The α-arrestin ARRDC3 mediates ALIX ubiquitination and G protein-coupled receptor lysosomal sorting.

The sorting of G protein-coupled receptors (GPCRs) to lysosomes is critical for proper signaling and cellular responses. We previously showed that the adaptor protein ALIX regulates lysosomal degradation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, independent of ubiquitin-binding ESCRTs and receptor ubiquitination. However, the mechanisms that regulate ALIX function during PAR1 lysosomal sorting are not known. Here we show that the mammalian α-arrestin arrestin domain-containing protein-3 (ARRDC3) regulates ALIX function in GPCR sorting via ubiquitination. ARRDC3 colocalizes with ALIX and is required for PAR1 sorting at late endosomes and degradation. Depletion of ARRDC3 by small interfering RNA disrupts ALIX interaction with activated PAR1 and the CHMP4B ESCRT-III subunit, suggesting that ARRDC3 regulates ALIX activity. We found that ARRDC3 is required for ALIX ubiquitination induced by activation of PAR1. A screen of nine mammalian NEDD4-family E3 ubiquitin ligases revealed a critical role for WWP2. WWP2 interacts with ARRDC3 and not ALIX. Depletion of WWP2 inhibited ALIX ubiquitination and blocked ALIX interaction with activated PAR1 and CHMP4B. These findings demonstrate a new role for the α-arrestin ARRDC3 and the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs

Source processes of large earthquakes along the Xianshuihe fault in southwestern China

The Xianshuihe fault is one of the most active faults in southwestern China. Recently, three large earthquakes occurred along it in 1967 (M_s = 6.1), 1973 (M_s = 7.5), and 1981 (M_s = 6.8). The 1981 event occurred near the central portion of the fault zone. Modeling of the body and surface waves indicates pure left-lateral strike-slip motion on a vertical fault striking N40°W consistent with the surface trend of the Xianshuihe fault. Two major ruptures are suggested for this source, with a total moment of 1.3 × 10^(26) dyne-cm. The 1973 event occurred about 65 km northwest of the 1981 event and ruptured about 90 km bilaterally along the fault. The body-wave synthetics indicate three main ruptures during faulting within 43 sec, with a total moment of 1.8 × 10^(27) dyne-cm. The mechanisms are similar to the 1981 event, and the average slip is determined to be 3.8 m. The largest aftershock (M_s = 5.9) occurred 1 day after the main event with a normal-fault mechanism striking almost perpendicular to the surface breakage. The 1967 event occurred at the northwestern end of the fault zone, with a strike of N65°E. It had a nearly normalfault mechanism with a seismic moment of 4.5 × 10^(25) dyne-cm. The largest aftershock (M_s = 5.1) occurred 7 hr later with a similar focal mechanism. The primary faulting along the Xianshuihe fault is left-lateral strike-slip, but the normal faulting with strike direction about perpendicular to the Xianshuihe fault trace is common, especially in the northwestern segment. The faulting pattern in this region is consistent with the regional stress field caused by the India-Tibet collision. The normal event which is not on the major fault seems to have more frequent foreshocks and aftershocks than those on the main fault

ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting.

The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)-0, -I, -II, and -III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor-regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III-dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4-ESCRT-III interacting protein, bound to a YPX(3)L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPX(n)L motifs

AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway

The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor–regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein–coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail–localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting

Causal associations between fluid intake patterns and dermatitis risk: a Mendelian randomization study

BackgroundDermatitis is one of the most common skin disorders across the world. Atopic dermatitis (AD) and contact dermatitis (CD) are its two primary types. Few studies have focused on the causal relationship between fluid intake and dermatitis. With an Mendelian Randomization (MR), this study investigated the potential causal effects of alcohol, coffee, tea, and water intake on the risk of AD and CD.MethodsUtilizing genetic variants as instrumental variables (IVs), a two-sample MR analysis was implemented based on data from the UK Biobank and FinnGen r9 consortium. Fluid intake was categorized into alcohol, coffee, tea, and water intake. Causal estimates were analyzed through Inverse Variance Weighted (IVW), MR-Egger, and weighted median methods. Cochran’s Q, MR-Egger intercept, and MR-PRESSO tests were conducted to assess potential heterogeneity and pleiotropy.ResultsWater intake exhibited a significant causal effect on raised CD risk (IVW OR = 2.92, 95% CI: 1.58–5.41, p = <0.01). Coffee intake was associated with increased CD risk (IVW OR = 2.16, 95% CI: 1.19–3.91, p = 0.01). Conversely, tea intake demonstrated a protective effect on AD risk (IVW OR = 0.71, 95% CI: 0.56–0.91, p = <0.01).ConclusionThis MR study suggests a potential association where water and coffee intake may be linked to an elevated risk of CD, while tea intake may potentially have a mitigating effect on AD risk. Modifying fluid intake patterns could be a targeted approach for dermatitis prevention, emphasizing the need for additional longitudinal studies to validate and expand upon these findings

