Characterization of three TRAPPC11 variants suggests a critical role for the extreme carboxy terminus of the protein

AbstractTRAPPC11 was identified as a component of the TRAPP III complex that functions in membrane trafficking and autophagy. Variants in TRAPPC11 have been reported to be associated with a broad spectrum of phenotypes but all affected individuals display muscular pathology. Identifying additional variants will further our understanding of the clinical spectrum of phenotypes and will reveal regions of the protein critical for its functions. Here we report three individuals from unrelated families that have bi-allellic TRAPPC11 variants. Subject 1 harbors a compound heterozygous variant (c.1287 + 5G > A and c.3379_3380insT). The former variant results in a partial deletion of the foie gras domain (p.Ala372_Ser429del), while the latter variant results in a frame-shift and extension at the carboxy terminus (p.Asp1127Valfs*47). Subjects 2 and 3 both harbour a homozygous missense variant (c.2938G > A; p.Gly980Arg). Fibroblasts from all three subjects displayed membrane trafficking defects manifested as delayed endoplasmic reticulum (ER)-to-Golgi transport and/or a delay in protein exit from the Golgi. All three individuals also show a defect in glycosylation of an ER-resident glycoprotein. However, only the compound heterozygous subject displayed an autophagic flux defect. Collectively, our characterization of these individuals with bi-allelic TRAPPC11 variants highlights the functional importance of the carboxy-terminal portion of the protein.

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Hyperpolarization-activated inward leakage currents caused by deletion or mutation of carboxy-terminal tyrosines of the Na+/K+-ATPase α subunit

The Journal of General Physiology ◽

10.1085/jgp.200910301 ◽

2010 ◽

Vol 135 (2) ◽

pp. 115-134 ◽

Cited By ~ 35

Author(s):

Susan Meier ◽

Neslihan N. Tavraz ◽

Katharina L. Dürr ◽

Thomas Friedrich

Keyword(s):

Binding Site ◽

Critical Role ◽

Aromatic Amino Acids ◽

Cation Binding ◽

Carboxy Terminus ◽

Leakage Currents ◽

Α Subunit ◽

Inward Currents ◽

The Common ◽

Carboxy Terminal

The Na+/K+-ATPase mediates electrogenic transport by exporting three Na+ ions in exchange for two K+ ions across the cell membrane per adenosine triphosphate molecule. The location of two Rb+ ions in the crystal structures of the Na+/K+-ATPase has defined two “common” cation binding sites, I and II, which accommodate Na+ or K+ ions during transport. The configuration of site III is still unknown, but the crystal structure has suggested a critical role of the carboxy-terminal KETYY motif for the formation of this “unique” Na+ binding site. Our two-electrode voltage clamp experiments on Xenopus oocytes show that deletion of two tyrosines at the carboxy terminus of the human Na+/K+-ATPase α2 subunit decreases the affinity for extracellular and intracellular Na+, in agreement with previous biochemical studies. Apparently, the ΔYY deletion changes Na+ affinity at site III but leaves the common sites unaffected, whereas the more extensive ΔKETYY deletion affects the unique site and the common sites as well. In the absence of extracellular K+, the ΔYY construct mediated ouabain-sensitive, hyperpolarization-activated inward currents, which were Na+ dependent and increased with acidification. Furthermore, the voltage dependence of rate constants from transient currents under Na+/Na+ exchange conditions was reversed, and the amounts of charge transported upon voltage pulses from a certain holding potential to hyperpolarizing potentials and back were unequal. These findings are incompatible with a reversible and exclusively extracellular Na+ release/binding mechanism. In analogy to the mechanism proposed for the H+ leak currents of the wild-type Na+/K+-ATPase, we suggest that the ΔYY deletion lowers the energy barrier for the intracellular Na+ occlusion reaction, thus destabilizing the Na+-occluded state and enabling inward leak currents. The leakage currents are prevented by aromatic amino acids at the carboxy terminus. Thus, the carboxy terminus of the Na+/K+-ATPase α subunit represents a structural and functional relay between Na+ binding site III and the intracellular cation occlusion gate.

