Biosynthesis of Surfactant Protein C (SP-C)

Wang, Wenjing; Russo, Scott J.; Mulugeta, Surafel; Beers, Michael F.

doi:10.1074/jbc.m201537200

Cited by 61 publications

(40 citation statements)

References 40 publications

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“…Within 48 h of introduction of plasmid cDNA, expression of the EGFP/SP-C fusion protein was detected in cytoplasmic vesicles (Fig. 2), which co-localizes with CD63, a marker of lysosomal-like organelles (24,25). By both deletion analysis (SP-C 19 -194 ) and polyalanine substitution (SP-C A11-18 ), EGFP/ proSP-C fusion proteins each lacking the PDDY domain were expressed in a diffuse reticular pattern that co-localizes with calnexin consistent with ER retention (24,25).…”

Section: Mutation Of the Nh 2 -Flanking Propeptide To Remove A Ppdy Mmentioning

confidence: 99%

“…2), which co-localizes with CD63, a marker of lysosomal-like organelles (24,25). By both deletion analysis (SP-C 19 -194 ) and polyalanine substitution (SP-C A11-18 ), EGFP/ proSP-C fusion proteins each lacking the PDDY domain were expressed in a diffuse reticular pattern that co-localizes with calnexin consistent with ER retention (24,25). The role of the PPDY domain in trafficking was then directly assessed by polyalanine substitution of only these four residues, which also resulted in disruption of normal trafficking of the EGFP fusion protein (Fig.…”

Section: Mutation Of the Nh 2 -Flanking Propeptide To Remove A Ppdy Mmentioning

confidence: 99%

“…Immunoblotting of transferred samples was done using successive incubations with primary immune sera and horseradish peroxidase-conjugated secondary antibodies as previously described (25). The resulting fluorescence images visualized by ECL were captured either by exposure to film or by direct acquisition using the Kodak 2000 Image System.…”

Section: Western Blottingmentioning

confidence: 99%

“…HA-tagged proSP-C -The hemagglutinin (HA) tag (YPYDVPDYA) was added to the NH 2 terminus of rat SP-C by PCR using pcDNA-SP-C as a template, as previously described (25,26). The 5Ј (forward) primer was an 82-mer and contained a KpnI site (bold), a Kozak sequence, and the HA coding sequence (underlined): TAC AAG GGT ACC ATG GAC TAC CCA TAC GAT GTT CCA GAT TAC GCT GCT GAC ATG GGT AGC AAA GAG GTA CTG ATG GAG AGC.…”

Section: Sp-c Cdna Expression Constructsmentioning

confidence: 99%

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Anterograde Transport of Surfactant Protein C Proprotein to Distal Processing Compartments Requires PPDY-mediated Association with Nedd4 Ubiquitin Ligases

Kotorashvili

Russo

Mulugeta

et al. 2009

Journal of Biological Chemistry

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Biosynthesis of surfactant protein C (SP-C) by alveolar type 2 cells requires proteolytic processing of a 21-kDa propeptide (proSP-C 21 ) in post-Golgi compartments to yield a 3.7-kDa mature form. Scanning alanine mutagenesis, binding assays, and co-immunoprecipitation were used to characterize the proSP-C targeting domain. Delivery of proSP-C 21 to distal processing organelles is dependent upon the NH 2 -terminal cytoplasmic SP-C propeptide, which contains a conserved PPDY motif. In A549 cells, transfection of EGFP/proSP-C 21 constructs containing polyalanine substitution for Glu 11 -Thr 18 , 13 PPDY 16 , or 14 P, 16 Y produced endoplasmic reticulum retention of the fusion proteins. Protein-protein interactions of proSP-C with known WW domains were screened using a solid-phase array that revealed binding of the proSP-C NH 2 terminus to several WW domains found in the Nedd4 family of E3 ligases. Specificity of the interaction was confirmed by co-immunoprecipitation of proSP-C and Nedd4 or Nedd4-2 in epithelial cell lines. By Western blotting and reverse transcription-PCR, both forms were detected in primary human type 2 cells. Knockdown of Nedd4-2 by small interference RNA transfection of cultured human type 2 cells blocked processing of 35 S-labeled proSP-C 21. Mutagenesis of potential acceptor sites for ubiquitination in the cytosolic domain of proSP-C (Lys 6 , Lys 34 , or both) failed to inhibit trafficking of EGFP/proSP-C 21 . These results indicate that PPDYmediated interaction with Nedd4 E3-ligases is required for trafficking of proSP-C. We speculate that the Nedd4/proSP-C tandem is part of a larger protein complex containing a ubiquitinated component that further directs its transport. Surfactant protein C (SP-C)3 is a hydrophobic lung-specific protein that co-isolates with the biophysically active phospholipid fraction of pulmonary surfactant (1). The 3.7-kDa alveolar form of SP-C (SP-C 3.7 or mature SP-C) is a 35-amino acid, valine-rich peptide that results from synthesis of a 197 amino acid precursor form (proSP-C 21 ). Within the endoplasmic reticulum (ER), proSP-C 21 exists as a type II bitopic transmembrane protein (N cyt /C lumen ), which remains integrally membrane-associated while it is extensively processed by four post-translational endoproteolytic cleavages that take place in post-Golgi compartments (2-6). The resulting mature protein is transferred into the lumen of lamellar bodies for secretion into the alveolar space together with surfactant phospholipid and SP-B (7). Thus, the complete biosynthesis of SP-C 3.7 is critically dependent upon oligomeric sorting and targeting of proSP-C 21 to subcellular processing compartments distal to the ER/Golgi.Using deletional mutagenesis, we had previously shown that the NH 2 -terminal propeptide flanking domain (NTP) of proSP-C, which is oriented into the cytosol, contained potential targeting motifs for post-Golgi trafficking (8, 9). The candidate region included the amino acid sequence Met 10 -Thr 18 and, when present, was sufficient to direct transf...

