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1.
Adv Exp Med Biol ; 1415: 263-267, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37440043

RESUMO

The formation of higher-order protein assemblies (commonly called protein aggregates) has long been associated with disease states, particularly in neurodegenerative disorders. Within the eye, protein aggregation has also been implicated in various retinal degenerative diseases ranging from retinitis pigmentosa (RP) to Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) to age-related macular degeneration (AMD). Yet, many essential cellular processes including transcription, translation, and the formation of non-membrane bound organelles require the formation of functional, non-pathologic protein aggregates to maintain cellular homeostasis. Thus, functional protein aggregates, also called condensates, likely play essential roles in maintaining normal retina function. However, currently, there is a critical gap in our knowledge: What proteins form higher-order assemblies under normal conditions within the retina and what function do these structures serve? Herein, we present data suggesting that protein aggregation is identifiable in multiple retinal layers of normal, healthy murine retina, and briefly discuss the potential contributions of aggregated proteins to normal retinal function, with a focus on the photoreceptor inner and outer segment.


Assuntos
Degeneração Macular , Degeneração Retiniana , Humanos , Camundongos , Animais , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Agregados Proteicos , Degeneração Macular/genética , Degeneração Macular/patologia , Retina/patologia , Proteínas Amiloidogênicas
2.
Hum Mutat ; 43(12): 1945-1955, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35998264

RESUMO

Fibulin-3 (F3 or EFEMP1) is a disulfide-rich, secreted glycoprotein necessary for maintaining extracellular matrix (ECM) and connective tissue integrity. Three studies have identified distinct autosomal recessive F3 mutations in individuals with Marfan Syndrome-like phenotypes. Herein, we characterize how one of these mutations, c.163T>C; p.Cys55Arg (C55R), disrupts F3 secretion, quaternary structure, and function by forming unique extracellular disulfide-linked homodimers. Dual cysteine mutants suggest that the C55R-induced disulfide species forms because of the new availability of Cys70 on adjacent F3 monomers. Surprisingly, mutation of single cysteines located near Cys55 (i.e., Cys29, Cys42, Cys48, Cys61, Cys70, Cys159, and Cys171) also produced similar extracellular disulfide-linked dimers, suggesting that this is not a phenomenon isolated to the C55R mutant. To assess C55R functionality, F3 knockout (KO) retinal pigmented epithelial (RPE) cells were generated, followed by reintroduction of wild-type (WT) or C55R F3. F3 KO cells produced lower levels of the ECM remodeling enzyme, matrix metalloproteinase 2, and reduced formation of collagen VI ECM filaments, both of which were partially rescued by WT F3 overexpression. However, C55R F3 was unable to compensate for these same ECM-related defects. Our results highlight the unique behavior of particular cysteine mutations in F3 and uncover potential routes to restore C55R F3 loss-of-function.


Assuntos
Cisteína , Dissulfetos , Humanos , Cisteína/genética , Metaloproteinase 2 da Matriz/genética , Proteínas da Matriz Extracelular/genética , Matriz Extracelular/genética , Mutação
3.
Mol Vis ; 27: 179-190, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33907372

