Clusterin (Apolipoprotein J), a Molecular Chaperone That Facilitates Degradation of the Copper-ATPases ATP7A and ATP7B

The copper-transporting P1B-type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and toxicity disorders, Menkes and Wilson diseases, respectively. This report describes the interaction between the Cu-ATPases and clusterin and demonstrates a chaperone-like role for clusterin in facilitating their degradation. Clusterin interacted with both ATP7A and ATP7B in mammalian cells. This interaction increased under conditions of oxidative stress and with mutations in ATP7B that led to its misfolding and mislocalization. A Wilson disease patient mutation (G85V) led to enhanced ATP7B turnover, which was further exacerbated when cells overexpressed clusterin. We demonstrated that clusterin-facilitated degradation of mutant ATP7B is likely to involve the lysosomal pathway. The knockdown and overexpression of clusterin increased and decreased, respectively, the Cu-ATPase-mediated copper export capacity of cells. These results highlight a new role for intracellular clusterin in mediating Cu-ATPase quality control and hence in the normal maintenance of copper homeostasis, and in promoting cell survival in the context of disease. Based on our findings, it is possible that variations in clusterin expression and function could contribute to the variable clinical expression of Menkes and Wilson diseases.

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PLEKHA5, PLEKHA6 and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis

10.1101/2021.06.17.448791 ◽

2021 ◽

Author(s):

Sandra Citi ◽

Sophie Sluysmans ◽

Isabelle Mean ◽

Tong Xiao ◽

Amina Boukhatemi ◽

...

Keyword(s):

Plasma Membrane ◽

Copper Homeostasis ◽

Cell Periphery ◽

Ww Domains ◽

C Terminus ◽

Menkes Atpase ◽

Junctional Protein ◽

Cellular Copper ◽

And Function

Copper homeostasis is crucial for cellular physiology and development, and its dysregulation leads to disease. The Menkes ATPase ATP7A plays a key role in copper efflux, by trafficking from the Golgi to the plasma membrane upon cell exposure to elevated copper, but the mechanisms that target ATP7A to the cell periphery are poorly understood. PDZD11 interacts with the C-terminus of ATP7A, which contains sequences involved in ATP7A trafficking, but the role of PDZD11 in ATP7A localization is unknown. Here we identify PLEKHA5 and PLEKHA6 as new interactors of PDZD11, which similarly to the junctional protein PLEKHA7 bind to PDZD11 N-terminus through their WW domains. Using CRISPR-KO kidney epithelial cells, we show by immunofluorescence that WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) recruit PDZD11 to distinct plasma membrane localizations, and that they are required for the efficient anterograde targeting of ATP7A to the cell periphery in elevated copper. Pulldown experiments show that WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. However, WW-PLEKHAs and PDZD11 are not necessary for ATP7A Golgi localization in basal copper, ATP7A copper-induced exit from the Golgi, and ATP7A retrograde trafficking to the Golgi. Finally, measuring bioavailable copper with the labile copper probe CF4 shows that WW-PLEKHAs and PDZD11 are required to maintain low intracellular copper levels when cells are exposed to elevated copper. These data indicate that WW-PLEKHAs-PDZD11 complexes regulate the localization and function of ATP7A to modulate cellular copper homeostasis.

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The Role of Centrosome Distal Appendage Proteins (DAPs) in Nephronophthisis and Ciliogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms222212253 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12253

Author(s):

Fatma Mansour ◽

Felix J. Boivin ◽

Iman B. Shaheed ◽

Markus Schueler ◽

Kai M. Schmidt-Ott

Keyword(s):

Primary Cilium ◽

Mammalian Cells ◽

Vital Role ◽

Vesicle Docking ◽

Genes Encoding ◽

Mother Centriole ◽

Stage Renal Disease ◽

End Stage ◽

And Function

The primary cilium is found in most mammalian cells and plays a functional role in tissue homeostasis and organ development by modulating key signaling pathways. Ciliopathies are a group of genetically heterogeneous disorders resulting from defects in cilia development and function. Patients with ciliopathic disorders exhibit a range of phenotypes that include nephronophthisis (NPHP), a progressive tubulointerstitial kidney disease that commonly results in end-stage renal disease (ESRD). In recent years, distal appendages (DAPs), which radially project from the distal end of the mother centriole, have been shown to play a vital role in primary ciliary vesicle docking and the initiation of ciliogenesis. Mutations in the genes encoding these proteins can result in either a complete loss of the primary cilium, abnormal ciliary formation, or defective ciliary signaling. DAPs deficiency in humans or mice commonly results in NPHP. In this review, we outline recent advances in our understanding of the molecular functions of DAPs and how they participate in nephronophthisis development.