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DRAM-3 modulates autophagy and promotes cell survival in the absence of glucose

Cell Death and Differentiation ◽

10.1038/cdd.2015.26 ◽

2015 ◽

Vol 22 (10) ◽

pp. 1714-1726 ◽

Cited By ~ 11

Author(s):

M Mrschtik ◽

J O'Prey ◽

L Y Lao ◽

J S Long ◽

F Beaumatin ◽

...

Keyword(s):

Cell Survival ◽

Membrane Trafficking ◽

Clonogenic Survival ◽

Autophagic Flux ◽

Normal Tissues ◽

Atg Proteins ◽

Dna Damaging Agents ◽

Evolutionarily Conserved ◽

Starvation Conditions

Abstract Macroautophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. The process operates under basal conditions as a mechanism to turnover damaged or misfolded proteins and organelles. As a result, it has a major role in preserving cellular integrity and viability. In addition to this basal function, macroautophagy can also be modulated in response to various forms of cellular stress, and the rate and cargoes of macroautophagy can be tailored to facilitate appropriate cellular responses in particular situations. The macroautophagy machinery is regulated by a group of evolutionarily conserved autophagy-related (ATG) proteins and by several other autophagy regulators, which either have tissue-restricted expression or operate in specific contexts. We report here the characterization of a novel autophagy regulator that we have termed DRAM-3 due to its significant homology to damage-regulated autophagy modulator (DRAM-1). DRAM-3 is expressed in a broad spectrum of normal tissues and tumor cells, but different from DRAM-1, DRAM-3 is not induced by p53 or DNA-damaging agents. Immunofluorescence studies revealed that DRAM-3 localizes to lysosomes/autolysosomes, endosomes and the plasma membrane, but not the endoplasmic reticulum, phagophores, autophagosomes or Golgi, indicating significant overlap with DRAM-1 localization and with organelles associated with macroautophagy. In this regard, we further proceed to show that DRAM-3 expression causes accumulation of autophagosomes under basal conditions and enhances autophagic flux. Reciprocally, CRISPR/Cas9-mediated disruption of DRAM-3 impairs autophagic flux confirming that DRAM-3 is a modulator of macroautophagy. As macroautophagy can be cytoprotective under starvation conditions, we also tested whether DRAM-3 could promote survival on nutrient deprivation. This revealed that DRAM-3 can repress cell death and promote long-term clonogenic survival of cells grown in the absence of glucose. Interestingly, however, this effect is macroautophagy-independent. In summary, these findings constitute the primary characterization of DRAM-3 as a modulator of both macroautophagy and cell survival under starvation conditions.

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Coronavirus Particle Assembly: Primary Structure Requirements of the Membrane Protein

Journal of Virology ◽

10.1128/jvi.72.8.6838-6850.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6838-6850 ◽

Cited By ~ 116

Author(s):

Cornelis A. M. de Haan ◽

Lili Kuo ◽

Paul S. Masters ◽

Harry Vennema ◽

Peter J. M. Rottier

Keyword(s):

Hepatitis Virus ◽

Critical Role ◽

Point Mutations ◽

Major Protein ◽

Dependent Manner ◽

Carboxy Terminus ◽

Concentration Dependent Manner ◽

Particle Assembly ◽

Virus Like Particles ◽

Carboxy Terminal

ABSTRACT Coronavirus-like particles morphologically similar to normal virions are assembled when genes encoding the viral membrane proteins M and E are coexpressed in eukaryotic cells. Using this envelope assembly assay, we have studied the primary sequence requirements for particle formation of the mouse hepatitis virus (MHV) M protein, the major protein of the coronavirion membrane. Our results show that each of the different domains of the protein is important. Mutations (deletions, insertions, point mutations) in the luminal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain had effects on the assembly of M into enveloped particles. Strikingly, the extreme carboxy-terminal residue is crucial. Deletion of this single residue abolished particle assembly almost completely; most substitutions were strongly inhibitory. Site-directed mutations in the carboxy terminus of M were also incorporated into the MHV genome by targeted recombination. The results supported a critical role for this domain of M in viral assembly, although the M carboxy terminus was more tolerant of alteration in the complete virion than in virus-like particles, likely because of the stabilization of virions by additional intermolecular interactions. Interestingly, glycosylation of M appeared not essential for assembly. Mutations in the luminal domain that abolished the normal O glycosylation of the protein or created an N-glycosylated form had no effect. Mutant M proteins unable to form virus-like particles were found to inhibit the budding of assembly-competent M in a concentration-dependent manner. However, assembly-competent M was able to rescue assembly-incompetent M when the latter was present in low amounts. These observations support the existence of interactions between M molecules that are thought to be the driving force in coronavirus envelope assembly.

