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His Domain Protein Tyrosine Phosphatase (HD-PTP) facilitates function of the endosomal sorting complexes required for transport (ESCRTs) during multivesicular body (MVB) formation. To uncover its role in physiological homeostasis, embryonic lethality caused by a complete lack of HD-PTP was bypassed through generation of hypomorphic mice expressing reduced protein, resulting in animals that are viable into adulthood. These mice exhibited marked lipodystrophy and decreased receptor-mediated signaling within white adipose tissue (WAT), involving multiple prominent pathways including RAS/MAPK, PI3K/AKT and RTKs such as EGFR. EGFR signaling was dissected in vitro to assess the nature of defective signaling, revealing decreased trans-autophosphorylation and downstream effector activation, despite normal EGF binding. This corresponds to decreased plasma membrane cholesterol and increased lysosomal cholesterol, likely resulting from defective endosomal maturation necessary for cholesterol trafficking and homeostasis. ESCRT components Vps4 and HRS have previously been implicated in cholesterol homeostasis, thus these findings expand knowledge on which ESCRT subunits are involved in cholesterol homeostasis and highlight a non-canonical role for HD-PTP in signal regulation and adipose tissue homeostasis.
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Optimizing the spin coating of silver nanowires to form transparent conducting electrodes (TCE) is guided by machine learning (ML). A good TCE has two competing characteristics: high transmittance and high conductance. Optimization using a scalar figure of merit, as often done in the field, cannot satisfy the independent requirements for transmittance and conductance imposed by specific applications. By performing a Pareto front analysis based on ML models, we show that the desired outcomes of transmittance ≥ 75% and sheet resistance ≤ 15 Ω/sq are challenging but can be achieved using processing parameters identified by ML analysis.
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Mutations in MPZ (myelin protein zero) can cause demyelinating early-onset Charcot-Marie-Tooth type 1B disease or later onset type 2I/J disease characterized by axonal degeneration, reflecting the diverse roles of MPZ in Schwann cells. MPZ holds apposing membranes of the myelin sheath together, with the adhesion role fulfilled by its extracellular immunoglobulin-like domain (IgMPZ), which oligomerizes. Models for how the IgMPZ might form oligomeric assemblies has been extrapolated from a protein crystal structure in which individual rat IgMPZ subunits are packed together under artificial conditions, forming three weak interfaces. One interface organizes the IgMPZ into tetramers, a second 'dimer' interface links tetramers together across the intraperiod line, and a third hydrophobic interface that mediates binding to lipid bilayers or the same hydrophobic surface on another IgMPZ domain. Presently, there are no data confirming whether the proposed IgMPZ interfaces actually mediate oligomerization in solution, whether they are required for the adhesion activity of MPZ, whether they are important for myelination, or whether their loss results in disease. We performed nuclear magnetic resonance spectroscopy and small angle X-ray scattering analysis of wild-type IgMPZ as well as mutant forms with amino acid substitutions designed to interrupt its presumptive oligomerization interfaces. Here, we confirm the interface that mediates IgMPZ tetramerization, but find that dimerization is mediated by a distinct interface that has yet to be identified. We next correlated different types of Charcot-Marie-Tooth disease symptoms to subregions within IgMPZ tetramers. Variants causing axonal late-onset disease (CMT2I/J) map to surface residues of IgMPZ proximal to the transmembrane domain. Variants causing early-onset demyelinating disease (CMT1B) segregate into two groups: one is described by variants that disrupt the stability of the Ig-fold itself and are largely located within the core of the IgMPZ domain; whereas another describes a region on the surface of IgMPZ tetramers, accessible to protein interactions. Computational docking studies predict that this latter disease-relevant subregion may potentially mediate dimerization of IgMPZ tetramers.
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Enfermedad de Charcot-Marie-Tooth , Animales , Ratas , Axones , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/diagnóstico , Dominios de Inmunoglobulinas , Mutación/genética , Proteína P0 de la Mielina/genética , HumanosRESUMEN
Voltage-gated sodium (NaV) channels drive the upstroke of the action potential and are comprised of a pore-forming α-subunit and regulatory ß-subunits. The ß-subunits modulate the gating, trafficking, and pharmacology of the α-subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous ß-subunits. To better reveal ß-subunit functions, we engineered a human cell line devoid of endogenous NaV ß-subunits and their immediate phylogenetic relatives. This new cell line, ß-subunit-eliminated eHAP expression (BeHAPe) cells, were derived from haploid eHAP cells by engineering inactivating mutations in the ß-subunits SCN1B, SCN2B, SCN3B, and SCN4B, and other subfamily members MPZ (myelin protein zero(P0)), MPZL1, MPZL2, MPZL3, and JAML. In diploid BeHAPe cells, the cardiac NaV α-subunit, NaV1.5, was highly sensitive to ß-subunit modulation and revealed that each ß-subunit and even MPZ imparted unique gating properties. Furthermore, combining ß1 and ß2 with NaV1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of ß-subunits to regulate NaV1.5 activity and can be used to improve the characterization of other α/ß NaV complexes.
