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1.
Nihon Yakurigaku Zasshi ; 159(2): 78-82, 2024.
Artigo em Japonês | MEDLINE | ID: mdl-38432923

RESUMO

Clioquinol was extensively used as an amebicide to treat indigestion and diarrhea in the mid-1900s. However, it was withdrawn from the market in Japan because its use was epidemiologically linked to an increase in the incidence of subacute myelo-optic neuropathy (SMON). SMON is characterized by the subacute onset of sensory and motor disturbances in the lower extremities with occasional visual impairments, which are preceded by abdominal symptoms. Although pathological studies demonstrated axonopathy of the spinal cord and optic nerves, the underlying mechanisms of clioquinol toxicity have not been elucidated in detail. We previously performed a global analysis of human neuroblastoma cells using DNA chips and demonstrated that clioquinol induced 1) DNA double-strand breaks and subsequent activation of ATM/p53 signaling; 2) the expression of VGF, the precursor of neuropeptides involved in pain reactions, by inducing c-Fos; 3) the expression of interleukin-8, which is reported to be involved in intestinal inflammation, optic neuropathy, and neuropathic pain, by down-regulating GATA-2 and GATA-3. We also demonstrated that clioquinol induced zinc influx and oxidation of the copper chaperone ATOX1, leading to the impairment of the functional maturation of a copper-dependent enzyme dopamine-ß-hydroxylase and the inhibition of noradrenaline biosynthesis. Thus, clioquinol-induced neurotoxicity in SMON seems to be mediated by multiple pathways.


Assuntos
Clioquinol , Doenças do Nervo Óptico , Humanos , Clioquinol/efeitos adversos , Cobre , Medula Espinal , Japão , Proteínas de Transporte de Cobre , Chaperonas Moleculares
2.
J Orthop Surg Res ; 19(1): 156, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38429742

RESUMO

Heat-shock protein beta1 (HSPB1) is a member of the small HSP family, downregulated in osteoarthritis (OA) chondrocytes and demonstrated the capacity to serve as an RNA-binding protein (RBP). This work aimed to explore the profile of HSPB1 bound RNA and reveal the potential regulation mechanism of HSPB1 in OA. In this work, we captured an unbiased HSPB1-RNA interaction map in Hela cells using the iRIP-seq. The results demonstrated that HSPB1 interacted with plentiful of mRNAs and genomic location toward the CDS region. Functional enrichment of HSPB1-related peaks showed the involvement in gene expression, translation initiation, cellular protein metabolic process, and nonsense-mediated decay. HOMER software analysis showed that HSPB1 bound peaks were over-represented in GAGGAG sequences. In addition, ABLIRC and CIMS algorithm indicated that HSPB1 bound to AU-rich motifs and the proportion of AU-rich peaks in 3' UTR were slightly higher than that in other regions. Moreover, HSPB1-binding targets analysis revealed several gens were associated with OA including EGFR, PLEC, COL5A1, and ROR2. The association of OA-related mRNAs to HSPB1 was additionally confirmed in OA tissues by the quantitative RIP-PCR experiments. Further experiment demonstrated the downregulation of HSPB1 in OA tissues. In conclusion, our current study confirmed HSPB1 as an RNA-binding protein and revealed its potential function in the pathological process of OA, providing a reliable insight to further investigate the molecular regulation mechanism of HSPB1 in OA.