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Comparison of thermosensitive alleles of the CDC25 gene involved in the cAMP metabolism of Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/124.4.797 ◽

1990 ◽

Vol 124 (4) ◽

pp. 797-806 ◽

Cited By ~ 2

Author(s):

A Petitjean ◽

F Hilger ◽

K Tatchell

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Product ◽

Critical Role ◽

Missense Mutations ◽

Nucleotide Exchange ◽

Carboxy Terminus ◽

Reading Frame ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Carboxy Terminal

Abstract The CDC25 gene from Saccharomyces cerevisiae is an essential component of the RAS-adenylate cyclase pathway. Genetic and biochemical evidence has led to the proposal that the gene product may act upstream of RAS, possibly as a guanine nucleotide exchange factor. We report here the cloning, sequencing and characterization of four mutations in the CDC25 gene. All four are missense mutations which reside within the carboxy-terminal quarter of the single open reading frame found within the gene. Three of the four are missense mutations in the same amino acid codon. A search of protein data bases reveals that the carboxy terminus of the putative CDC25 gene product is similar to that of LTE1, a gene required for growth at low temperature and SCD25, a suppressor of cdc25. Taken together these data indicate that the carboxy terminus of CDC25 plays a critical role in the function of the CDC25 gene product and that other proteins, such as LTE1 or SCD25, may have related activities.

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Characterization of Immunodominant Linear B-Cell Epitopes on the Carboxy Terminus of the Rinderpest Virus Nucleocapsid Protein

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.4.658-664.2004 ◽

2004 ◽

Vol 11 (4) ◽

pp. 658-664 ◽

Cited By ~ 11

Author(s):

Kang-Seuk Choi ◽

Jin-Ju Nah ◽

Young-Joon Ko ◽

Shien-Young Kang ◽

Kyoung-Jin Yoon ◽

...

Keyword(s):

Antigenic Site ◽

Enzyme Linked Immunosorbent Assay ◽

Carboxy Terminus ◽

Rinderpest Virus ◽

N Protein ◽

Amino Terminal ◽

Immunogenic Epitope ◽

Carboxy Terminal ◽

Gst Fusion Proteins

ABSTRACT The nucleocapsid (N) protein of rinderpest virus (RPV) is one of the most abundant and immunogenic viral proteins expressed during natural or experimental infection. To identify immunogenic epitopes on the N protein, different forms of RPV N protein, including the full-length protein (N1-525), an amino-terminal construct (N1-179), and a carboxy-terminal construct (N414-496), were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The antigenicity of each recombinant protein was evaluated by Western immunoblotting. All recombinants were recognized by hyperimmune RPV bovine antisera, indicating that immunoreactive epitopes may be present at both ends of the N protein. However, GST-N414-496 was much more antigenic than GST-N1-179 when tested with sera from vaccinated cattle, suggesting that an immunodominant or highly immunogenic epitope(s) may be located at the carboxy terminus of the N protein. Epitope mapping with overlapping peptides representing different regions of the carboxy terminus (amino acids 415 to 524) revealed three nonoverlapping antigenic sites in regions containing the residues 440VPQVRKETRASSR452 (site 1), 479PEADTDPL486 (site 2), and 520DKDLL524 (site 3). Among these, antigenic site 2 showed the strongest reactivity with hyperimmune anti-RPV bovine sera in a peptide enzyme-linked immunosorbent assay but did not react with hyperimmune caprine sera raised against peste-des-petits-ruminants virus, which is antigenically closely related to RPV. Identification of an immunodominant linear antigenic site at the carboxy terminus of the N protein may provide an antigen basis for designing diagnostics specific for RPV.

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Characterization of dominant and recessive assembly-defective mutations in mouse neurofilament NF-M.

