Stomatin-like Protein-1 Interacts with Stomatin and Is Targeted to Late Endosomes

The human stomatin-like protein-1 (SLP-1) is a membrane protein with a characteristic bipartite structure containing a stomatin domain and a sterol carrier protein-2 (SCP-2) domain. This structure suggests a role for SLP-1 in sterol/lipid transfer and transport. Because SLP-1 has not been investigated, we first studied the molecular and cell biological characteristics of the expressed protein. We show here that SLP-1 localizes to the late endosomal compartment, like stomatin. Unlike stomatin, SLP-1 does not localize to the plasma membrane. Overexpression of SLP-1 leads to the redistribution of stomatin from the plasma membrane to late endosomes suggesting a complex formation between these proteins. We found that the targeting of SLP-1 to late endosomes is caused by a GYXXΦ (Φ being a bulky, hydrophobic amino acid) sorting signal at the N terminus. Mutation of this signal results in plasma membrane localization. SLP-1 and stomatin co-localize in the late endosomal compartment, they co-immunoprecipitate, thus showing a direct interaction, and they associate with detergent-resistant membranes. In accordance with the proposed lipid transfer function, we show that, under conditions of blocked cholesterol efflux from late endosomes, SLP-1 induces the formation of enlarged, cholesterol-filled, weakly LAMP-2-positive, acidic vesicles in the perinuclear region. This massive cholesterol accumulation clearly depends on the SCP-2 domain of SLP-1, suggesting a role for this domain in cholesterol transfer to late endosomes.Human stomatin-like protein-1 (SLP-1),³ also known as STOML-1, STORP (1), slipin-1 (2), or hUNC-24 (3), is the human orthologue of Caenorhabditis elegans UNC-24 and a member of the stomatin protein family that comprises 5 human members: stomatin (4–6), SLP-1 (1, 7), SLP-2 (8), SLP-3 (9, 10), and podocin (11). SLP-1 is predominantly expressed in the brain, heart, and skeletal muscle (7, 8) and can be identified in most other tissues (1). Its structure contains a hydrophilic N terminus, a 30-residue hydrophobic domain that is thought to anchor the protein to the cytoplasmic side of the membrane, followed by a stomatin/prohibitin/flotillin/HflK/C (SPFH) domain (12) that is also known as prohibitin (PHB) domain (13), and a C-terminal sterol carrier protein-2 (SCP-2)/nonspecific lipid transfer protein domain (14, 15). This unique structure that was first revealed in C. elegans UNC-24 (16) suggests that SLP-1 may be involved in lipid transfer and transport (17).The founder of the family, stomatin, is a major protein of the red blood cell membrane (band 7.2) and is ubiquitously expressed (18). It is missing in red cells of patients with overhydrated hereditary stomatocytosis, a pathological condition characterized by increased permeability of the red cells for monovalent ions and stomatocytic morphology (19, 20). However, the lack of stomatin is not due to a mutation in its gene but rather to a transport defect (21, 22). Stomatin is a monotopic, oligomeric, palmitoylated, cholesterol-binding membrane protein (18) that is associated with lipid rafts (23, 24) or raft-like detergent-resistant membranes (DRMs) (25), serving as a respective marker (26–28). Other stomatin family members like podocin (29, 30) and SLP-3 (9) are also enriched in DRMs. Many SPFH/PHB proteins share this property suggesting that the SPFH/PHB domain plays an important role in lipid raft/DRM targeting (13, 31). Several interactions of stomatin with membrane proteins have been revealed, notably with the acid sensing ion channels (32) and the glucose transporter GLUT1 (33, 34). Interestingly, stomatin functions as a switch of GLUT1 specificity from glucose to dehydroascorbate in the human red blood cell thus increasing vitamin C recycling and compensating the human inability to synthesize vitamin C (35).The C. elegans genome contains 10 members of the stomatin family. Defects in three of these genes (mec-2, unc-1, and unc-24) cause distinct neuropathologic phenotypes, namely uncoordinated movement and defect in mechanosensation, respectively (36, 37). These are explained by dysfunction of the respective stomatin-like proteins in complex with degenerin/epithelial sodium channels that also affects the sensitivity to volatile anesthetics (38, 39). Importantly, MEC-2 and human podocin bind cholesterol and form large supercomplexes with various ion channels thus modulating channel activity (40). The biological functions of the SLP-1 orthologue UNC-24 and stomatin orthologue UNC-1 are associated, because the unc-24 gene controls the distribution or stability of the UNC-1 protein (41). In addition, UNC-24 co-localizes and interacts with MEC-2 and is essential for touch sensitivity (36). Based on these observations, we hypothesize that human stomatin and SLP-1 similarly interact and modify the distribution of each other. These proteins may have important functions in regulating the activity of ion channels in the human brain and muscle tissues. Despite its putative role in cellular lipid distribution, SLP-1 has not been studied to date.In this work, we characterized human SLP-1 as a late endosomal protein and identified an N-terminal GYXXΦ motif as the targeting signal. We found that SLP-1 interacts with stomatin in vitro and in vivo and associates with DRMs. Regarding the proposed lipid transfer function, we showed that SLP-1 induces the formation of large, cholesterol-rich vesicles or vacuoles when cholesterol trafficking from the late endosomes is blocked suggesting a net cholesterol transfer to the late endosomes and/or lysosomes. This effect was clearly attributed to the SCP-2/nonspecific lipid transfer protein domain of SLP-1, in line with the original hypothesis.     

