RESUMO
Plakin proteins form connections that link the cell membrane to the intermediate filament cytoskeleton. Their interactions are mediated by a highly conserved linker domain through an unresolved mechanism. Here analysis of the human periplakin linker domain structure reveals a bi-lobed module transected by an electropositive groove. Key basic residues within the periplakin groove are vital for co-localization with vimentin in human cells and compromise direct binding which also requires acidic residues D176 and E187 in vimentin. We propose a model whereby basic periplakin linker domain residues recognize acidic vimentin side chains and form a complementary binding groove. The model is shared amongst diverse linker domains and can be used to investigate the effects of pathogenic mutations in the desmoplakin linker associated with arrhythmogenic right ventricular cardiomyopathy. Linker modules either act solely or collaborate with adjacent plakin repeat domains to create strong and adaptable tethering within epithelia and cardiac muscle.
Assuntos
Plaquinas/química , Plaquinas/metabolismo , Vimentina/química , Vimentina/metabolismo , Sequência de Aminoácidos , Aminoácidos Acídicos/química , Aminoácidos Acídicos/genética , Aminoácidos Acídicos/metabolismo , Ácido Aspártico/metabolismo , Ácido Glutâmico/metabolismo , Células HeLa , Humanos , Filamentos Intermediários/química , Filamentos Intermediários/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Plaquinas/genética , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Estrutura Quaternária de Proteína , Vimentina/genéticaRESUMO
BACKGROUND: Chronic lymphocytic leukemia (CLL) is frequently accompanied by immune dysregulation. AIMS: In this multicenter prospective study, we investigated whether heavy + light chains (HLC: IgGκ, IgGλ, IgAκ, IgAκ, IgMκ, IgMλ) and IgG subclasses (IgG1, IgG2, IgG3, and IgG4) could be used as novel prognostic markers of immunoparesis in 105 treatment-naïve patients with CLL. RESULTS: Heavy + light chains immunoparesis of ≥1, ≥2, and ≥3 isotypes was evident in 74 (70%), 58 (55%), and 36 (34%) patients, respectively. Severe HLC immunoparesis was identified in 40 (38%) patients. Of the IgG subclasses, IgG1 and IgG2 were most frequently suppressed, affecting 46 (44%) and 36 (34%) patients, respectively; 63 (60%) patients had low levels of at least one IgG subclass. In multivariate analysis, severe HLC immunoparesis (hazard ratio [HR]: 36.5; P = .010) and ΣFLC ≥ 70 mg/L (HR: 13.2; P = .004) were the only factors independently associated with time to first treatment (TTFT). A risk model including these variables identified patients with 0, 1, and 2 risk factors and significantly different TTFT (P < .001). Patients with two factors represented an ultra-high-risk group with a median TTFT of only 1.3 months. CONCLUSION: The above findings demonstrate the potential for the use of HLC immunoparesis, together with sFLC measurements, as future prognostic biomarkers in CLL.
Assuntos
Cadeias Pesadas de Imunoglobulinas/sangue , Cadeias Leves de Imunoglobulina/sangue , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/epidemiologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Masculino , Pessoa de Meia-Idade , Modelos Teóricos , Prognóstico , Modelos de Riscos Proporcionais , Tempo para o TratamentoRESUMO
Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. Envoplakin has a single plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoplakin's complete PRD fold, revealing binding determinants within its electropositive binding groove. Four of its five internal repeats recognize negatively charged patches within vimentin via five basic determinants that are identified by nuclear magnetic resonance spectroscopy. Mutations of the Lys1901 or Arg1914 binding determinants delocalize heterodimeric envoplakin from intracellular vimentin and keratin filaments in cultured cells. Recognition of vimentin is abolished when its residues Asp112 or Asp119 are mutated. The latter slot intermediate filament rods into basic PRD domain grooves through electrosteric complementarity in a widely applicable mechanism. Together this reveals how plakin family members form dynamic linkages with cytoskeletal frameworks.