RESUMO

Purpose: Retinitis pigmentosa (RP) is an inherited retinal disorder that results in the degeneration of photoreceptor cells, ultimately leading to severe visual impairment. We characterized a consanguineous family from Southern India wherein a 25 year old individual presented with night blindness since childhood. The purpose of this study was to identify the causative mutation for RP in this individual as well as characterize how the mutation may ultimately affect protein function. Methods: We performed a complete ophthalmologic examination of the proband followed by exome sequencing. The likely causative mutation was identified and modeled in cultured cells, evaluating its expression, solubility (both with western blotting), subcellular distribution, (confocal microscopy), and testing whether this variant induced endoplasmic reticulum (ER) stress (quantitative PCR [qPCR] and western blotting). Results: The proband presented with generalized and parafoveal retinal pigmented epithelium (RPE) atrophy with bone spicule-like pigmentation in the midperiphery and arteriolar attenuation. Optical coherence tomography scans through the macula of both eyes showed atrophy of the outer retinal layers with loss of the ellipsoid zone, whereas the systemic examination of this individual was normal. The proband's parents and sibling were asymptomatic and had normal funduscopic examinations. We discovered a novel homozygous p.Pro388Ser mutation in the tubby-like protein 1 (TULP1) gene in the individual with RP. In cultured cells, the P388S mutation does not alter the subcellular distribution of TULP1 or induce ER stress when compared to wild-type TULP1, but instead significantly lowers protein stability as indicated with steady-state and cycloheximide-chase experiments. Conclusions: These results add to the list of known mutations in TULP1 identified in individuals with RP and suggest a possible unique pathogenic mechanism in TULP1-induced RP, which may be shared among select mutations in TULP1.


Assuntos
Proteínas do Olho/genética , Mutação de Sentido Incorreto/genética , Retinose Pigmentar/genética , Adulto , Western Blotting , Consanguinidade , Eletrorretinografia , Homozigoto , Humanos , Índia , Masculino , Microscopia Confocal , Linhagem , Reação em Cadeia da Polimerase em Tempo Real , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/fisiopatologia , Tomografia de Coerência Óptica , Acuidade Visual/fisiologia , Sequenciamento do Exoma
4.
Biochemistry ; 52(42): 7512-21, 2013 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-24060347

RESUMO

Thermobifida fusca o-succinylbenzoate synthase (OSBS), a member of the enolase superfamily that catalyzes a step in menaquinone biosynthesis, has an amino acid sequence that is 22 and 28% identical with those of two previously characterized OSBS enzymes from Escherichia coli and Amycolatopsis sp. T-1-60, respectively. These values are considerably lower than typical levels of sequence identity among homologous proteins that have the same function. To determine how such divergent enzymes catalyze the same reaction, we determined the structure of T. fusca OSBS and identified amino acids that are important for ligand binding. We discovered significant differences in structure and conformational flexibility between T. fusca OSBS and other members of the enolase superfamily. In particular, the 20s loop, a flexible loop in the active site that permits ligand binding and release in most enolase superfamily proteins, has a four-amino acid deletion and is well-ordered in T. fusca OSBS. Instead, the flexibility of a different region allows the substrate to enter from the other side of the active site. T. fusca OSBS was more tolerant of mutations at residues that were critical for activity in E. coli OSBS. Also, replacing active site amino acids found in one protein with the amino acids that occur at the same place in the other protein reduces the catalytic efficiency. Thus, the extraordinary divergence between these proteins does not appear to reflect a higher tolerance of mutations. Instead, large deletions outside the active site were accompanied by alteration of active site size and electrostatic interactions, resulting in small but significant differences in ligand binding.


Assuntos
Actinomycetales/enzimologia , Evolução Biológica , Carbono-Carbono Liases/metabolismo , Escherichia coli/enzimologia , Magnésio/metabolismo , Sítios de Ligação , Carbono-Carbono Liases/química , Carbono-Carbono Liases/genética , Catálise , Domínio Catalítico , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação/genética , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
5.
iScience ; 25(5): 104206, 2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35521529

RESUMO

The Escherichia coli dihydrofolate reductase (DHFR) destabilizing domain (DD) serves as a promising approach to conditionally regulate protein abundance in a variety of tissues. To test whether this approach could be effectively applied to a wide variety of aged and disease-related ocular mouse models, we evaluated the DHFR DD system in the eyes of aged mice (up to 24 months), a light-induced retinal degeneration (LIRD) model, and two genetic models of retinal degeneration (rd2 and Abca4 -/- mice). The DHFR DD was effectively degraded in all model systems, including rd2 mice, which showed significant defects in chymotrypsin proteasomal activity. Moreover, trimethoprim (TMP) administration stabilized the DHFR DD in all mouse models. Thus, the DHFR DD-based approach allows for control of protein abundance in a variety of mouse models, laying the foundation to use this strategy for the conditional control of gene therapies to potentially treat multiple eye diseases.