The Journal of Cell Biology ◽

10.1083/jcb.111.5.1987 ◽

1990 ◽

Vol 111 (5) ◽

pp. 1987-2003 ◽

Cited By ~ 57

Author(s):

P C Wong ◽

D W Cleveland

Keyword(s):

Wild Type ◽

Carboxy Terminus ◽

M Gene ◽

Amino Terminal ◽

Filament Assembly ◽

Carboxy Terminal ◽

Filament Network ◽

Dominant Mutants ◽

Amino Acid Epitope

We have generated a set of amino- and carboxy-terminal deletions of the neurofilament NF-M gene and determined the molecular consequences of forced expression of these mutant constructs in mouse fibroblasts. To follow the expression of mutant NF-M subunits in transfected cells, a 12 amino acid epitope (from the human c-myc protein) was expressed at the carboxy terminus of each mutant. We show that NF-M molecules missing up to 90 or 70% of the nonhelical carboxy-terminal tail or amino-terminal head domains, respectively, incorporate readily into an intermediate filament network comprised either of vimentin or NF-L, whereas deletions into either the amino- or carboxy-terminal alpha-helical rod region generate assembly-incompetent polypeptides. Carboxy-terminal deletions into the rod domain invariably yield dominant mutants which rapidly disrupt the array of filaments comprised of NF-L or vimentin. Accumulation of these mutant NF-M subunits disrupts vimentin filament arrays even when present at approximately 1% the level of the wild-type subunits. In contrast, the amino-terminal deletions into the rod produce pseudo-recessive mutants that perturb the wild-type NF-L or vimentin arrays only modestly. The inability of such amino-terminal mutants to disrupt wild-type subunits defines a region near the amino-terminal alpha-helical rod domain (residues 75-126) that is required for the earliest steps in filament assembly.

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Biallelic VPS35L pathogenic variants cause 3C/Ritscher-Schinzel-like syndrome through dysfunction of retriever complex

Journal of Medical Genetics ◽

10.1136/jmedgenet-2019-106213 ◽

2019 ◽

Vol 57 (4) ◽

pp. 245-253 ◽

Cited By ~ 3

Author(s):

Kohji Kato ◽

Yasuyoshi Oka ◽

Hideki Muramatsu ◽

Filipp F Vasilev ◽

Takanobu Otomo ◽

...

Keyword(s):

Core Protein ◽

Early Stage ◽

Critical Role ◽

Missense Variant ◽

Compound Heterozygous ◽

Chondrodysplasia Punctata ◽

Loss Of Function ◽

Skeletal Malformation ◽

Endosomal Membrane ◽

Pathogenic Variants

Background3C/Ritscher-Schinzel syndrome is characterised by congenital cranio-cerebello-cardiac dysplasia, where CCDC22 and WASHC5 are accepted as the causative genes. In combination with the retromer or retriever complex, these genes play a role in endosomal membrane protein recycling. We aimed to identify the gene abnormality responsible for the pathogenicity in siblings with a 3C/Ritscher-Schinzel-like syndrome, displaying cranio-cerebello-cardiac dysplasia, coloboma, microphthalmia, chondrodysplasia punctata and complicated skeletal malformation.MethodsExome sequencing was performed to identify pathogenic variants. Cellular biological analyses and generation of knockout mice were carried out to elucidate the gene function and pathophysiological significance of the identified variants.ResultsWe identified compound heterozygous pathogenic variants (c.1097dup; p.Cys366Trpfs*28 and c.2755G>A; p.Ala919Thr) in the VPS35L gene, which encodes a core protein of the retriever complex. The identified missense variant lacked the ability to form the retriever complex, and the frameshift variant induced non-sense-mediated mRNA decay, thereby confirming biallelic loss of function of VPS35L. In addition, VPS35L knockout cells showed decreased autophagic function in nutrient-rich and starvation conditions, as well as following treatment with Torin 1. We also generated Vps35l−/− mice and demonstrated that they were embryonic lethal at an early stage, between E7.5 and E10.5.ConclusionsOur results suggest that biallelic loss-of-function variants in VPS35L underlies 3C/Ritscher-Schinzel-like syndrome. Furthermore, VPS35L is necessary for autophagic function and essential for early embryonic development. The data presented here provide a new insight into the critical role of the retriever complex in fetal development.

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Publisher Correction: Characterization of three TRAPPC11 variants suggests a critical role for the extreme carboxy terminus of the protein

Scientific Reports ◽

10.1038/s41598-020-76436-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Miroslav P. Milev ◽

Daniela Stanga ◽

Anne Schänzer ◽

Andrés Nascimento ◽

Djenann Saint-Dic ◽

...