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PLEKHA5, PLEKHA6 and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis

Molecular Biology of the Cell ◽

10.1091/mbc.e21-07-0355 ◽

2021 ◽

Author(s):

Sophie Sluysmans ◽

Isabelle Méan ◽

Tong Xiao ◽

Amina Boukhatemi ◽

Flavio Ferreira ◽

...

Keyword(s):

Plasma Membrane ◽

Copper Homeostasis ◽

Cell Periphery ◽

Ww Domains ◽

C Terminus ◽

Menkes Atpase ◽

Junctional Protein ◽

Cellular Copper ◽

And Function

Copper homeostasis is crucial for cellular physiology and development, and its dysregulation leads to disease. The Menkes ATPase ATP7A plays a key role in copper efflux, by trafficking from the Golgi to the plasma membrane upon cell exposure to elevated copper, but the mechanisms that target ATP7A to the cell periphery are poorly understood. PDZD11 interacts with the C-terminus of ATP7A, which contains sequences involved in ATP7A trafficking, but the role of PDZD11 in ATP7A localization is unknown. Here we identify PLEKHA5 and PLEKHA6 as new interactors of PDZD11, which bind to PDZD11 N-terminus through their WW domains similarly to the junctional protein PLEKHA7. Using CRISPR-KO kidney epithelial cells, we show by immunofluorescence microscopy that WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) recruit PDZD11 to distinct plasma membrane localizations, and that they are required for the efficient anterograde targeting of ATP7A to the cell periphery in elevated copper conditions. Pulldown experiments show that WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. However, WW-PLEKHAs and PDZD11 are not necessary for ATP7A Golgi localization in basal copper, ATP7A copper-induced exit from the Golgi, and ATP7A retrograde trafficking to the Golgi. Finally, measuring bioavailable and total cellular copper, metallothionein-1 expression and cell viability shows that WW-PLEKHAs and PDZD11 are required to maintain low intracellular copper levels when cells are exposed to elevated copper. These data indicate that WW-PLEKHAs-PDZD11 complexes regulate the localization and function of ATP7A to promote copper extrusion in elevated copper.

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A Metzincin and TIMP-Like Protein Pair of a Phage Origin Sensitize Listeria monocytogenes to Phage Lysins and Other Cell Wall Targeting Agents

Microorganisms ◽

10.3390/microorganisms9061323 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1323

Author(s):

Etai Boichis ◽

Nadejda Sigal ◽

Ilya Borovok ◽

Anat A. Herskovits

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Mammalian Cells ◽

Protein Pair ◽

Growth Conditions ◽

Wall Structure ◽

Antibiotic Resistant ◽

Extracellular Milieu ◽

Virion Release ◽

Genes Encoding

Infection of mammalian cells by Listeria monocytogenes (Lm) was shown to be facilitated by its phage elements. In a search for additional phage remnants that play a role in Lm’s lifecycle, we identified a conserved locus containing two XRE regulators and a pair of genes encoding a secreted metzincin protease and a lipoprotein structurally similar to a TIMP-family metzincin inhibitor. We found that the XRE regulators act as a classic CI/Cro regulatory switch that regulates the expression of the metzincin and TIMP-like genes under intracellular growth conditions. We established that when these genes are expressed, their products alter Lm morphology and increase its sensitivity to phage mediated lysis, thereby enhancing virion release. Expression of these proteins also sensitized the bacteria to cell wall targeting compounds, implying that they modulate the cell wall structure. Our data indicate that these effects are mediated by the cleavage of the TIMP-like protein by the metzincin, and its subsequent release to the extracellular milieu. While the importance of this locus to Lm pathogenicity remains unclear, the observation that this phage-associated protein pair act upon the bacterial cell wall may hold promise in the field of antibiotic potentiation to combat antibiotic resistant bacterial pathogens.

