A fungal ubiquitin ligase and arrestin binding partner contribute to pathogenesis and survival during cellular stress.

Cellular responses to external stress allow microorganisms to adapt to a vast array of environmental conditions, including infection sites. The molecular mechanisms behind these responses are studied to gain insight into microbial pathogenesis, which could lead to new antimicrobial therapies. Here, we explore a role for arrestin protein-mediated ubiquitination in stress response and pathogenesis in the pathogenic fungus Cryptococcus neoformans. In a previous study, we identified four arrestin-like proteins in C. neoformans and found that one of these is required for efficient membrane synthesis, likely by directing interaction between fatty acid synthases and the Rsp5 E3 ubiquitin ligase. Here, we further explore Cn Rsp5 function and determine that this single Ub ligase is absolutely required for pathogenesis and survival in the presence of cellular stress. Additionally, we show that a second arrestin-like protein, Ali2, similarly facilitates interaction between Rsp5 and some of its protein targets. Of the four postulated C. neoformans arrestin-like proteins, Ali2 appears to contribute the most to C. neoformans pathogenesis, likely by directing Rsp5 to pathogenesis-related ubiquitination targets. A proteomics-based differential ubiquitination screen revealed that several known cell surface proteins are ubiquitinated by Rsp5 and a subset also requires Ali2 for their ubiquitination. Rsp5-mediated ubiquitination alters the stability and the localization of these proteins. A loss of Rsp5-mediated ubiquitination results in cell wall defects that increase susceptibility to external stresses. These findings support a model in which arrestin-like proteins guide Rsp5 to ubiquitinate specific target proteins, some of which are required for survival during stress. IMPORTANCE: Microbial proteins involved in human infectious diseases often need to be modified by specific chemical additions to be fully functional. Here, we explore the role of a particular protein modification, ubiquitination, in infections due to the human fungal pathogen Cryptococcus neoformans. We identified a complex of proteins responsible for adding ubiquitin groups to fungal proteins, and this complex is required for virulence. These proteins are fungal specific and might be targets for novel anti-infection therapy.

Differential pathogenetic mechanisms of mutations in helix 2 and helix 6 of rhodopsin.

Variants in rhodopsin (RHO) have been linked to autosomal dominant congenital stationary night blindness (adCSNB), which affects the ability to see in dim light, and the pathogenetic mechanism is still not well understood. In this study we report two novel RHO variants found in adCSNB families, p.W265R and p.A269V, that map in the sixth transmembrane domain of RHO protein. We applied in silico molecular simulation and in vitro biochemical and molecular studies to characterize the two new variants and compare the molecular determinants to two previously characterized adCSNB variants, p.G90D and p.T94I, that map in the second transmembrane domain of the RHO protein. We demonstrate that W265R and A269V cause constitutive activation of RHO with light-independent G protein coupling and impaired binding to arrestin. Differently, G90D and T94I are characterized by slow kinetics of RHO activation and deactivation. This study provides new evidence on the differential contribution of transmembrane α-helixes two and six to the interaction with intracellular transducers of RHO and mutations in these helixes result in a similar phenotype in patients but with distinct molecular effects.

Pupillary Light Reflex Reveals Melanopsin System Alteration in the Background of Myopia-26, the Female Limited Form of Early-Onset High Myopia.

