

An evolutionary learning-based method for identifying a circulating miRNA signature for breast cancer diagnosis prediction.

Breast cancer (BC) is one of the most commonly diagnosed cancers worldwide. As key regulatory molecules in several biological processes, microRNAs (miRNAs) are potential biomarkers for cancer. Understanding the miRNA markers that can detect BC may improve survival rates and develop new targeted therapeutic strategies. To identify a circulating miRNA signature for diagnostic prediction in patients with BC, we developed an evolutionary learning-based method called BSig. BSig established a compact set of miRNAs as potential markers from 1280 patients with BC and 2686 healthy controls retrieved from the serum miRNA expression profiles for the diagnostic prediction. BSig demonstrated outstanding prediction performance, with an independent test accuracy and area under the receiver operating characteristic curve were 99.90% and 0.99, respectively. We identified 12 miRNAs, including hsa-miR-3185, hsa-miR-3648, hsa-miR-4530, hsa-miR-4763-5p, hsa-miR-5100, hsa-miR-5698, hsa-miR-6124, hsa-miR-6768-5p, hsa-miR-6800-5p, hsa-miR-6807-5p, hsa-miR-642a-3p, and hsa-miR-6836-3p, which significantly contributed towards diagnostic prediction in BC. Moreover, through bioinformatics analysis, this study identified 65 miRNA-target genes specific to BC cell lines. A comprehensive gene-set enrichment analysis was also performed to understand the underlying mechanisms of these target genes. BSig, a tool capable of BC detection and facilitating therapeutic selection, is publicly available at https://github.com/mingjutsai/BSig.

Genetic and clinical determinants of telomere length.

Many epidemiologic studies have identified important relationships between leukocyte telomere length (LTL) with genetics and health. Most of these studies have been significantly limited in scope by focusing predominantly on individual diseases or restricted to GWAS analysis. Using two large patient populations derived from Vanderbilt University and Marshfield Clinic biobanks linked to genomic and phenomic data from medical records, we investigated the inter-relationship between LTL, genomics, and human health. Our GWAS confirmed 11 genetic loci previously associated with LTL and two novel loci in SCNN1D and PITPNM1. PheWAS of LTL identified 67 distinct clinical phenotypes associated with both short and long LTL. We demonstrated that several diseases associated with LTL were related to one another but were largely independent from LTL genetics. Age of death was correlated with LTL independent of age. Those with very short LTL (<-1.5 standard deviation [SD]) died 10.4 years (p < 0.0001) younger than those with average LTL (±0.5 SD; mean age of death = 74.2 years). Likewise, those with very long LTL (>1.5 SD) died 1.9 years (p = 0.0175) younger than those with average LTL. This is consistent with the PheWAS results showing diseases associating with both short and long LTL. Finally, we estimated that the genome (12.8%) and age (8.5%) explain the largest proportion of LTL variance, whereas the phenome (1.5%) and sex (0.9%) explained a smaller fraction. In total, 23.7% of LTL variance was explained. These observations provide the rationale for expanded research to understand the multifaceted correlations between TL biology and human health over time, leading to effective LTL usage in medical applications.

Survival estimation in patients with stomach and esophageal carcinoma using miRNA expression profiles.

Identifying a miRNA signature associated with survival will open a new window for developing miRNA-targeted treatment strategies in stomach and esophageal cancers (STEC). Here, using data from The Cancer Genome Atlas on 516 patients with STEC, we developed a Genetic Algorithm-based Survival Estimation method, GASE, to identify a miRNA signature that could estimate survival in patients with STEC. GASE identified 27 miRNAs as a survival miRNA signature and estimated the survival time with a mean squared correlation coefficient of 0.80 ± 0.01 and a mean absolute error of 0.44 ± 0.25 years between actual and estimated survival times, and showed a good estimation capability on an independent test cohort. The miRNAs of the signature were prioritized and analyzed to explore their roles in STEC. The diagnostic ability of the identified miRNA signature was analyzed, and identified some critical miRNAs in STEC. Further, miRNA-gene target enrichment analysis revealed the involvement of these miRNAs in various pathways, including the somatotrophic axis in mammals that involves the growth hormone and transforming growth factor beta signaling pathways, and gene ontology annotations. The identified miRNA signature provides evidence for survival-related miRNAs and their involvement in STEC, which would aid in developing miRNA-target based therapeutics.