Assuntos
Proteínas de Choque Térmico , Osteoartrite , Humanos , Proteínas de Choque Térmico/genética , Células HeLa , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/genética , Osteoartrite/metabolismo , Chaperonas Moleculares/genética
3.
Cells ; 13(5)2024 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-38474334

RESUMO

The integrity and permeability of epithelial and endothelial barriers depend on the formation of tight junctions, adherens junctions, and a junction-associated cytoskeleton. The establishment of this junction-cytoskeletal module relies on the correct folding and oligomerization of its protein components. Molecular chaperones are known regulators of protein folding and complex formation in different cellular compartments. Mammalian cells possess an elaborate chaperone network consisting of several hundred chaperones and co-chaperones. Only a small part of this network has been linked, however, to the regulation of intercellular adhesions, and the systematic analysis of chaperone functions at epithelial and endothelial barriers is lacking. This review describes the functions and mechanisms of the chaperone-assisted regulation of intercellular junctions. The major focus of this review is on heat shock protein chaperones, their co-chaperones, and chaperonins since these molecules are the focus of the majority of the articles published on the chaperone-mediated control of tissue barriers. This review discusses the roles of chaperones in the regulation of the steady-state integrity of epithelial and vascular barriers as well as the disruption of these barriers by pathogenic factors and extracellular stressors. Since cytoskeletal coupling is essential for junctional integrity and remodeling, chaperone-assisted assembly of the actomyosin cytoskeleton is also discussed.


Assuntos
Citoesqueleto , Junções Intercelulares , Animais , Citoesqueleto/metabolismo , Junções Intercelulares/metabolismo , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Chaperonas Moleculares/metabolismo , Mamíferos/metabolismo
4.
Methods Mol Biol ; 2778: 259-272, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478283

RESUMO

Chemical crosslinking-mass spectrometry (XL-MS) is an established tool that can be used to study the architecture and dynamics of proteins and protein assemblies. Here the application of XL-MS to study outer membrane proteins (OMPs) and their interactions with periplasmic chaperones is described, to inform on the molecular mechanisms underpinning OMP assembly. XL-MS data are especially powerful when used to complement high-resolution structural data, data from structural prediction or to drive molecular modeling of proteins and protein assemblies. The approach described here could be applied to the study of any protein assembly (including other membrane proteins).


Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Chaperonas Moleculares/metabolismo , Periplasma/metabolismo , Dobramento de Proteína
5.
Int J Mol Sci ; 25(5)2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38474073

RESUMO

Alpha-B-crystallin, a member of the small heat shock family of proteins, has been implicated in a variety of cardiomyopathies and in normal cardiac homeostasis. It is known to function as a molecular chaperone, particularly for desmin, but also interacts with a wide variety of additional proteins. The molecular chaperone function is also enhanced by signal-dependent phosphorylation at specific residues under stress conditions. Naturally occurring mutations in CRYAB, the gene that encodes alpha-B-crystallin, have been suggested to alter ionic intermolecular interactions that affect dimerization and chaperone function. These mutations have been associated with myofibrillar myopathy, restrictive cardiomyopathy, and hypertrophic cardiomyopathy and promote pathological hypertrophy through different mechanisms such as desmin aggregation, increased reductive stress, or activation of calcineurin-NFAT signaling. This review will discuss the known mechanisms by which alpha-B-crystallin functions in cardiac homeostasis and the pathogenesis of cardiomyopathies and provide insight into potential future areas of exploration.


Assuntos
Cardiomiopatias , Cardiomiopatia Restritiva , Humanos , Desmina/genética , Cardiomiopatias/patologia , Mutação , Cardiomiopatia Restritiva/complicações , Chaperonas Moleculares/genética
6.
Int J Mol Sci ; 25(5)2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38474102