Purpose: The purpose of this study was to evaluate pupillary light reflex (PLR) to chromatic flashes in patients with early-onset high-myopia (eoHM) without (myopic controls = M-CTRL) and with (female-limited myopia-26 = MYP-26) genetic mutations in the ARR3 gene encoding the cone arrestin. Methods: Participants were 26 female subjects divided into 3 groups: emmetropic controls (E-CTRL, N = 12, mean age = 28.6 ± 7.8 years) and 2 myopic (M-CTRL, N = 7, mean age = 25.7 ± 11.5 years and MYP-26, N = 7, mean age = 28.3 ± 15.4 years) groups. In addition, one hemizygous carrier and one control male subject were examined. Direct PLRs were recorded after 10-minute dark adaptation. Stimuli were 1-second red (peak wavelength = 621 nm) and blue (peak wavelength = 470 nm) flashes at photopic luminance of 250 cd/m². A 2-minute interval between the flashes was introduced. Baseline pupil diameter (BPD), peak pupil constriction (PPC), and postillumination pupillary response (PIPR) were extracted from the PLR. Group comparisons were performed with ANOVAs. Results: Dark-adapted BPD was comparable among the groups, whereas PPC to the red light was slightly reduced in patients with myopia (P = 0.02). PIPR at 6 seconds elicited by the blue flash was significantly weaker (P < 0.01) in female patients with MYP-26, whereas it was normal in the M-CTRL group and the asymptomatic male carrier. Conclusions: L/M-cone abnormalities due to ARR3 gene mutation is currently claimed to underlie the pathological eye growth in MYP-26. Our results suggest that malfunction of the melanopsin system of intrinsically photosensitive retinal ganglion cells (ipRGCs) is specific to patients with symptomatic MYP-26, and may therefore play an additional role in the pathological eye growth of MYP-26.

The causal mutation in ARR3 gene for high myopia and progressive color vision defect.

The ARR3 gene, also known as cone arrestin, belongs to the arrestin family and is expressed in cone cells, inactivating phosphorylated-opsins and preventing cone signals. Variants of ARR3 reportedly cause X-linked dominant female-limited early-onset (age < 7 years old) high myopia (< - 6D). Here, we reveal a new mutation (c.228T>A, p.Tyr76*) in ARR3 gene that can cause early-onset high myopia (eoHM) limited to female carriers. Protan/deutan color vision defects were also found in family members, affecting both genders. Using ten years of clinical follow-up data, we identified gradually worsening cone dysfunction/color vision as a key feature among affected individuals. We present a hypothesis that higher visual contrast due to the mosaic of mutated ARR3 expression in cones contributes to the development of myopia in female carriers.

PD 102807 Induces M3 mAChR-Dependent GRK-/Arrestin-Biased Signaling in Airway Smooth Muscle Cells.

G protein-coupled receptors (GPCRs) not only are turned on or off to control canonical G protein signaling but also may be fine-tuned to promote qualitative/biased signaling. Qualitative signaling by M3 muscarinic acetylcholine receptors (mAChRs) has been proposed, but its impact on physiologic systems remains unclear, and currently no biased M3 mAChR ligands have been described. Herein, we identify PD 102807 as a biased M3 ligand and delineate its signaling and function in human airway smooth muscle (ASM) cells. PD 102807 induced M3-mediated ß-arrestin recruitment but not calcium mobilization. PD 102807 inhibited methacholine (MCh)-induced calcium mobilization in (M3-expressing) ASM cells. PD 102807 induced phosphorylation of AMP-activated protein kinase (AMPK) and the downstream effector acetyl-coenzyme A carboxylase (ACC). PD 102807- induced phosphorylated (p)-AMPK levels were greatly reduced in ASM cells with minimal M3 expression and were not inhibited by the Gq inhibitor YM-254890. Induction of p-AMPK and p-ACC was inhibited by ß-arrestin 1 or GRK2/3 knockdown. Similarly, MCh induced phosphorylation of AMPK/ACC, but these effects were Gq dependent and unaffected by GRK2/3 knockdown. Consistent with the known ability of AMPK to inhibit transforming growth factor ß (TGF-ß)-mediated functions, PD 102807 inhibited TGF-ß-induced SMAD-Luc activity, sm-α-actin expression, actin stress fiber formation, and ASM cell hypercontractility. These findings reveal that PD 102807 is a biased M3 ligand that inhibits M3-transduced Gq signaling but promotes Gq protein-independent, GRK-/arrestin-dependent, M3-mediated AMPK signaling, which in turn regulates ASM phenotype and contractile function. Consequently, biased M3 ligands hold significant promise as therapeutic agents capable of exploiting the pleiotropic nature of M3 signaling.

