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1.
Curr Opin Cell Biol ; 85: 102266, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37866019

RESUMO

Neurofilaments (NFs) and GFAP are cytoskeletal intermediate filaments (IFs) that support cellular processes unfolding within the uniquely complex environments of neurons and astrocytes, respectively. This review highlights emerging concepts on the transitions between stable and destabilized IF networks in the nervous system. While self-association between transiently structured low-complexity IF domains promotes filament assembly, the opposing destabilizing actions of phosphorylation-mediated filament severing facilitate faster intracellular transport. Cellular proteases, including caspases and calpains, produce a variety of IF fragments, which may interact with N-degron and C-degron pathways of the protein degradation machinery. The rapid adoption of NF and GFAP-based clinical biomarker tests is contrasted with the lagging understanding of the dynamics between the native IF proteins and their fragments.


Assuntos
Citoesqueleto , Filamentos Intermediários , Filamentos Intermediários/metabolismo , Citoesqueleto/metabolismo , Sistema Nervoso , Neurônios , Fosforilação
2.
Mol Biol Cell ; : mbcE23030094, 2023 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-37672338

RESUMO

Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by KLHL16 mutations. KLHL16 encodes gigaxonin, which regulates intermediate filament (IF) turnover. Previous neuropathological studies and examination of postmortem brain tissue in the current study revealed involvement of astrocytes in GAN. To develop a clinically-relevant model, we reprogrammed skin fibroblasts from seven GAN patients to pluripotent stem cells (iPSCs), which were used to generate neural progenitor cells (NPCs), astrocytes, and brain organoids. Multiple isogenic control clones were derived via CRISPR/Cas9 gene editing of one patient line carrying the G332R gigaxonin mutation. All GAN iPSCs were deficient for gigaxonin and displayed patient-specific increased vimentin expression. GAN NPCs had lower nestin expression and fewer nestin-positive cells compared to isogenic controls, but nestin morphology was unaffected. GAN brain organoids were marked by the presence of neurofilament and GFAP aggregates. GAN iPSC-astrocytes displayed striking dense perinuclear vimentin and GFAP accumulations and abnormal nuclear morphology. In over-expression systems, GFAP oligomerization and perinuclear aggregation were augmented in the presence of vimentin. GAN patient cells with large perinuclear vimentin aggregates accumulated significantly more nuclear KLHL16 mRNA compared to cells without vimentin aggregates. As an early effector of KLHL16 mutations, vimentin may be a potential target in GAN.

3.
bioRxiv ; 2023 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-36993491

RESUMO

Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by KLHL16 mutations. KLHL16 encodes gigaxonin, a regulator of intermediate filament (IF) protein turnover. Previous neuropathological studies and our own examination of postmortem GAN brain tissue in the current study revealed astrocyte involvement in GAN. To study the underlying mechanisms, we reprogrammed skin fibroblasts from seven GAN patients carrying different KLHL16 mutations to iPSCs. Isogenic controls with restored IF phenotypes were derived via CRISPR/Cas9 editing of one patient carrying a homozygous missense mutation (G332R). Neural progenitor cells (NPCs), astrocytes, and brain organoids were generated through directed differentiation. All GAN iPSC lines were deficient for gigaxonin, which was restored in the isogenic control. GAN iPSCs displayed patient-specific increased vimentin expression, while GAN NPCs had decreased nestin expression compared to isogenic control. The most striking phenotypes were observed in GAN iPSC-astrocytes and brain organoids, which exhibited dense perinuclear IF accumulations and abnormal nuclear morphology. GAN patient cells with large perinuclear vimentin aggregates accumulated nuclear KLHL16 mRNA. In over-expression studies, GFAP oligomerization and perinuclear aggregation were potentiated in the presence of vimentin. As an early effector of KLHL16 mutations, vimentin may serve as a potential therapeutic target in GAN.

5.
Molecules ; 23(12)2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30563071

RESUMO

Numerous proteins are involved in the multiple pathways of the DNA damage response network and play a key role to protect the genome from the wide variety of damages that can occur to DNA. An example of this is the structure-specific endonuclease ERCC1-XPF. This heterodimeric complex is in particular involved in nucleotide excision repair (NER), but also in double strand break repair and interstrand cross-link repair pathways. Here we review the function of ERCC1-XPF in various DNA repair pathways and discuss human disorders associated with ERCC1-XPF deficiency. We also overview our molecular and structural understanding of XPF-ERCC1.


Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Sequência de Aminoácidos , Animais , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Proteínas de Ligação a DNA/química , Endonucleases/química , Sequências Hélice-Alça-Hélice , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais
6.
Hum Mutat ; 39(2): 255-265, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29105242

RESUMO

Pathogenic variants in genes, which encode DNA repair and damage response proteins, result in a number of genomic instability syndromes with features of accelerated aging. ERCC4 (XPF) encodes a protein that forms a complex with ERCC1 and is required for the 5' incision during nucleotide excision repair. ERCC4 is also FANCQ, illustrating a critical role in interstrand crosslink repair. Pathogenic variants in this gene cause xeroderma pigmentosum, XFE progeroid syndrome, Cockayne syndrome (CS), and Fanconi anemia. We performed massive parallel sequencing for 42 unsolved cases submitted to the International Registry of Werner Syndrome. Two cases, each carrying two novel heterozygous ERCC4 variants, were identified. The first case was a compound heterozygote for: c.2395C > T (p.Arg799Trp) and c.388+1164_792+795del (p.Gly130Aspfs*18). Further molecular and cellular studies indicated that the ERCC4 variants in this patient are responsible for a phenotype consistent with a variant of CS. The second case was heterozygous for two variants in cis: c.[1488A > T; c.2579C > A] (p.[Gln496His; Ala860Asp]). While the second case also had several phenotypic features of accelerated aging, we were unable to provide biological evidence supporting the pathogenic roles of the associated ERCC4 variants. Precise genetic causes and disease mechanism of the second case remains to be determined.