PhD

dissertationIron is an essential nutrient for all eukaryotes and involved in many biological processes such as energy production, oxygen transport and DNA synthesis. Both iron deficiency and iron overload lead to human diseases. All organisms from yeast to humans have no regulated iron excretory pathway. Consequently, once iron enters cells it is detoxified by iron storage. In Saccharomyces cerevisiae, iron is stored in the vacuolar compartment mediated by the vacuolar iron transporter Cccl. Cells with a deletion of CCC1 are sensitive to high concentrations of iron. To understand the nature and origin of iron toxicity, we employed genetic screens to identify suppressors of high iron toxicity in \cccl cells. Our genetic analysis identified genes that reduced iron toxicity by decreasing cytosolic iron through increased iron sequestration in intracellular organelles. We identified that mutations in Zrcl, a vacuolar zinc and cobalt transporter, resulted in the ability to transport iron into the vacuole. We took advantage of these gain-of-function mutations to define amino acids and structural features important for substrate selection in the Zrcl family of cation diffusion facilitators. We also identified that overexpression of mitochondrial iron transporters Mrs3 or Mrs4 protected A cccl from high concentrations of iron by storing iron in mitochondria. Our genetic screen also identified Rim2 as a homologue of Mrs3 and Mrs4 and showed that Rim2 could also affect mitochondrial iron transport. Our studies identified novel forms of regulation of Mrs3/Mrs4 mediated iron transport. These genetic results suggest that iron induced damage occurs in the cytosol and iron sequestration in organelles can alleviate the toxic effect of high concentrations of iron

Chapter Twenty-Two Endosomal Signaling by Protease-Activated Receptors

Protease-activated receptors (PARs) are a family of G protein-coupled receptors (GPCRs) that are uniquely activated by proteolysis. There are four members of the PAR family including: PAR1, PAR2, PAR3, and PAR4. PARs are expressed primarily in the cells of the vasculature and elicit cellular responses to coagulant and anticoagulant proteases. PAR1 exemplifies the unusual proteolytic mechanism of receptor activation. Thrombin binds to and cleaves the N-terminal exodomain of PAR1, generating a new N-terminus that functions as a tethered ligand. The N-terminal tethered ligand domain of PAR1 binds intramolecularly to the receptor to trigger transmembrane signaling and cannot diffuse away. Similar to other GPCRs, activation of PARs promotes coupling to heterotrimeric G proteins at the plasma membrane. After activation, PARs are rapidly internalized to endosomes and then sorted to lysosomes and degraded. Internalization functions to uncouple PARs from heterotrimeric G proteins at the cell surface. However, recent studies indicate that activated internalized PARs signal from endosomes through the recruitment of β-arrestins and potentially other pathways. Here, we provide an overview of methods and strategies used to examine endosomal signaling by PARs

Endosomal signaling by protease-activated receptors.

Protease-activated receptors (PARs) are a family of G protein-coupled receptors (GPCRs) that are uniquely activated by proteolysis. There are four members of the PAR family including: PAR1, PAR2, PAR3, and PAR4. PARs are expressed primarily in the cells of the vasculature and elicit cellular responses to coagulant and anticoagulant proteases. PAR1 exemplifies the unusual proteolytic mechanism of receptor activation. Thrombin binds to and cleaves the N-terminal exodomain of PAR1, generating a new N-terminus that functions as a tethered ligand. The N-terminal tethered ligand domain of PAR1 binds intramolecularly to the receptor to trigger transmembrane signaling and cannot diffuse away. Similar to other GPCRs, activation of PARs promotes coupling to heterotrimeric G proteins at the plasma membrane. After activation, PARs are rapidly internalized to endosomes and then sorted to lysosomes and degraded. Internalization functions to uncouple PARs from heterotrimeric G proteins at the cell surface. However, recent studies indicate that activated internalized PARs signal from endosomes through the recruitment of β-arrestins and potentially other pathways. Here, we provide an overview of methods and strategies used to examine endosomal signaling by PARs