Solo vs. Chorus: Monomers and Oligomers of Arrestin Proteins.

Three out of four subtypes of arrestin proteins expressed in mammals self-associate, each forming oligomers of a distinct kind. Monomers and oligomers have different subcellular localization and distinct biological functions. Here we summarize existing evidence regarding arrestin oligomerization and discuss specific functions of monomeric and oligomeric forms, although too few of the latter are known. The data on arrestins highlight biological importance of oligomerization of signaling proteins. Distinct modes of oligomerization might be an important contributing factor to the functional differences among highly homologous members of the arrestin protein family.

Arrestin-Coding Genes Regulate Endocytosis, Sporulation, Pathogenicity, and Stress Resistance in Arthrobotrys oligospora.

Arrestins are a family of scaffold proteins that play a crucial role in regulating numerous cellular processes, such as GPCR signaling. The Arthrobotrys oligospora arrestin family contains 12 members, which have highly conserved N-terminal and C-terminal domains. In the presence of ammonia, A. oligospora can change its lifestyle from saprotrophic to carnivorous. During this transition, the expression pattern of arrestin-coding (AoArc) genes was markedly upregulated. Therefore, we disrupted seven AoArc genes from A. oligospora to identify their functions. Although individual arrestin mutant strains display similar pathogenesis, phenotypes, and stress resistance, the fundamental data on the roles of AoArc genes in A. oligospora are obtained in this study. Membrane endocytosis in AoArc mutants was significantly reduced. Meanwhile, the capacity of trap device formation against nematodes and ammonia was impaired due to AoArc deletions. We also found that AoArc genes could regulate conidial phenotypes, cell nuclear distribution, pH response, and stress resistance. Results of qRT-PCR assays revealed that sporulation-regulated genes were affected after the deletion of AoArc genes. In particular, among the 12 arrestins, AoArc2 mediates pH signaling in the fungus A. oligospora. Notably, combined with the classical paradigm of arrestin-GPCR signal transduction, we suggest that arrestin-regulated trap formation in A. oligospora may be directly linked to the receptor endocytosis pathway.

Identification, tissue specific expression analysis and functional characterization of arrestin gene (ARRDC) in the earthworm Eudrilus eugeniae: a molecular hypothesis behind worm photoreception.

BACKGROUND: The arrestin domain containing proteins (ARRDCs) are crucial adaptor proteins assist in signal transduction and regulation of sensory physiology. The molecular localization of the ARRDC gene has been confined mainly to the mammalian system while in invertebrates the expression pattern was not addressed significantly. The present study reports the identification, tissue specific expression and functional characterization of an ARRDC transcript in earthworm, Eudrilus eugeniae. METHODS AND RESULTS: The coding region of earthworm ARRDC transcript was 1146 bp in length and encoded a protein of 381 amino acid residues. The worm ARRDC protein consists of conserved N-terminal and C-terminal regions and showed significant homology with the ARRDC3 sequence of other species. The tissue specific expression analysis through whole mount in-situ hybridization denoted the expression of ARRDC transcript in the central nervous system of the worm which includes cerebral ganglion and ventral nerve cord. Besides, the expression of ARRDC gene was observed in the epidermal region of earthworm skin. The functional characterization of ARRDC gene was assessed through siRNA silencing and the gene was found to play key role in the light sensing ability and photophobic movement of the worm. CONCLUSIONS: The neuronal and dermal expression patterns of ARRDC gene and its functional characterization hypothesized the role of the gene in assisting the photosensory cells to regulate the process of photoreception and phototransduction in the worm.

Structural Basis of Arrestin Selectivity for Active Phosphorylated G Protein-Coupled Receptors.