Keyword(s):

Critical Role ◽

Carboxy Terminus

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Expanding the Clinical and Genetic Spectrum of RAB28-Related Cone-Rod Dystrophy: Pathogenicity of Novel Variants in Italian Families

International Journal of Molecular Sciences ◽

10.3390/ijms22010381 ◽

2020 ◽

Vol 22 (1) ◽

pp. 381

Author(s):

Giancarlo Iarossi ◽

Valerio Marino ◽

Paolo Enrico Maltese ◽

Leonardo Colombo ◽

Fabiana D’Esposito ◽

...

Keyword(s):

Membrane Trafficking ◽

Missense Variant ◽

Photoreceptor Cells ◽

Compound Heterozygous ◽

Loss Of Function ◽

Nonsense Mutations ◽

Structural Mechanism ◽

Splicing Variants ◽

Dynamics Simulations ◽

Retinal Thinning

The small Ras-related GTPase Rab-28 is highly expressed in photoreceptor cells, where it possibly participates in membrane trafficking. To date, six alterations in the RAB28 gene have been associated with autosomal recessive cone-rod dystrophies. Confirmed variants include splicing variants, missense and nonsense mutations. Here, we present a thorough phenotypical and genotypical characterization of five individuals belonging to four Italian families, constituting the largest cohort of RAB28 patients reported in literature to date. All probands displayed similar clinical phenotype consisting of photophobia, decreased visual acuity, central outer retinal thinning, and impaired color vision. By sequencing the four probands, we identified: a novel homozygous splicing variant; two novel nonsense variants in homozygosis; a novel missense variant in compound heterozygous state with a previously reported nonsense variant. Exhaustive molecular dynamics simulations of the missense variant p.(Thr26Asn) in both its active and inactive states revealed an allosteric structural mechanism that impairs the binding of Mg2+, thus decreasing the affinity for GTP. The impaired GTP-GDP exchange ultimately locks Rab-28 in a GDP-bound inactive state. The loss-of-function mutation p.(Thr26Asn) was present in a compound heterozygosis with the nonsense variant p.(Arg137*), which does not cause mRNA-mediated decay, but is rather likely degraded due to its incomplete folding. The frameshift p.(Thr26Valfs4*) and nonsense p.(Leu13*) and p.(Trp107*) variants, if translated, would lack several key structural components necessary for the correct functioning of the encoded protein.

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α-synuclein impairs autophagosome maturation through abnormal actin stabilization

PLoS Genetics ◽

10.1371/journal.pgen.1009359 ◽

2021 ◽

Vol 17 (2) ◽

pp. e1009359

Author(s):

Souvarish Sarkar ◽

Abby L. Olsen ◽

Katja Sygnecka ◽

Kelly M. Lohr ◽

Mel B. Feany

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Actin Cytoskeleton ◽

Membrane Trafficking ◽

Transgenic Animals ◽

Autophagic Flux ◽

Pathological Mechanism ◽

Abnormal Mitochondria ◽

Trafficking Defects ◽

Autophagosome Maturation

Vesicular trafficking defects, particularly those in the autophagolysosomal system, have been strongly implicated in the pathogenesis of Parkinson’s disease and related α-synucleinopathies. However, mechanisms mediating dysfunction of membrane trafficking remain incompletely understood. Using a Drosophila model of α-synuclein neurotoxicity with widespread and robust pathology, we find that human α-synuclein expression impairs autophagic flux in aging adult neurons. Genetic destabilization of the actin cytoskeleton rescues F-actin accumulation, promotes autophagosome clearance, normalizes the autophagolysosomal system, and rescues neurotoxicity in α-synuclein transgenic animals through an Arp2/3 dependent mechanism. Similarly, mitophagosomes accumulate in human α-synuclein-expressing neurons, and reversal of excessive actin stabilization promotes both clearance of these abnormal mitochondria-containing organelles and rescue of mitochondrial dysfunction. These results suggest that Arp2/3 dependent actin cytoskeleton stabilization mediates autophagic and mitophagic dysfunction and implicate failure of autophagosome maturation as a pathological mechanism in Parkinson’s disease and related α-synucleinopathies.

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