Casein Kinase 1δ Stabilizes Mature Axons by Inhibiting Transcription Termination of Ankyrin.

After axon outgrowth and synapse formation, the nervous system transitions to a stable architecture. In C. elegans, this transition is marked by the appearance of casein kinase 1δ (CK1δ) in the nucleus. In CK1δ mutants, neurons continue to sprout growth cones into adulthood, leading to a highly ramified nervous system. Nervous system architecture in these mutants is completely restored by suppressor mutations in ten genes involved in transcription termination. CK1δ prevents termination by phosphorylating and inhibiting SSUP-72. SSUP-72 would normally remodel the C-terminal domain of RNA polymerase in anticipation of termination. The antitermination activity of CK1δ establishes the mature state of a neuron by promoting the expression of the long isoform of a single gene, the cytoskeleton protein Ankyrin.

Connecdenn, a novel DENN domain-containing protein of neuronal clathrin-coated vesicles functioning in synaptic vesicle endocytosis.

Clathrin-coated vesicles (CCVs) are responsible for the endocytosis of multiple cargo, including synaptic vesicle membranes. We now describe a new CCV protein, termed connecdenn, that contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a poorly characterized protein module found in multiple proteins of unrelated function and a C-terminal peptide motif domain harboring three distinct motifs for binding the alpha-ear of the clathrin adaptor protein 2 (AP-2). Connecdenn coimmunoprecipitates and partially colocalizes with AP-2, and nuclear magnetic resonance and peptide competition studies reveal that all three alpha-ear-binding motifs contribute to AP-2 interactions. In addition, connecdenn contains multiple Src homology 3 (SH3) domain-binding motifs and coimmunoprecipitates with the synaptic SH3 domain proteins intersectin and endophilin A1. Interestingly, connecdenn is enriched on neuronal CCVs and is present in the presynaptic compartment of neurons. Moreover, connecdenn has a uniquely stable association with CCV membranes because it resists extraction with Tris and high-salt buffers, unlike most other CCV proteins, but it is not detected on purified synaptic vesicles. Together, these observations suggest that connecdenn functions on the endocytic limb of the synaptic vesicle cycle. Accordingly, disruption of connecdenn interactions with its binding partners through overexpression of the C-terminal peptide motif domain or knock down of connecdenn through lentiviral delivery of small hairpin RNA both lead to defects in synaptic vesicle endocytosis in cultured hippocampal neurons. Thus, we identified connecdenn as a component of the endocytic machinery functioning in synaptic vesicle endocytosis, providing the first evidence of a role for a DENN domain-containing protein in endocytosis.

Analysis of connecdenn 1-3 (DENN1A-C) GEF activity for Rab35.

Rabs (Ras-related proteins in brain) form the largest family of small GTPases and control numerous aspects of membrane trafficking at multiple cellular sites. Rab GTPases toggle between an inactive GDP-bound state and an active GTP-bound state. Activation of Rab GTPases requires guanine nucleotide exchange factors (GEFs) that interact with inactive GDP-bound Rabs and catalyze the removal of GDP, allowing GTP to bind. The largest single family of GEFs for Rabs is comprised of proteins bearing a DENN (differentially expressed in normal and neoplastic cells) domain. In this chapter we describe a biochemical method that directly measures the exchange activity of DENN domains by monitoring loading of GTP onto a Rab GTPase. Rabs are first purified from bacterial or mammalian sources and are then loaded with GDP. Purified DENN domains or DENN domain-bearing proteins are added in the presence of [(35)S]GTPγS and the transfer of [(35)S]GTPγS to the Rab is measured by filtering the reaction over nitrocellulose membranes to trap the Rab and thus the associated [(35)S]GTPγS.

The NECAP PHear domain increases clathrin accessory protein binding potential.