具有广泛抗病毒活性的SAMHD1蛋白的研究进展

SAMHD1蛋白是2011年首次被认定为一种独特的天然抗病毒因子,它主要在树突状细胞、巨噬细胞等髓系细胞中表达。它通过降解细胞内dNTPs的水平,使细胞内的dNTPs的水平低于病毒复制所需的水平,从而抑制髓系细胞中反转录病毒和DNA病毒的复制。HIV-2产生的病毒蛋白X(Vpx)可将泛素连接酶与SAMHD1相结合,使SAMHD1分子最后被蛋白酶体降解。最近还发现SAMHD1蛋白活性受多种因子影响,具有调节肿瘤细胞中LINE-1活性的功能。结合最新研究成果对SAMHD1的结构、功能、抗病毒机制以及影响因子进行综述。     

Fusion of Lysosomes with Late Endosomes Produces a Hybrid Organelle of Intermediate Density and Is NSF Dependent

Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37°C, late endosome–lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide–sensitive factor– dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.     

A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.     

Withaferin A Targets Intermediate Filaments Glial Fibrillary Acidic Protein and Vimentin in a Model of Retinal Gliosis

Gliosis is a biological process that occurs during injury repair in the central nervous system and is characterized by the overexpression of the intermediate filaments (IFs) glial fibrillary acidic protein (GFAP) and vimentin. A common thread in many retinal diseases is reactive Müller cell gliosis, an untreatable condition that leads to tissue scarring and even blindness. Here, we demonstrate that the vimentin-targeting small molecule withaferin A (WFA) is a novel chemical probe of GFAP. Using molecular modeling studies that build on the x-ray crystal structure of tetrameric vimentin rod 2B domain we reveal that the WFA binding site is conserved in the corresponding domain of tetrameric GFAP. Consequently, we demonstrate that WFA covalently binds soluble recombinant tetrameric human GFAP at cysteine 294. In cultured primary astrocytes, WFA binds to and down-regulates soluble vimentin and GFAP expression to cause cell cycle G₀/G₁ arrest. Exploiting a chemical injury model that overexpresses vimentin and GFAP in retinal Müller glia, we demonstrate that systemic delivery of WFA down-regulates soluble vimentin and GFAP expression in mouse retinas. This pharmacological knockdown of soluble IFs results in the impairment of GFAP filament assembly and inhibition of cell proliferative response in Müller glia. We further show that a more severe GFAP filament assembly deficit manifests in vimentin-deficient mice, which is partly rescued by WFA. These findings illustrate WFA as a chemical probe of type III IFs and illuminate this class of withanolide as a potential treatment for diverse gliosis-dependent central nervous system traumatic injury conditions and diseases, and for orphan IF-dependent pathologies.     