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A Family of Genes Clustered at the Triplo-lethal Locus of Drosophila melanogaster Has an Unusual Evolutionary History and Significant Synteny With Anopheles gambiae

Genetics ◽

10.1093/genetics/165.2.613 ◽

2003 ◽

Vol 165 (2) ◽

pp. 613-621 ◽

Cited By ~ 2

Author(s):

Douglas R Dorer ◽

Jamie A Rudnick ◽

Etsuko N Moriyama ◽

Alan C Christensen

Keyword(s):

Phylogenetic Analysis ◽

Drosophila Melanogaster ◽

Anopheles Gambiae ◽

Evolutionary History ◽

Codon Bias ◽

Good Target ◽

The Family ◽

Genes Encoding ◽

And Function ◽

Gambiae Genome

Abstract Within the unique Triplo-lethal region (Tpl) of the Drosophila melanogaster genome we have found a cluster of 20 genes encoding a novel family of proteins. This family is also present in the Anopheles gambiae genome and displays remarkable synteny and sequence conservation with the Drosophila cluster. The family is also present in the sequenced genome of D. pseudoobscura, and homologs have been found in Aedes aegypti mosquitoes and in four other insect orders, but it is not present in the sequenced genome of any noninsect species. Phylogenetic analysis suggests that the cluster evolved prior to the divergence of Drosophila and Anopheles (250 MYA) and has been highly conserved since. The ratio of synonymous to nonsynonymous substitutions and the high codon bias suggest that there has been selection on this family both for expression level and function. We hypothesize that this gene family is Tpl, name it the Osiris family, and consider possible functions. We also predict that this family of proteins, due to the unique dosage sensitivity and the lack of homologs in noninsect species, would be a good target for genetic engineering or novel insecticides.

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Spine impairment in mice high-expressing neuregulin 1 due to LIMK1 activation

Cell Death and Disease ◽

10.1038/s41419-021-03687-8 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Peng Chen ◽

Hongyang Jing ◽

Mingtao Xiong ◽

Qian Zhang ◽

Dong Lin ◽

...

Keyword(s):

Neural Development ◽

Protein Level ◽

Brain Regions ◽

Postmortem Brain ◽

Pathophysiological Mechanism ◽

Brain Disorders ◽

Spine Density ◽

Genes Encoding ◽

High Level ◽

And Function

AbstractThe genes encoding for neuregulin1 (NRG1), a growth factor, and its receptor ErbB4 are both risk factors of major depression disorder and schizophrenia (SZ). They have been implicated in neural development and synaptic plasticity. However, exactly how NRG1 variations lead to SZ remains unclear. Indeed, NRG1 levels are increased in postmortem brain tissues of patients with brain disorders. Here, we studied the effects of high-level NRG1 on dendritic spine development and function. We showed that spine density in the prefrontal cortex and hippocampus was reduced in mice (ctoNrg1) that overexpressed NRG1 in neurons. The frequency of miniature excitatory postsynaptic currents (mEPSCs) was reduced in both brain regions of ctoNrg1 mice. High expression of NRG1 activated LIMK1 and increased cofilin phosphorylation in postsynaptic densities. Spine reduction was attenuated by inhibiting LIMK1 or blocking the NRG1–LIMK1 interaction, or by restoring NRG1 protein level. These results indicate that a normal NRG1 protein level is necessary for spine homeostasis and suggest a pathophysiological mechanism of abnormal spines in relevant brain disorders.

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ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization

Molecular Biology of the Cell ◽

10.1091/mbc.e02-02-0078 ◽

2002 ◽

Vol 13 (9) ◽

pp. 3078-3095 ◽

Cited By ~ 30

Author(s):

Annette L. Boman ◽

Paul D. Salo ◽

Melissa J. Hauglund ◽

Nicole L. Strand ◽

Shelly J. Rensink ◽

...

Keyword(s):

Membrane Trafficking ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Protein Localization ◽

Carboxypeptidase Y ◽

Minor Role ◽

Temperature Sensitive ◽

Adp Ribosylation ◽

And Function ◽

Adp Ribosylation Factor

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

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Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5620 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5620-5631 ◽

Cited By ~ 159

Author(s):

B Shan ◽

X Zhu ◽

P L Chen ◽

T Durfee ◽

Y Yang ◽

...