Assuntos
Filamentos Intermediários/metabolismo , Proteínas de Membrana/metabolismo , Precursores de Proteínas/metabolismo , Vimentina/metabolismo , Motivos de Aminoácidos , Animais , Clonagem Molecular , Escherichia coli/metabolismo , Humanos , Filamentos Intermediários/química , Proteínas de Membrana/química , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Precursores de Proteínas/química , Vimentina/químicaRESUMO
The plakin repeat domain is a distinctive hallmark of the plakin superfamily of proteins, which are found within all epithelial tissues. Plakin repeat domains mediate the interactions of these proteins with the cell cytoskeleton and are critical for the maintenance of tissue integrity. Despite their biological importance, no solution state resonance assignments are available for any homologue. Here we report the essentially complete (1)H, (13)C and (15)N backbone chemical shift assignments of the singular 22 kDa plakin repeat domain of human envoplakin, providing the means to investigate its interactions with ligands including intermediate filaments.
Assuntos
Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular , Plaquinas/química , Precursores de Proteínas/química , Sequências Repetitivas de Aminoácidos , Sequência de Aminoácidos , HumanosRESUMO
Ms1 (also known as STARS and ABRA) has been shown to act as an early stress response gene in processes as different as hypertrophy in skeletal and cardiac muscle and growth of collateral blood vessels. It is important for cardiac development in zebrafish and is upregulated in mouse models for cardiac hypertrophy as well as in human failing hearts. Ms1 possesses actin binding sites at its C-terminus and is usually found in the cell bound to actin filaments in the cytosol or in sarcomeres. We determined the NMR structure of the only folded domain of Ms1 comprising the second actin binding site called actin binding domain 2 (ABD2, residues 294-375), and found that it is similar to the winged helix-turn-helix fold adopted mainly by DNA binding domains of transcriptional factors. In vitro experiments show specific binding of this domain, in combination with a newly discovered AT-hook motif located N-terminally, to the sequence (A/C/G)AAA(C/A). NMR and fluorescence titration experiments confirm that this motif is indeed bound specifically by the recognition helix. In neonatal rat cardiomyocytes endogenous Ms1 is found in the nucleus in a spotted pattern, reminiscent of PML bodies. In adult rat cardiomyocytes Ms1 is exclusively found in the sarcomere. A nuclear localisation site in the N-terminus of the protein is required for nuclear localisation. This suggests that Ms1 has the potential to act directly in the nucleus through specific interaction with DNA in development and potentially as a response to stress in adult tissues.
Assuntos
Núcleo Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miócitos Cardíacos/metabolismo , Motivos AT-Hook , Animais , Sítios de Ligação , Células HeLa , Humanos , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Ratos , Sarcômeros/metabolismoRESUMO
MS1 is a protein predominantly expressed in cardiac and skeletal muscle that is upregulated in response to stress and contributes to development of hypertrophy. In the aortic banding model of left ventricular hypertrophy, its cardiac expression was significantly upregulated within 1 h. Its function is postulated to depend on its F-actin binding ability, located to the C-terminal half of the protein, which promotes stabilization of F-actin in the cell thus releasing myocardin-related transcription factors to the nucleus where they stimulate transcription in cooperation with serum response factor. Initial attempts to purify the protein only resulted in heavily degraded samples that showed distinct bands on SDS gels, suggesting the presence of stable domains. Using a combination of combinatorial domain hunting and sequence analysis, a set of potential domains was identified. The C-terminal half of the protein actually contains two independent F-actin binding domains. The most C-terminal fragment (294-375), named actin binding domain 2 (ABD2), is independently folded while a proximal fragment called ABD1 (193-296) binds to F-actin with higher affinity than ABD2 (KD 2.21 ± 0.47 µM vs. 10.61 ± 0.7 µM), but is not structured by itself in solution. NMR interaction experiments show that it binds and folds in a cooperative manner to F-actin, justifying the label of domain. The architecture of the MS1 C-terminus suggests that ABD1 alone could completely fulfill the F-actin binding function opening up the intriguing possibility that ABD2, despite its high level of conservation, could have developed other functions.