Neurospora crassa NKIN2, a Kinesin-3 Motor,Transports Early Endosomes and Is Required for Polarized Growth

Biological motors are molecular nanomachines, which convert chemical energy into mechanical forces. The combination of mechanoenzymes with structural components, such as the cytoskeleton, enables eukaryotic cells to overcome entropy, generate molecular gradients, and establish polarity. Hyphae of filamentous fungi are among the most polarized cells, and polarity defects are most obvious. Here, we studied the role of the kinesin-3 motor, NKIN2, in Neurospora crassa. We found that NKIN2 localizes as fast-moving spots in the cytoplasm of mature hyphae. To test whether the spots represented early endosomes, the Rab5 GTPase YPT52 was used as an endosomal marker. NKIN2 colocalized with YPT52. Deletion of nkin2 caused strongly reduced endosomal movement. Combined, these results confirm the involvement of NKIN2 in early endosome transport. Introduction of a rigor mutation into NKIN2 labeled with green fluorescent protein (GFP) resulted in decoration of microtubules. Interestingly, NKIN2^rigor was associated with a subpopulation of microtubules, as had been shown earlier for the Aspergillus nidulans orthologue UncA. Other kinesins did not show this specificity.     

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the “long loop” recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the β₂-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation.     

Hv1 Proton Channel Opening Is Preceded by a Voltage-independent Transition

The voltage sensing domain (VSD) of the voltage-gated proton channel Hv1 mediates a H⁺-selective conductance that is coordinately controlled by the membrane potential (V) and the transmembrane pH gradient (ΔpH). Allosteric control of Hv1 channel opening by ΔpH (V-ΔpH coupling) is manifested by a characteristic shift of approximately 40 mV per ΔpH unit in the activation. To further understand the mechanism for V-ΔpH coupling in Hv1, H⁺ current kinetics of activation and deactivation in excised membrane patches were analyzed as a function of the membrane potential and the pH in the intracellular side of the membrane (pH_I). In this study, it is shown for the first time to our knowledge that the opening of Hv1 is preceded by a voltage-independent transition. A similar process has been proposed to constitute the step involving coupling between the voltage-sensing and pore domains in tetrameric voltage-gated channels. However, for Hv1, the VSD functions as both the voltage sensor and the conduction pathway, suggesting that the voltage independent transition is intrinsic to the voltage-sensing domain. Therefore, this article proposes that the underlying mechanism for the activation of Hv1 involves a process similar to VSD relaxation, a process previously described for voltage-gated channels and voltage-controlled enzymes. Finally, deactivation seemingly occurs as a strictly voltage dependent process, implying that the kinetic event leading to opening of the proton conductance are different than those involved in the closing. Thus, from this work it is proposed that Hv1 activity displays hysteresis.     

Hv1 Proton Channel Opening Is Preceded by a Voltage-independent Transition

The voltage sensing domain (VSD) of the voltage-gated proton channel Hv1 mediates a H⁺-selective conductance that is coordinately controlled by the membrane potential (V) and the transmembrane pH gradient (ΔpH). Allosteric control of Hv1 channel opening by ΔpH (V-ΔpH coupling) is manifested by a characteristic shift of approximately 40 mV per ΔpH unit in the activation. To further understand the mechanism for V-ΔpH coupling in Hv1, H⁺ current kinetics of activation and deactivation in excised membrane patches were analyzed as a function of the membrane potential and the pH in the intracellular side of the membrane (pH_I). In this study, it is shown for the first time to our knowledge that the opening of Hv1 is preceded by a voltage-independent transition. A similar process has been proposed to constitute the step involving coupling between the voltage-sensing and pore domains in tetrameric voltage-gated channels. However, for Hv1, the VSD functions as both the voltage sensor and the conduction pathway, suggesting that the voltage independent transition is intrinsic to the voltage-sensing domain. Therefore, this article proposes that the underlying mechanism for the activation of Hv1 involves a process similar to VSD relaxation, a process previously described for voltage-gated channels and voltage-controlled enzymes. Finally, deactivation seemingly occurs as a strictly voltage dependent process, implying that the kinetic event leading to opening of the proton conductance are different than those involved in the closing. Thus, from this work it is proposed that Hv1 activity displays hysteresis.     