Assuntos
Síndrome de Cockayne/genética , Proteínas de Ligação a DNA/genética , Xeroderma Pigmentoso/genética , Actinas/genética , Idoso , Reparo do DNA/genética , Proteínas de Ligação a DNA/química , Anemia de Fanconi/genética , Feminino , Predisposição Genética para Doença/genética , Humanos , Lamina Tipo A/genética , Masculino , Pessoa de Meia-Idade , Linhagem
7.
J Biol Chem ; 292(7): 2842-2853, 2017 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-28028171

RESUMO

The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the helix-hairpin-helix (HhH) domains of XPF and ERCC-XPF and show that the binding to single-stranded DNA (ssDNA)/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences but with a clear preference for guanine-containing substrates. NMR titration experiments and in vitro DNA binding assays also show that, within the heterodimeric ERCC1-XPF complex, XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-overlapping DNA binding domains in the ERCC1-XPF heterodimer jointly bind to an ssDNA/dsDNA substrate and, thereby, at least partially dictate the incision position during damage removal. Based on structural models, NMR titrations, DNA-binding studies, site-directed mutagenesis, charge distribution, and sequence conservation, we propose that the HhH domain of ERCC1 binds to dsDNA upstream of the damage, and XPF binds to the non-damaged strand within a repair bubble.


Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/química , Dimerização , Sequências Hélice-Alça-Hélice , Humanos , Ligação Proteica , Especificidade por Substrato
8.
J Theor Biol ; 383: 130-7, 2015 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-26231416

RESUMO

During years 2007 and 2008, we published three papers (Jahandideh, 2007a, JTB, 246, 159-166; Jahandideh, 2007b, JTB, 248, 721-726; Jahandideh, 2008, JTB, 255, 113-118) investigating sequence and structural parameters in adaptation of proteins to low temperatures. Our studies revealed important features in cold-adaptation of proteins. Here, we calculate values of a new set of physico-chemical parameters and perform a comparative systematic analysis on a more comprehensive database of psychrophilic-mesophilic homologous protein pairs. Our obtained results confirm that psychrophilicity rules are not merely the inverse rules of thermostability; for instance, although contact order is reported as a key feature in thermostability, our results have shown no significant difference between contact orders of psychrophilic proteins compared to mesophilic proteins. We are optimistic that these findings would help future efforts to propose a strategy for designing cold-adapted proteins.


Assuntos
Temperatura Baixa , Proteínas/química , Aclimatação/fisiologia , Adaptação Fisiológica , Fenômenos Químicos , Bases de Dados de Proteínas , Ligação de Hidrogênio , Peso Molecular , Conformação Proteica , Estrutura Secundária de Proteína
9.
J Biol Chem ; 290(33): 20541-55, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26085086

RESUMO

The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe(231), Leu(231) lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation.


Assuntos
Síndrome de Cockayne/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Mutação Puntual , Sequência de Aminoácidos , Proteínas de Ligação a DNA/química , Dimerização , Endonucleases/química , Humanos , Modelos Químicos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
10.
J Biomol Struct Dyn ; 33(6): 1315-25, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25027605

RESUMO

Human prion diseases are associated with misfolding or aggregation of the Human Prion Protein (HuPrP). Missense mutations in the HuPrP gene, contribute to conversion of HuPrP(C) to HuPrP(Sc) and amyloid formation. Based on our previous comprehensive study, three missense mutations, from two different functional groups, i.e. disease-related mutations, and protective mutations, were selected and extensive molecular dynamics simulations were performed on these three mutants to compare their dynamics and conformations with those of the wildtype HuPrP. In addition to simulations of monomeric forms of mutants, in order to study the dominant-negative effect of protective mutation (E219K), 30-ns simulations were performed on E219K-wildtype and wildtype-wildtype dimeric forms. Our results indicate that, although after 30-ns simulations the global three-dimensional structure of models remain fairly intact, the disease-related mutations (V210I and Q212P) introduce local structural changes, i.e. close contact changes and secondary structure changes, in addition to global flexibility changes. Furthermore, our results support the loss of hydrophobic interaction due to the mutations in hydrophobic core that has been reported by previous NMR and computational studies. On the other hand, this protective mutation (E219K) results in helix elongation, and significant increases of overall flexibility of E219K mutant during 30-ns simulation. In conclusion, the simulations of dimeric forms suggest that the dominant-negative effect of this protective mutation (E219K) is due to the incompatible structures and dynamics of allelic variants during conversion process.


Assuntos
Simulação de Dinâmica Molecular , Príons/química , Sequência de Aminoácidos , Códon , Genes Dominantes , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Polimorfismo Genético , Príons/genética , Conformação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína
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