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1.
FEBS Lett ; 598(7): 801-817, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38369616

RESUMEN

Secretory proteins of Plasmodium exhibit differential spatial and functional activity within the host cell nucleus. However, the nuclear localization signals (NLSs) for these proteins remain largely uncharacterized. In this study, we have identified and characterized two NLSs in the circumsporozoite protein of Plasmodium falciparum (Pf-CSP). Both NLSs in the Pf-CSP contain clusters of lysine and arginine residues essential for specific interactions with the conserved tryptophan and asparagine residues of importin-α, facilitating nuclear translocation of Pf-CSP. While the two NLSs of Pf-CSP function independently and are both crucial for nuclear localization, a single NLS of Pf-CSP leads to weak nuclear localization. These findings shed light on the mechanism of nuclear penetrability of secretory proteins of Plasmodium proteins.


Asunto(s)
Señales de Localización Nuclear , Plasmodium falciparum , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/química , Señales de Localización Nuclear/metabolismo , alfa Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Núcleo Celular/metabolismo
2.
Clin Genet ; 105(3): 329-334, 2024 03.
Artículo en Inglés | MEDLINE | ID: mdl-38014644

RESUMEN

Osteogenesis imperfecta (OI) is a group of genetic disorders of bone formation characterized by soft and shorter brittle bones in affected individuals. OI is generally considered a collagenopathy resulting from abnormal expression of type I collagen. As assay system to detect the cellular level and quality of type I collagen would help in rapid and correct detection of OI from the diagnostic perspectives. Here, we report an immunofluorescence assay for detection of type I collagen in fibroblast models of OI and represented them into two broad categories based on the expression level and aggregation characteristics of pro-α1(I). Cell phenotypic assays of pro-α1(I) in OI-related gene knocked down fibroblasts revealed aggregates of pro-α1(I) in conditions with knockdown of SERPINF1, CRTAP, P3H1, PPIB, SERPINH1, FKBP10, TMEM38B, MESD, and KDELR2, whereas pro-α1(I) expression was very low in fibroblasts which had knockdown of IFITM5, SP7, BMP1, WNT1, CREB3L1, MBTPS2, and CCDC134. The expression of pro-α1(I) showed abundant and non-aggregated distribution in the fibroblasts with knockdown of non-OI skeletal disorder-related genes (RAB33B and IFT52). The in vitro assay accurately detected abnormally expressed pro-α1(I) levels in cellular models of various types of OI. Thus, this procedure represents a promising point-of-detection assay for potential diagnosis and therapeutic decisions in OI.


Asunto(s)
Colágeno Tipo I , Osteogénesis Imperfecta , Humanos , Colágeno Tipo I/genética , Osteogénesis Imperfecta/diagnóstico , Osteogénesis Imperfecta/genética , Genes Recesivos , Fibroblastos/metabolismo , Mutación , Proteínas de Transporte Vesicular/genética , Proteínas de la Membrana/genética
3.
Biochim Biophys Acta Mol Basis Dis ; 1869(6): 166741, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37146916

RESUMEN

Genetic mutations are involved in Mendelian disorders. Unbuffered intronic mutations in gene variants can generate aberrant splice sites in mutant transcripts, resulting in mutant isoforms of proteins with modulated expression, stability, and function in diseased cells. Here, we identify a deep intronic variant, c.794_1403A>G, in CRTAP by genome sequencing of a male fetus with osteogenesis imperfecta (OI) type VII. The mutation introduces cryptic splice sites in intron-3 of CRTAP, resulting in two mature mutant transcripts with cryptic exons. While transcript-1 translates to a truncated isoform (277 amino acids) with thirteen C-terminal non-wild-type amino acids, transcript-2 translates to a wild-type protein sequence, except that this isoform contains an in-frame fusion of non-wild-type twenty-five amino acids in a tetratricopeptide repeat sequence. Both mutant isoforms of CRTAP are unstable due to the presence of a unique 'GWxxI' degron, which finally leads to loss of proline hydroxylation and aggregation of type I collagen. Although type I collagen aggregates undergo autophagy, the overall proteotoxicity resulted in death of the proband cells by senescence. In summary, we present a genetic disease pathomechanism by linking a novel deep intronic mutation in CRTAP to unstable mutant isoforms of the protein in lethal OI type VII.


Asunto(s)
Colágeno Tipo I , Osteogénesis Imperfecta , Masculino , Humanos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Chaperonas Moleculares/genética , Mutación , Isoformas de Proteínas/genética , Aminoácidos
4.
Matrix Biol ; 115: 81-106, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36526215

RESUMEN

Aberrant forms of endoplasmic reticulum (ER)-resident chaperones are implicated in loss of protein quality control in rare diseases. Here we report a novel mutation (p.Asp233Asn) in the ER retention signal of MESD by whole exome sequencing of an individual diagnosed with osteogenesis imperfecta (OI) type XX. While MESDD233N has similar stability and chaperone activity as wild-type MESD, its mislocalization to cytoplasm leads to imbalance of ER proteostasis, resulting in improper folding and aggregation of proteins, including LRP5 and type I collagen. Aggregated LRP5 loses its plasma membrane localization to disrupt the expression of WNT-responsive genes, such as BMP2, BMP4, in proband fibroblasts. We show that MESD is a direct chaperone of pro-α1(I) [COL1A1], and absence of MESDD233N in ER results in cytosolic type I collagen aggregates that remain mostly not secreted. While cytosolic type I collagen aggregates block the intercellular nanotubes, decreased extracellular type I collagen also results in loss of interaction of ITGB1 with type I collagen and weaker attachment of fibroblasts to matrix. Although proband fibroblasts show increased autophagy to degrade the aggregated type I collagen, an overall cellular stress overwhelms the proband fibroblasts. In summary, we present an essential chaperone function of MESD for LRP5 and type I collagen and demonstrating how the D233N mutation in MESD correlates with impaired WNT signaling and proteostasis in OI.


Asunto(s)
Colágeno Tipo I , Osteogénesis Imperfecta , Humanos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Osteogénesis Imperfecta/genética , Osteogénesis Imperfecta/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Membrana Celular/metabolismo
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