Targets for the Antiviral and Antitumor Activities of Nucleoside,Nucleotide and Oligonucleotide Analogues

Abstract

The following targets can be considered in the development of antiviral agents: (i) DNA polymerase via dThd kinase, (ii) S-adenosylhomocysteine hydrolase; and in the development of antitumor agents: (iii) dTMP synthetase and (iv) protein synthesis via the 2–5A pathway.     

Autophagy Is a Protective Mechanism for Human Melanoma Cells under Acidic Stress

Cyclic hypoxia and alterations in oncogenic signaling contribute to switch cancer cell metabolism from oxidative phosphorylation to aerobic glycolysis. A major consequence of up-regulated glycolysis is the increased production of metabolic acids responsible for the presence of acidic areas within solid tumors. Tumor acidosis is an important determinant of tumor progression and tumor pH regulation is being investigated as a therapeutic target. Autophagy is a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, currently considered an important survival mechanism in cancer cells under metabolic stress or subjected to chemotherapy. We investigated the response of human melanoma cells cultured in acidic conditions in terms of survival and autophagy regulation. Melanoma cells exposed to acidic culture conditions (7.0 < pH < 6.2) promptly accumulated LC3+ autophagic vesicles. Immunoblot analysis showed a consistent increase of LC3-II in acidic culture conditions as compared with cells at normal pH. Inhibition of lysosomal acidification by bafilomycin A1 further increased LC3-II accumulation, suggesting an active autophagic flux in cells under acidic stress. Acute exposure to acidic stress induced rapid inhibition of the mammalian target of rapamycin signaling pathway detected by decreased phosphorylation of p70S6K and increased phosphorylation of AMP-activated protein kinase, associated with decreased ATP content and reduced glucose and leucine uptake. Inhibition of autophagy by knockdown of the autophagic gene ATG5 consistently reduced melanoma cell survival in low pH conditions. These observations indicate that induction of autophagy may represent an adaptation mechanism for cancer cells exposed to an acidic environment. Our data strengthen the validity of therapeutic strategies targeting tumor pH regulation and autophagy in progressive malignancies.     

MEIOB Targets Single-Strand DNA and Is Necessary for Meiotic Recombination

Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB). This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 ^−/− spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob ^−/− meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.     

Influenza B Virus Ribonucleoprotein Is a Potent Activator of the Antiviral Kinase PKR

Activation of the latent kinase PKR is a potent innate defense reaction of vertebrate cells towards viral infections, which is triggered by recognition of viral double-stranded (ds) RNA and results in a translational shutdown. A major gap in our understanding of PKR''s antiviral properties concerns the nature of the kinase activating molecules expressed by influenza and other viruses with a negative strand RNA genome, as these pathogens produce little or no detectable amounts of dsRNA. Here we systematically investigated PKR activation by influenza B virus and its impact on viral pathogenicity. Biochemical analysis revealed that PKR is activated by viral ribonucleoprotein (vRNP) complexes known to contain single-stranded RNA with a 5′-triphosphate group. Cell biological examination of recombinant viruses showed that the nucleo-cytoplasmic transport of vRNP late in infection is a strong trigger for PKR activation. In addition, our analysis provides a mechanistic explanation for the previously observed suppression of PKR activation by the influenza B virus NS1 protein, which we show here to rely on complex formation between PKR and NS1''s dsRNA binding domain. The high significance of this interaction for pathogenicity was revealed by the finding that attenuated influenza viruses expressing dsRNA binding-deficient NS1 proteins were rescued for high replication and virulence in PKR-deficient cells and mice, respectively. Collectively, our study provides new insights into an important antiviral defense mechanism of vertebrates and leads us to suggest a new model of PKR activation by cytosolic vRNP complexes, a model that may also be applicable to other negative strand RNA viruses.