6.
Sci Rep ; 11(1): 2998, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33542268

RESUMO

Distinct mutations in the secreted extracellular matrix protein, fibulin-3 (F3), have been associated with a number of ocular diseases ranging from primary open angle glaucoma to cuticular age-related macular degeneration to a rare macular dystrophy, Malattia Leventinese (ML). The R345W F3 mutation that causes ML leads to F3 misfolding, inefficient secretion and accumulation at higher intracellular steady state levels in cultured cells. Herein, we determined whether fifteen other clinically-identified F3 mutations also led to similar levels of misfolding and secretion defects, which might provide insight into their potential pathogenicity. Surprisingly, we found that only a single F3 variant, L451F, presented with a significant secretion defect (69.5 ± 2.4% of wild-type (WT) F3 levels) and a corresponding increase in intracellular levels (226.8 ± 25.4% of WT F3 levels). Upon follow-up studies, when this conserved residue (L451) was mutated to a charged (Asp or Arg) or bulky (Pro, Trp, Tyr) residue, F3 secretion was also compromised, indicating the importance of small side chains (Leu, Ala, or Gly) at this residue. To uncover potential inherent F3 instability not easily observed under typical culture conditions, we genetically eliminated the sole stabilizing N-linked glycosylation site (N249) from select clinically-identified F3 mutants. This removal exacerbated R345W and L451F secretion defects (19.8 ± 3.0% and 12.4 ± 1.2% of WT F3 levels, respectively), but also revealed a previously undiscovered secretion defect in another C-terminal variant, Y397H (42.0 ± 10.1% of WT F3 levels). Yet, glycan removal did not change the relative secretion of the N-terminal mutants tested (D49A, R140W, I220F). These results highlight the uniqueness and molecular similarities between the R345W and L451F variants and also suggest that previously identified disease-associated mutations (e.g., R140W) are indistinguishable from WT with respect to secretion, hinting that they may lead to disease by an alternative mechanism.


Assuntos
Proteínas da Matriz Extracelular/genética , Glaucoma de Ângulo Aberto/genética , Degeneração Macular/genética , Linhagem Celular , Proteínas da Matriz Extracelular/ultraestrutura , Glaucoma de Ângulo Aberto/patologia , Humanos , Degeneração Macular/patologia , Mutação/genética , Drusas do Disco Óptico/congênito , Drusas do Disco Óptico/genética , Drusas do Disco Óptico/patologia , Dobramento de Proteína , Estabilidade Proteica , Retina/metabolismo , Retina/patologia
7.
Nat Commun ; 12(1): 2949, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011959

RESUMO

The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.


Assuntos
Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Resistência a Trimetoprima/genética , Trimetoprima/análogos & derivados , Substituição de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Cristalografia por Raios X , Evolução Molecular Direcionada , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Genes Bacterianos , Genótipo , Humanos , Modelos Moleculares , Mutação , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/química , Trimetoprima/farmacologia
8.
Cell Chem Biol ; 27(5): 622-634.e6, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32330442

RESUMO

Destabilizing domains (DDs), such as a mutated form of Escherichia coli dihydrofolate reductase (ecDHFR), confer instability and promote protein degradation. However, when combined with small-molecule stabilizers (e.g., the antibiotic trimethoprim), DDs allow positive regulation of fusion protein abundance. Using a combinatorial screening approach, we identified and validated 17 unique 2,4-diaminopyrimidine/triazine-based ecDHFR DD stabilizers, at least 15 of which were ineffective antibiotics against E. coli and S. aureus. Identified stabilizers functioned in vivo to control an ecDHFR DD-firefly luciferase in the mouse eye and/or the liver. Next, stabilizers were leveraged to perform synergistic dual functions in vitro (HeLa cell death sensitization) and in vivo (repression of ocular inflammation) by stabilizing a user-defined ecDHFR DD while also controlling endogenous signaling pathways. Thus, these newly identified pharmacological chaperones allow for simultaneous control of compound-specific endogenous and user-defined genetic pathways, the combination of which may provide synergistic effects in complex biological scenarios.