Arrestins are a small family of proteins that bind G protein-coupled receptors (GPCRs). Arrestin binds to active phosphorylated GPCRs with higher affinity than to all other functional forms of the receptor, including inactive phosphorylated and active unphosphorylated. The selectivity of arrestins suggests that they must have two sensors, which detect receptor-attached phosphates and the active receptor conformation independently. Simultaneous engagement of both sensors enables arrestin transition into a high-affinity receptor-binding state. This transition involves a global conformational rearrangement that brings additional elements of the arrestin molecule, including the middle loop, in contact with a GPCR, thereby stabilizing the complex. Here, we review structural and mutagenesis data that identify these two sensors and additional receptor-binding elements within the arrestin molecule. While most data were obtained with the arrestin-1-rhodopsin pair, the evidence suggests that all arrestins use similar mechanisms to achieve preferential binding to active phosphorylated GPCRs.

High-mass MALDI-MS unravels ligand-mediated G protein-coupling selectivity to GPCRs.

G protein-coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, we developed a high-sensitivity, high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) method to interrogate the first stage of signal transduction. GPCR-G protein complex formation is detected as a proxy for the effect of ligands on GPCR conformation and on coupling selectivity. Over 70 ligand-GPCR-partner protein combinations were studied using as little as 1.25 pmol protein per sample. We determined the selectivity profile and binding affinities of three GPCRs (rhodopsin, beta-1 adrenergic receptor [ß1AR], and angiotensin II type 1 receptor) to engineered Gα-proteins (mGs, mGo, mGi, and mGq) and nanobody 80 (Nb80). We found that GPCRs in the absence of ligand can bind mGo, and that the role of the G protein C terminus in GPCR recognition is receptor-specific. We exemplified our quantification method using ß1AR and demonstrated the allosteric effect of Nb80 binding in assisting displacement of nadolol to isoprenaline. We also quantified complex formation with wild-type heterotrimeric Gαißγ and ß-arrestin-1 and showed that carvedilol induces an increase in coupling of ß-arrestin-1 and Gαißγ to ß1AR. A normalization strategy allows us to quantitatively measure the binding affinities of GPCRs to partner proteins. We anticipate that this methodology will find broad use in screening and characterization of GPCR-targeting drugs.

DeSiphering receptor core-induced and ligand-dependent conformational changes in arrestin via genetic encoded trimethylsilyl 1H-NMR probe.

Characterization of the dynamic conformational changes in membrane protein signaling complexes by nuclear magnetic resonance (NMR) spectroscopy remains challenging. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion. Crystallographic analysis revealed structural changes that reshaped the TMSiPhe-specific amino-acyl tRNA synthetase active site to selectively accommodate the trimethylsilyl (TMSi) group. The unique up-field 1H-NMR chemical shift and the highly efficient incorporation of TMSiPhe enabled the characterization of multiple conformational states of a phospho-ß2 adrenergic receptor/ß-arrestin-1(ß-arr1) membrane protein signaling complex, using only 5 µM protein and 20 min of spectrum accumulation time. We further showed that extracellular ligands induced conformational changes located in the polar core or ERK interaction site of ß-arr1 via direct receptor transmembrane core interactions. These observations provided direct delineation and key mechanism insights that multiple receptor ligands were able to induce distinct functionally relevant conformational changes of arrestin.

Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids.

How cells adjust nutrient transport across their membranes is incompletely understood. Previously, we have shown that S. cerevisiae broadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that the selective endocytosis of four AATs during starvation required the α-arrestin family protein Art2/Ecm21, an adaptor for the ubiquitin ligase Rsp5, and its induction through the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in AATs and thereby instructs Rsp5 to ubiquitinate proximal lysine residues. When amino acids are in excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis. Thus, amino acid excess or starvation activate complementary α-arrestin-Rsp5-complexes to control selective endocytosis and adapt nutrient acquisition.

G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.