AP-2 is a key regulator of the endocytic protein machinery driving clathrin-coated vesicle (CCV) formation. One critical function, mediated primarily by the AP-2 alpha-ear, is the recruitment of accessory proteins. NECAPs are alpha-ear-binding proteins that enrich on CCVs. Here, we have solved the structure of the conserved N-terminal region of NECAP 1, revealing a unique module in the pleckstrin homology (PH) domain superfamily, which we named the PHear domain. The PHear domain binds accessory proteins bearing FxDxF motifs, which were previously thought to bind exclusively to the AP-2 alpha-ear. Structural analysis of the PHear domain reveals the molecular surface for FxDxF motif binding, which was confirmed by site-directed mutagenesis. The reciprocal analysis of the FxDxF motif in amphiphysin I identified distinct binding requirements for binding to the alpha-ear and PHear domain. We show that NECAP knockdown compromises transferrin uptake and establish a functional role for NECAPs in clathrin-mediated endocytosis. Our data uncover a striking convergence of two evolutionarily and structurally distinct modules to recognize a common peptide motif and promote efficient endocytosis.

Non-stoichiometric relationship between clathrin heavy and light chains revealed by quantitative comparative proteomics of clathrin-coated vesicles from brain and liver.

We used tandem mass spectrometry with peptide counts to identify and to determine the relative levels of expression of abundant protein components of highly enriched clathrin-coated vesicles (CCVs) from rat liver. The stoichiometry of stable protein complexes including clathrin heavy chain and clathrin light chain dimers and adaptor protein (AP) heterotetramers was assessed. We detected a deficit of clathrin light chain compared with clathrin heavy chain in non-brain tissues, suggesting a level of regulation of clathrin cage formation specific to brain. The high ratio of AP-1 to AP-2 in liver CCVs is reversed compared with brain where there is more AP-2 than AP-1. Despite this, general endocytic cargo proteins were readily detected in liver but not in brain CCVs, consistent with the previous demonstration that a major function for brain CCVs is recycling synaptic vesicles. Finally we identified 21 CCV-associated proteins in liver not yet characterized in mammals. Our results further validate the peptide accounting approach, reveal new information on the properties of CCVs, and allow for the use of quantitative proteomics to compare abundant components of organelles under different experimental and pathological conditions.

Isolation of clathrin-coated vesicles by differential and density gradient centrifugation.

Clathrin-coated vesicles (CCVs) are an important class of transport organelles that mediate the endocytosis of proteins and lipids at the plasma membrane and the transport of proteins from the trans-Golgi network to the endosomal/lysosomal system. The authors describe a protocol for isolating CCVs from adult rat brain using differential centrifugation, Ficoll and D(2)O-sucrose density gradient centrifugation, and velocity sedimentation in linear sucrose gradients. The application of this basic method to the isolation of CCVs from developing rat brains and to the generation of relatively crude CCVs from cultured cells is also described. Furthermore, we describe a protocol in which differential centrifugation and a series of discontinuous sucrose gradients are used to isolate CCVs from rat liver. An approach to analyzing CCV purity by electron microscopy is also described.

Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling.

Tandem MS has identified 209 proteins of clathrin-coated vesicles (CCVs) isolated from rat brain. An overwhelming abundance of peptides were assigned to the clathrin coat with a 1:1 stoichiometry observed for clathrin heavy and light chains and a 2:1 stoichiometry of clathrin heavy chain with clathrin adaptor protein heterotetramers. Thirty-two proteins representing many of the known components of synaptic vesicles (SVs) were identified, supporting that a main function for brain CCVs is to recapture SVs after exocytosis. A ratio of vesicle-N-ethylmaleimide-sensitive factor attachment protein receptors to target-N-ethylmaleimide-sensitive factor attachment protein receptors, similar to that previously detected on SVs, supports a single-step model for SV sorting during CCV-mediated recycling of SVs. The uncovering of eight previously undescribed proteins, four of which have to date been linked to clathrin-mediated trafficking, further attests to the value of the current organelle-based proteomics strategy.