Assuntos
Antibacterianos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Antagonistas do Ácido Fólico/farmacologia , Pirimidinas/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Antibacterianos/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Feminino , Antagonistas do Ácido Fólico/química , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pirimidinas/química , Tetra-Hidrofolato Desidrogenase/química , Triazinas/química , Triazinas/farmacologia , Trimetoprima/análogos & derivados , Trimetoprima/farmacologia
9.
Nat Commun ; 10(1): 2493, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175300

RESUMO

Tauopathies are neurodegenerative diseases characterized by intracellular amyloid deposits of tau protein. Missense mutations in the tau gene (MAPT) correlate with aggregation propensity and cause dominantly inherited tauopathies, but their biophysical mechanism driving amyloid formation is poorly understood. Many disease-associated mutations localize within tau's repeat domain at inter-repeat interfaces proximal to amyloidogenic sequences, such as 306VQIVYK311. We use cross-linking mass spectrometry, recombinant protein and synthetic peptide systems, in silico modeling, and cell models to conclude that the aggregation-prone 306VQIVYK311 motif forms metastable compact structures with its upstream sequence that modulates aggregation propensity. We report that disease-associated mutations, isomerization of a critical proline, or alternative splicing are all sufficient to destabilize this local structure and trigger spontaneous aggregation. These findings provide a biophysical framework to explain the basis of early conformational changes that may underlie genetic and sporadic tau pathogenesis.


Assuntos
Agregação Patológica de Proteínas/genética , Tauopatias/genética , Proteínas tau/genética , Motivos de Aminoácidos/genética , Simulação por Computador , Células HEK293 , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Mutação de Sentido Incorreto , Agregação Patológica de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas tau/metabolismo , Proteínas tau/ultraestrutura
10.
Elife ; 72018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30526844

RESUMO

Tauopathies have diverse presentation, progression, and neuropathology. They are linked to tau prion strains, self-replicating assemblies of unique quaternary conformation, whose origin is unknown. Strains can be propagated indefinitely in cultured cells, and induce unique patterns of transmissible neuropathology upon inoculation into mice. DS9 and DS10 cell lines propagate different synthetic strains that derive from recombinant tau. We previously observed that tau monomer adopts two conformational states: one that is inert (Mi) and one that is seed-competent (Ms) (Mirbaha et al., 2018). We have now found that Ms itself is comprised of multiple stable ensembles that encode unique strains. DS9 monomer inoculated into naive cells encoded only DS9, whereas DS10 monomer encoded multiple sub-strains. Sub-strains each induced distinct pathology upon inoculation into a tauopathy mouse model (PS19). Ms purified from an Alzeimer's disease brain encoded a single strain. Conversely, Ms from a corticobasal degeneration brain encoded three sub-strains, in which monomer from any one re-established all three upon inoculation into cells. Seed competent tau monomer thus adopts multiple, stable seed-competent conformations, each of which encodes a limited number of strains. This provides insight into the emergence of distinct tauopathies, and may improve diagnosis and therapy.


Assuntos
Encéfalo/metabolismo , Linfócitos/química , Príons/química , Tauopatias/metabolismo , Proteínas tau/química , Animais , Encéfalo/patologia , Química Encefálica , Mapeamento Encefálico , Linhagem Celular Tumoral , Proliferação de Células , Células Clonais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Linfócitos/metabolismo , Linfócitos/patologia , Masculino , Camundongos , Camundongos Transgênicos , Príons/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Tauopatias/classificação , Tauopatias/patologia , Proteínas tau/metabolismo
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