Quantification of Molecular Interactions in Living Cells in Real Time using a Membrane Protein Nanopattern.

Molecular processes within cells have traditionally been studied with biochemical methods due to their high degree of specificity and ease of use. In recent years, cell-based assays have gained more and more popularity since they facilitate the extraction of mode of action, phenotypic, and toxicity information. However, to provide specificity, cellular assays rely heavily on biomolecular labels and tags while label-free cell-based assays only offer holistic information about a bulk property of the investigated cells. Here, we introduce a cell-based assay for protein-protein interaction analysis. We achieve specificity by spatially ordering a membrane protein of interest into a coherent pattern of fully functional membrane proteins on the surface of an optical sensor. Thereby, molecular interactions with the coherently ordered membrane proteins become visible in real time, while nonspecific interactions and holistic changes within the living cell remain invisible. Due to its unbiased nature, this new cell-based detection method presents itself as an invaluable tool for cell signaling research and drug discovery.

Oguchi disease caused by a homozygous novel SAG splicing alteration associated with the multiple evanescent white dot syndrome: A 15-month follow-up.

PURPOSE: We report a 15-month follow-up case on a Chinese patient with Oguchi disease associated with the multiple evanescent white dot syndrome (MEWDS). METHODS: The patient's clinical presentation and follow-up visits were documented via decimal best-corrected visual acuity, fundus photography, fundus autofluorescence (FAF) imaging, near-infrared FAF, spectral domain optical coherence tomography, Humphrey's visual fields, microperimetry, and multifocal electroretinography. We also performed whole exome sequencing for screening variation in the patient and her relatives. RESULTS: The patient had typical clinical characteristic of Oguchi disease, including night blindness, the Mizuo-Nakamura phenomenon (a golden yellow discoloration of the fundus that disappears in the prolonged dark adaptation [DA]) and typical full-field electroretinogram changes (nearly undetected b-wave in 0.01 and 0.03 ERGs that can partially recover only after prolonged DA). Aside from Oguchi disease, the patient was also diagnosed with the MEWDS based on clinical detections, including suddenly reduced visual acuity, appeared white dots, blurred ellipsoid zone and disrupted interdigitation zone, enlarged blind spot, and reduced macular sensitivity. A series of investigations revealed that along with the 15-month follow-up after onset, the visual acuity enhanced, the numerous white dots disappeared, and the macular structure returned to normal. Moreover, the novel homozygous splicing alteration c.181 + 1G > A was identified in the SAG gene. CONCLUSIONS: This work is the first long-term case study of a patient with Oguchi disease associated with the MEWDS. The recovery period of symptoms caused by the MEWDS was much longer than that in typical patients with MEWDS. Molecular genetics demonstrate that this is the first case of Oguchi disease caused by splicing alterations in the SAG gene.

An arrestin-1 surface opposite of its interface with photoactivated rhodopsin engages with enolase-1.

Arrestin-1 is the arrestin family member responsible for inactivation of the G protein-coupled receptor rhodopsin in photoreceptors. Arrestin-1 is also well-known to interact with additional protein partners and to affect other signaling cascades beyond phototransduction. In this study, we investigated one of these alternative arrestin-1 binding partners, the glycolysis enzyme enolase-1, to map the molecular contact sites between these two proteins and investigate how the binding of arrestin-1 affects the catalytic activity of enolase-1. Using fluorescence quench protection of strategically placed fluorophores on the arrestin-1 surface, we observed that arrestin-1 primarily engages enolase-1 along a surface that is opposite of the side of arrestin-1 that binds photoactivated rhodopsin. Using this information, we developed a molecular model of the arrestin-1-enolase-1 complex, which was validated by targeted substitutions of charge-pair interactions. Finally, we identified the likely source of arrestin's modulation of enolase-1 catalysis, showing that selective substitution of two amino acids in arrestin-1 can completely remove its effect on enolase-1 activity while still remaining bound to enolase-1. These findings open up opportunities for examining the functional effects of arrestin-1 on enolase-1 activity in photoreceptors and their surrounding cells.