RESUMO

Histone deacetylase SIRT1 represses gene expression through the deacetylation of histones and transcription factors and is involved in the protective cell response to stress and aging. However, upon endoplasmic reticulum (ER) stress, SIRT1 impairs the IRE1α branch of the unfolded protein response (UPR) through the inhibition of the transcriptional activity of XBP-1 and SIRT1 deficiency is beneficial under these conditions. We hypothesized that SIRT1 deficiency may unlock the blockade of transcription factors unrelated to the UPR promoting the synthesis of chaperones and improving the stability of immature proteins or triggering the clearance of unfolded proteins. SIRT1+/+ and SIRT1-/- fibroblasts were exposed to the ER stress inducer tunicamycin and cell survival and expression of heat shock proteins were analyzed 24 h after the treatment. We observed that SIRT1 loss significantly reduced cell sensitivity to ER stress and showed that SIRT1-/- but not SIRT1+/+ cells constitutively expressed high levels of phospho-STAT3 and heat shock proteins. Hsp70 silencing in SIRT1-/- cells abolished the resistance to ER stress. Furthermore, accumulation of ubiquitinated proteins was lower in SIRT1-/- than in SIRT1+/+ cells. Our data showed that SIRT1 deficiency enabled chaperones upregulation and boosted the proteasome activity, two processes that are beneficial for coping with ER stress.


Assuntos
Proteínas de Choque Térmico , Sirtuína 1 , Proteínas de Choque Térmico/metabolismo , Regulação para Cima , Sirtuína 1/metabolismo , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estresse do Retículo Endoplasmático , Resposta a Proteínas não Dobradas , Chaperonas Moleculares/metabolismo , Fatores de Transcrição/metabolismo
7.
Commun Biol ; 7(1): 260, 2024 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-38431713

RESUMO

RAF kinases are integral to the RAS-MAPK signaling pathway, and proper RAF1 folding relies on its interaction with the chaperone HSP90 and the cochaperone CDC37. Understanding the intricate molecular interactions governing RAF1 folding is crucial for comprehending this process. Here, we present a cryo-EM structure of the closed-state RAF1-HSP90-CDC37 complex, where the C-lobe of the RAF1 kinase domain binds to one side of the HSP90 dimer, and an unfolded N-lobe segment of the RAF1 kinase domain threads through the center of the HSP90 dimer. CDC37 binds to the kinase C-lobe, mimicking the N-lobe with its HxNI motif. We also describe structures of HSP90 dimers without RAF1 and CDC37, displaying only N-terminal and middle domains, which we term the semi-open state. Employing 1 µs atomistic simulations, energetic decomposition, and comparative structural analysis, we elucidate the dynamics and interactions within these complexes. Our quantitative analysis reveals that CDC37 bridges the HSP90-RAF1 interaction, RAF1 binds HSP90 asymmetrically, and that HSP90 structural elements engage RAF1's unfolded region. Additionally, N- and C-terminal interactions stabilize HSP90 dimers, and molecular interactions in HSP90 dimers rearrange between the closed and semi-open states. Our findings provide valuable insight into the contributions of HSP90 and CDC37 in mediating client folding.


Assuntos
Proteínas de Ciclo Celular , Chaperoninas , Humanos , Proteínas de Ciclo Celular/metabolismo , Ligação Proteica , Chaperoninas/química , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP90
8.
Cell Mol Life Sci ; 81(1): 119, 2024 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-38456949

RESUMO

Activated small ubiquitin-like modifiers (SUMOs) have been implicated in neuropathological processes following ischemic stroke. However, the target proteins of SUMOylation and their contribution to neuronal injury remain to be elucidated. MLK3 (mixed-lineage kinase 3), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, is a critical regulator of neuronal lesions following cerebral ischemia. Here, we found that SUMOylation of MLK3 increases in both global and focal ischemic rodent models and primary neuronal models of oxygen and glucose deprivation (OGD). SUMO1 conjugation at the Lys401 site of MLK3 promoted its activation, stimulated its downstream p38/c-Jun N-terminal kinase (JNK) cascades, and led to cell apoptosis. The interaction of MLK3 with PIAS3, a SUMO ligase, was elevated following ischemia and reperfusion. The PINIT domain of PIAS3 was involved in direct interactions with MLK3. Overexpression of the PINIT domain of PIAS3 disrupted the MLK3-PIAS3 interaction, inhibited SUMOylation of MLK3, suppressed downstream signaling, and reduced cell apoptosis and neurite damage. In rodent ischemic models, the overexpression of the PINIT domain reduced brain lesions and alleviated deficits in learning, memory, and sensorimotor functions. Our findings demonstrate that brain ischemia-induced MLK3 SUMOylation by PIAS3 is a potential target against poststroke neuronal lesions and behavioral impairments.