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Canal de Sodio Activado por Voltaje NAV1.5 , Subunidades de Proteína , Subunidades beta de Canales de Sodio Activados por Voltaje , Humanos , Potenciales de Acción , Línea Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/química , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Fosfoproteínas/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/deficiencia , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Subunidades beta de Canales de Sodio Activados por Voltaje/química , Subunidades beta de Canales de Sodio Activados por Voltaje/deficiencia , Subunidades beta de Canales de Sodio Activados por Voltaje/genética , Subunidades beta de Canales de Sodio Activados por Voltaje/metabolismo , MutaciónAsunto(s)
Fenómenos Bioquímicos , Diabetes Mellitus , Humanos , Dineínas/metabolismo , Transporte de ProteínasRESUMEN
PMP22 and MPZ are major myelin proteins in the peripheral nervous system. MPZ is a single pass integral membrane protein with an extracellular immunoglobulin (Ig)-like domain and works as an adhesion protein to hold myelin wraps together across the intraperiod line. Loss of MPZ causes severe demyelinating Charcot-Marie-Tooth (CMT) peripheral neuropathy. PMP22 is an integral membrane tetraspan protein belonging to the Claudin superfamily. Homozygous loss of PMP22 also leads to severe demyelinating neuropathy, and duplication of wildtype PMP22 causes the most common form of CMT, CMT1A. Yet the molecular functions provided by PMP22 and how its alteration causes CMT are unknown. Here we find that these abundant myelin proteins form a strong and specific complex. Mutagenesis and domain swapping experiments reveal that these proteins interact through interfaces within their transmembrane domains. We also find that the PMP22 A67T patient variant that causes an HNPP (Hereditary neuropathy with pressure palsies) phenotype, reflecting a heterozygous loss-of-function, maps to this interface. The PMP22 A67T variant results in the specific loss of MPZ association with PMP22 without affecting PMP22 localization to the plasma membrane or its interactions with other proteins. These data define the molecular basis for the MPZâ¼PMP22 interaction and indicate that the MPZâ¼PMP22 complex fulfills an important function in myelinating cells.
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The Endosomal Sorting Complexes Required for Transport (ESCRTs) drive membrane remodeling in a variety of cellular processes that include the formation of endosomal intralumenal vesicles (ILVs) during multivesicular body (MVB) biogenesis. During MVB sorting, ESCRTs recognize ubiquitin (Ub) attached to membrane protein cargo and execute ILV formation by controlling the activities of ESCRT-III polymers regulated by the AAA-ATPase Vps4. Exactly how these events are coordinated to ensure proper cargo loading into ILVs remains unclear. Here we discuss recent work documenting the ability of Bro1, an ESCRT-associated Ub-binding protein, to coordinate ESCRT-III and Vps4-dependent ILV biogenesis with upstream events such as cargo recognition.
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Cuerpos Multivesiculares , Proteínas de Saccharomyces cerevisiae , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Endosomas/metabolismo , Cuerpos Multivesiculares/metabolismo , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismoRESUMEN
AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission, and the plastic modulation of their surface levels determines synaptic strength. AMPARs of different subunit compositions fulfill distinct roles in synaptic long-term potentiation (LTP) and depression (LTD) to enable learning. Largely unknown endocytic mechanisms mediate the subunit-selective regulation of the surface levels of GluA1-homomeric Ca2+-permeable (CP) versus heteromeric Ca2+-impermeable (CI) AMPARs. Here, we report that the Alzheimer's disease risk factor CALM controls the surface levels of CP-AMPARs and thereby reciprocally regulates LTP and LTD in vivo to modulate learning. We show that CALM selectively facilitates the endocytosis of ubiquitinated CP-AMPARs via a mechanism that depends on ubiquitin recognition by its ANTH domain but is independent of clathrin. Our data identify CALM and related ANTH domain-containing proteins as the core endocytic machinery that determines the surface levels of CP-AMPARs to bidirectionally control synaptic plasticity and modulate learning in the mammalian brain.