Revealing Conformational Transitions in G-Protein-Coupled Receptor Rhodopsin upon Phosphorylation.

G-protein-coupled receptors (GPCRs) have evolved as highly specialized cellular machinery that can dictate biological outcomes in response to diverse stimuli. Specifically, they induce multiple pathway responses upon structural perturbations induced at local protein sites. GPCRs utilize a concurrent strategy involving a central transmembrane topology and biochemical modifications for precise functional implementation. However, the specific role of the latter is not known due to the lack of precise probing techniques that can characterize receptor dynamics upon biochemical modifications. Phosphorylation is known to be one of the critical biochemical modifications in GPCRs that aids in receptor desensitization via arrestin binding. Here, we carry out all-atom molecular dynamics simulations of rhodopsin in a membrane environment to study its conformational dynamics induced upon phosphorylation. Interestingly, our comparative analysis of non-phosphorylated and phosphorylated rhodopsin structure demonstrated enhanced receptor stability upon phosphorylation at the C-terminal region that leads to the opening of the extracellular part of the transmembrane helices. In addition, monitoring the distinct number of phosphorylation states showed that having fewer phosphorylated residues does not bring about appropriate conformational changes in the extracellular region. Since phosphorylation results in receptor desensitization and recycling of the ligand, our findings provide significant insights into the conformational dynamics of the mechanism of ligand exit from the receptor.

A Fungal Arrestin Protein Contributes to Cell Cycle Progression and Pathogenesis.

Arrestins, a structurally specialized and functionally diverse group of proteins, are central regulators of adaptive cellular responses in eukaryotes. Previous studies on fungal arrestins have demonstrated their capacity to modulate diverse cellular processes through their adaptor functions, facilitating the localization and function of other proteins. However, the mechanisms by which arrestin-regulated processes are involved in fungal virulence remain unexplored. We have identified a small family of four arrestins, Ali1, Ali2, Ali3, and Ali4, in the human fungal pathogen Cryptococcus neoformans Using complementary microscopy, proteomic, and reverse genetics techniques, we have defined a role for Ali1 as a novel contributor to cytokinesis, a fundamental cell cycle-associated process. We observed that Ali1 strongly interacts with proteins involved in lipid synthesis, and that ali1Δ mutant phenotypes are rescued by supplementation with lipid precursors that are used to build cellular membranes. From these data, we hypothesize that Ali1 contributes to cytokinesis by serving as an adaptor protein, facilitating the localization of enzymes that modify the plasma membrane during cell division, specifically the fatty acid synthases Fas1 and Fas2. Finally, we assessed the contributions of the C. neoformans arrestin family to virulence to better understand the mechanisms by which arrestin-regulated adaptive cellular responses influence fungal infection. We observed that the C. neoformans arrestin family contributes to virulence, and that the individual arrestin proteins likely fulfill distinct functions that are important for disease progression.IMPORTANCE To survive under unpredictable conditions, all organisms must adapt to stressors by regulating adaptive cellular responses. Arrestin proteins are conserved regulators of adaptive cellular responses in eukaryotes. Studies that have been limited to mammals and model fungi have demonstrated that the disruption of arrestin-regulated pathways is detrimental for viability. The human fungal pathogen Cryptococcus neoformans causes more than 180,000 infection-related deaths annually, especially among immunocompromised patients. In addition to being genetically tractable, C. neoformans has a small arrestin family of four members, lending itself to a comprehensive characterization of its arrestin family. This study serves as a functional analysis of arrestins in a pathogen, particularly in the context of fungal fitness and virulence. We investigate the functions of one arrestin protein, Ali1, and define its novel contributions to cytokinesis. We additionally explore the virulence contributions of the C. neoformans arrestin family and find that they contribute to disease establishment and progression.