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Section: Mutation Of the Nh 2 -Flanking Propeptide To Remove A Ppdy Mmentioning

confidence: 99%

Section: Mutation Of the Nh 2 -Flanking Propeptide To Remove A Ppdy Mmentioning

confidence: 99%

Section: Western Blottingmentioning

confidence: 99%

Section: Sp-c Cdna Expression Constructsmentioning

confidence: 99%

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Anterograde Transport of Surfactant Protein C Proprotein to Distal Processing Compartments Requires PPDY-mediated Association with Nedd4 Ubiquitin Ligases

Kotorashvili

Russo

Mulugeta

et al. 2009

Journal of Biological Chemistry

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“…ABCA3 mediates uptake of choline-phospholipids into LAMP3-positive vesicles to convert lysosomal organelles into lamellar body-like organelles (7,18). The lipid transport function of ABCA3 protein can therefore be evaluated by comparing endogenous content of choline-phospholipid-rich vesicles and the level of LAMP3, a marker of lysosomal organelles such as multivesicular bodies and lamellar bodies (34,35). Expression of wild-type ABCA3-GFP in HEK-293 cells was found to increase the level of LAMP3, as well as the ABCA3 is constituted by 2 transmembrane domains (TMD), each of which contains 6 putative transmembrane segments (TM), 2 extracellular domains (ECD), and 2 nucleotide binding domains (NBD).…”

Section: Subcellular Localization and Glycosylation Of Abca3-gfp And mentioning

confidence: 99%

Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease

Matsumura¹,

Ban²,

Inagaki³

2008

American Journal of Physiology-Lung Cellular and Molecular Phys

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However, the mechanisms underlying the less severe phenotype of patients with ABCA3 mutation are unclear. In this study, we characterized ABCA3 mutant proteins identified in pediatric interstitial lung disease (pILD). E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Vanadate-induced nucleotide trapping and photoaffinity labeling of wild-type and mutant proteins using 8-azido-[ 32 P]ATP revealed an aberrant catalytic cycle in these mutant proteins. These results demonstrate the importance of a functional catalytic cycle in lipid transport of ABCA3 and suggest a pathophysiological mechanism of pILD due to ABCA3 mutation.

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DNA with Different Local Torsional States Affects RecA‐Mediated Recombination Progression

2017

ChemPhysChem

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DNA topology is thought to affect DNA enzyme activity. The helical structure of duplex DNA dictates the change of topological states during strand separation when DNA is constrained. During the repair of DNA double-stranded breaks, the RecA nucleoprotein filament invades DNA and carries out consecutive strand exchange reactions coupled with duplex DNA strand separation. It has been suggested that torsional strain could be generated and its accumulation could inhibit strand exchange. We used hairpin and nicked DNA substrates to test how torsional strain alters the RecA-mediated strand exchange efficiency. Single-molecule tethered particle motion (TPM) experiments showed that torsionally constrained hairpin DNA substrates returned nearly no successful strand exchange events catalyzed by RecA. Surprisingly, the strand exchange efficiencies increase in the presence of DNA nicks or loop disruption. The dwell time of transient RecA events in hairpin is shorter compared to those found in nicked or fork DNA substrates, which suggests a limited strand exchange progression in hairpin substrates. Our observation shows that RecA generates local torsional strain during strand exchange, and the inability to dissipate this torsional strain inhibits homologous recombination progression. DNA topological states are thus important regulation measures of DNA recombination.

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Biosynthesis of Surfactant Protein C (SP-C)

Abstract: Rat surfactant protein C (SP-C

Cited by 61 publications

References 40 publications

Anterograde Transport of Surfactant Protein C Proprotein to Distal Processing Compartments Requires PPDY-mediated Association with Nedd4 Ubiquitin Ligases

Anterograde Transport of Surfactant Protein C Proprotein to Distal Processing Compartments Requires PPDY-mediated Association with Nedd4 Ubiquitin Ligases

Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease

DNA with Different Local Torsional States Affects RecA‐Mediated Recombination Progression

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