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Enfermedad de Alzheimer , Enfermedad de Alzheimer/etiología , Animales , Endocitosis , Mamíferos/metabolismo , Plasticidad Neuronal/fisiología , Receptores AMPA/metabolismo , Factores de RiesgoRESUMEN
Attachment of ubiquitin (Ub) to cell surface proteins serves as a signal for internalization via clathrin-mediated endocytosis (CME). How ubiquitinated membrane proteins engage the internalization apparatus remains unclear. The internalization apparatus contains proteins such as Epsin and Eps15, which bind Ub, potentially acting as adaptors for Ub-based internalization signals. Here, we show that additional components of the endocytic machinery including CALM, HIP1R, and Sla2 bind Ub via their N-terminal ANTH domain, a domain belonging to the superfamily of ENTH and VHS domains. Structural studies revealed that Ub binds with µM affinity to a unique C-terminal region within the ANTH domain not found in ENTH domains. Functional studies showed that combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the yeast internalization apparatus caused defects in the Ub-dependent internalization of the GPCR Ste2 that was engineered to rely exclusively on Ub as an internalization signal. In contrast, these mutations had no effect on the internalization of Ste2 engineered to use an alternate Ub-independent internalization signal. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.
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Proteínas de la Membrana/metabolismo , Dominios Proteicos , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Clatrina/metabolismo , Endocitosis , Proteínas de la Membrana/genética , Mutación , Unión Proteica , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The family of Bro1 proteins coordinates the activity of the Endosomal Sorting Complexes Required for Transport (ESCRTs) to mediate a number of membrane remodeling events. These events culminate in membrane scission catalyzed by ESCRT-III, whose polymerization and disassembly is controlled by the AAA-ATPase, Vps4. Bro1-family members Alix and HD-PTP as well as yeast Bro1 have central "V" domains that noncovalently bind Ub and connect ubiquitinated proteins to ESCRT-driven functions such as the incorporation of ubiquitinated membrane proteins into intralumenal vesicles of multivesicular bodies. Recently, it was discovered that the V domain of yeast Bro1 binds the MIT domain of Vps4 to stimulate its ATPase activity. Here we determine the structural basis for how the V domain of human HD-PTP binds ubiquitin. The HD-PTP V domain also binds the MIT domain of Vps4, and ubiquitin binding to the HD-PTP V domain enhances its ability to stimulate Vps4 ATPase activity. Additionally, we found that V domains of both HD-PTP and Bro1 bind CHMP5 and Vps60, respectively, providing another potential molecular mechanism to alter Vps4 activity. These data support a model whereby contacts between ubiquitin, ESCRT-III, and Vps4 by V domains of the Bro1 family may coordinate late events in ESCRT-driven membrane remodeling events.
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ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras/química , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Ubiquitina/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Sitios de Unión , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Humanos , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Proteínas Tirosina Fosfatasas no Receptoras/genética , Dispersión del Ángulo Pequeño , Técnicas del Sistema de Dos Híbridos , ATPasas de Translocación de Protón Vacuolares/genética , Difracción de Rayos XRESUMEN
A hallmark of functionally significant interactions between Rab proteins and their targets is whether that binding depends on the type of nucleotide bound to the Rab GTPase. A system that can directly compare those sets of interactions mediated by a Rab in its GTP-bound conformation versus its GDP bound conformation would provide a direct route to finding biologically relevant partners. Comprehensive large-scale yeast 2-hybrid assays allow a potential method to compare one interactome against another provided that the same set of potentially interacting partners is interrogated between samples. Here we describe the use of such a yeast 2-hybrid system that lends itself toward comparing pairs of Rab mutants, locked in either their GTP or GDP conformation. Importantly, using a complex library of protein fragments as potential binding ("prey") partners, identification of interacting proteins as well as the domain(s) mediating those interactions can be determined using a series of sequence analyses and binary validation experiments.
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Técnicas del Sistema de Dos Híbridos , Biblioteca de Genes , Guanosina Trifosfato/metabolismo , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT-driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III-dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1-Vps4-ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.