Keyword(s):

Transcription Factor ◽

Mammalian Cells ◽

Leucine Zipper ◽

T Antigen ◽

Transactivation Domain ◽

Recognition Sequence ◽

Cellular Genes ◽

Genes Encoding ◽

Associated Proteins ◽

Transcription Factor E2f

The retinoblastoma protein interacts with a number of cellular proteins to form complexes which are probably crucial for its normal physiological function. To identify these proteins, we isolated nine distinct clones by direct screening of cDNA expression libraries using purified RB protein as a probe. One of these clones, Ap12, is expressed predominantly at the G1-S boundary and in the S phase of the cell cycle. The nucleotide sequence of Ap12 has features characteristic of transcription factors. The C-terminal region binds to unphosphorylated RB in regions similar to those to which T antigen binds and contains a transactivation domain. A region containing a potential leucine zipper flanked by basic residues is able to bind an E2F recognition sequence specifically. Expression of Ap12 in mammalian cells significantly enhances E2F-dependent transcriptional activity. These results suggest that Ap12 encodes a protein with properties known to be characteristic of transcription factor E2F.

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RegA, an AraC-Like Protein, Is a Global Transcriptional Regulator That Controls Virulence Gene Expression in Citrobacter rodentium

Infection and Immunity ◽

10.1128/iai.00770-08 ◽

2008 ◽

Vol 76 (11) ◽

pp. 5247-5256 ◽

Cited By ~ 44

Author(s):

Emily Hart ◽

Ji Yang ◽

Marija Tauschek ◽

Michelle Kelly ◽

Matthew J. Wakefield ◽

...

Keyword(s):

Mammalian Cells ◽

Virulence Gene ◽

Virulence Determinants ◽

Intestinal Colonization ◽

Citrobacter Rodentium ◽

Promoter Regions ◽

Reading Frame ◽

Virulence Gene Expression ◽

Genes Encoding ◽

Colonization Factors

ABSTRACT Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5α, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization.

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Secreted CXCL12 (SDF-1) forms dimers under physiological conditions

Biochemical Journal ◽

10.1042/bj20111341 ◽

2012 ◽

Vol 442 (2) ◽

pp. 433-442 ◽

Cited By ~ 35

Author(s):

Paramita Ray ◽

Sarah A. Lewin ◽

Laura Anne Mihalko ◽

Sasha-Cai Lesher-Perez ◽

Shuichi Takayama ◽

...

Keyword(s):

Breast Cancer ◽

Extracellular Space ◽

Mammalian Cells ◽

Cell Signalling ◽

Xenograft Model ◽

Physiological Conditions ◽

Cxc Chemokine ◽

Chemokine Ligand ◽

Chemokine Cxcl12 ◽

And Function

Chemokine CXCL12 (CXC chemokine ligand 12) signalling through CXCR (CXC chemokine receptor) 4 and CXCR7 has essential functions in development and underlies diseases including cancer, atherosclerosis and autoimmunity. Chemokines may form homodimers that regulate receptor binding and signalling, but previous studies with synthetic CXCL12 have produced conflicting evidence for homodimerization. We used bioluminescence imaging with GL (Gaussia luciferase) fusions to investigate dimerization of CXCL12 secreted from mammalian cells. Using column chromatography and GL complementation, we established that CXCL12 was secreted from mammalian cells as both monomers and dimers. Secreted CXCL12 also formed homodimers in the extracellular space. Monomeric CXCL12 preferentially activated CXCR4 signalling through Gαi and Akt, whereas dimeric CXCL12 more effectively promoted recruitment of β-arrestin 2 to CXCR4 and chemotaxis of CXCR4-expressing breast cancer cells. We also showed that CXCR7 preferentially sequestered monomeric CXCL12 from the extracellular space and had minimal effects on dimeric CXCL12 in cell-based assays and an orthotopic tumour xenograft model of human breast cancer. These studies establish that CXCL12 secreted from mammalian cells forms homodimers under physiological conditions. Since monomeric and dimeric CXCL12 have distinct effects on cell signalling and function, our results have important implications for ongoing efforts to target CXCL12 pathways for therapy.

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