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Adenosina Trifosfatasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Cuerpos Multivesiculares/enzimología , Biogénesis de Organelos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Adenosina Trifosfatasas/genética , Sitios de Unión , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Activación Enzimática , Microscopía Fluorescente , Modelos Moleculares , Cuerpos Multivesiculares/genética , Mutación , Dominios Proteicos , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Patients with malignant melanoma have a 5-year survival rate of only 15-20% once the tumor has metastasized to distant tissues. While MAP kinase pathway inhibitors (MAPKi) are initially effective for the majority of patients with melanoma harboring BRAFV600E mutation, over 90% of patients relapse within 2 years. Thus, there is a critical need for understanding MAPKi resistance mechanisms. In this manuscript, we performed a forward genetic screen using a whole genome shRNA library to identify negative regulators of vemurafenib resistance. We identified loss of NF1 and CUL3 as drivers of vemurafenib resistance. NF1 is a known driver of vemurafenib resistance in melanoma through its action as a negative regulator of RAS. However, the mechanism by which CUL3, a key protein in E3 ubiquitin ligase complexes, is involved in vemurafenib resistance was unknown. We found that loss of CUL3 was associated with an increase in RAC1 activity and MEKS298 phosphorylation. However, the addition of the Src family inhibitor saracatinib prevented resistance to vemurafenib in CUL3KD cells and reversed RAC1 activation. This finding suggests that inhibition of the Src family suppresses MAPKi resistance in CUL3KD cells by inactivation of RAC1. Our results also indicated that the loss of CUL3 does not promote the activation of RAC1 through stabilization, suggesting that CUL3 is involved in the stability of upstream regulators of RAC1. Collectively, our study identifies the loss of CUL3 as a driver of MAPKi resistance through activation of RAC1 and demonstrates that inhibition of the Src family can suppress the MAPKi resistance phenotype in CUL3KD cells by inactivating RAC1 protein.
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In yeast, the main ubiquitin ligase responsible for the sorting of proteins to the lysosomal vacuole is Rsp5, a member of the Nedd4 family of ligases whose distinguishing features are a catalytic homologous to E6AP C terminus (HECT) domain and 3 central WW domains that bind PY motifs in target proteins. Many substrates do not bind Rsp5 directly and instead rely on PY-containing adaptor proteins that interact with Rsp5. Recent studies indicate that the activities of these adaptors are elevated when they undergo ubiquitination, yet the mechanism whereby ubiquitination activates the adaptors and how this process is regulated remain unclear. Here, we report on a mechanism that explains how ubiquitination stimulates adaptor function and how this process can be regulated by the Rsp5-associated deubiquitinase, Ubp2. Our overexpression experiments revealed that several adaptors compete for Rsp5 in vivo. We found that the ability of the adaptors to compete effectively was enhanced by their ubiquitination and diminished by a block of their ubiquitination. Ubiquitination-dependent adaptor activation required a ubiquitin-binding surface within the Rsp5 catalytic HECT domain. Finally, like constitutively ubiquitinated adaptors, a Ubp2 deficiency increased both the adaptor activity and the ability to compete for Rsp5. Our data support a model whereby ubiquitinated Rsp5 adaptors are more active when "locked" onto Rsp5 via its N-lobe ubiquitin-binding surface and less active when they are "unlocked" by Ubp2-mediated deubiquitination.
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Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Ubiquitinación , Endopeptidasas/deficiencia , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Microorganismos Modificados Genéticamente/genética , Microorganismos Modificados Genéticamente/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
The synaptotagmin (syt) proteins have been widely studied for their role in regulating fusion of intracellular vesicles with the plasma membrane. Here we report that syt-17, an unusual isoform of unknown function, plays no role in exocytosis, and instead plays multiple roles in intracellular membrane trafficking. Syt-17 is localized to the Golgi complex in hippocampal neurons, where it coordinates import of vesicles from the endoplasmic reticulum to support neurite outgrowth and facilitate axon regrowth after injury. Further, we discovered a second pool of syt-17 on early endosomes in neurites. Loss of syt-17 disrupts endocytic trafficking, resulting in the accumulation of excess postsynaptic AMPA receptors and defective synaptic plasticity. Two distinct pools of syt-17 thus control two crucial, independent membrane trafficking pathways in neurons. Function of syt-17 appears to be one mechanism by which neurons have specialized their secretory and endosomal systems to support the demands of synaptic communication over sprawling neurite arbors.
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Endosomas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuritas/fisiología , Proyección Neuronal , Transmisión Sináptica , Sinaptotagminas/metabolismo , Animales , Células Cultivadas , Retículo Endoplásmico/metabolismo , Exocitosis , Femenino , Aparato de Golgi/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal , Cultivo Primario de Células , Sinaptotagminas/genéticaRESUMEN
Genetic screens using shRNA, CRISPR, or cDNA libraries rely on adequately transferring the library into cells for further assay. These libraries can have many different elements and each element can be present at different copy numbers within a given pooled library. Calculating how many recipient cells are needed to adequately sample all or most of the different elements within a library is important, especially if one wants to compare the outcomes of different genetic screens that rely on accurately reproducing the starting population of library-containing cells. Here we present a simple application that starts with a list of library elements and their abundance and calculates the minimum sampling number to achieve full transfer of the library to an acceptor cell population to a user-specified level of probability. Users can adjust several input parameters including designating a subpopulation over which the calculation is made. Finally, the program performs a series of Monte Carlo simulations of a user-specified number of picks to produce an empirically determined distribution of each library element.
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We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.