Assuntos
Isquemia Encefálica , Sumoilação , Humanos , MAP Quinase Quinase Quinases/genética , Transdução de Sinais/fisiologia , Isquemia Encefálica/metabolismo , Cognição , Chaperonas Moleculares/metabolismo , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo
9.
Mol Cell Biol ; 44(2): 72-85, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38482865

RESUMO

ANP32e, a chaperone of H2A.Z, is receiving increasing attention because of its association with cancer growth and progression. An unanswered question is whether ANP32e regulates H2A.Z dynamics during the cell cycle; this could have clear implications for the proliferation of cancer cells. We confirmed that ANP32e regulates the growth of human U2OS cancer cells and preferentially interacts with H2A.Z during the G1 phase of the cell cycle. Unexpectedly, ANP32e does not mediate the removal of H2A.Z from chromatin, is not a stable component of the p400 remodeling complex and is not strongly associated with chromatin. Instead, most ANP32e is in the cytoplasm. Here, ANP32e preferentially interacts with H2A.Z in the G1 phase in response to an increase in H2A.Z protein abundance and regulates its protein stability. This G1-specific interaction was also observed in the nucleoplasm but was unrelated to any change in H2A.Z abundance. These results challenge the idea that ANP32e regulates the abundance of H2A.Z in chromatin as part of a chromatin remodeling complex. We propose that ANP32e is a molecular chaperone that maintains the soluble pool of H2A.Z by regulating its protein stability and acting as a buffer in response to cell cycle-dependent changes in H2A.Z abundance.


Assuntos
Histonas , Nucleossomos , Humanos , Histonas/metabolismo , Cromatina , Núcleo Celular/metabolismo , Chaperonas Moleculares/metabolismo , Ciclo Celular , Estabilidade Proteica
10.
Orphanet J Rare Dis ; 19(1): 125, 2024 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-38500130

RESUMO

BACKGROUND: CLN3 disease (also known as CLN3 Batten disease or Juvenile Neuronal Ceroid Lipofuscinosis) is a rare pediatric neurodegenerative disorder caused by biallelic mutations in CLN3. While extensive efforts have been undertaken to understand CLN3 disease etiology, pathology, and clinical progression, little is known about the impact of CLN3 disease on parents and caregivers. Here, we investigated CLN3 disease progression, clinical care, and family experiences using semi-structured interviews with 39 parents of individuals with CLN3 disease. Analysis included response categorization by independent observers and quantitative methods. RESULTS: Parents reported patterns of disease progression that aligned with previous reports. Insomnia and thought- and mood-related concerns were reported frequently. "Decline in visual acuity" was the first sign/symptom noticed by n = 28 parents (70%). A minority of parents reported "behavioral issues" (n = 5, 12.5%), "communication issues" (n = 3, 7.5%), "cognitive decline" (n = 1, 2.5%), or "seizures" (n = 1, 2.5%) as the first sign/symptom. The mean time from the first signs or symptoms to a diagnosis of CLN3 disease was 2.8 years (SD = 4.1). Misdiagnosis was common, being reported by n = 24 participants (55.8%). Diagnostic tests and treatments were closely aligned with observed symptoms. Desires for improved or stabilized vision (top therapeutic treatment concern for n = 14, 32.6%), cognition (n = 8, 18.6%), and mobility (n = 3, 7%) dominated parental concerns and wishes for therapeutic correction. Family impacts were common, with n = 34 (81%) of respondents reporting a financial impact on the family and n = 20 (46.5%) reporting marital strain related to the disease. CONCLUSIONS: Collectively, responses demonstrated clear patterns of disease progression, a strong desire for therapies to treat symptoms related to vision and cognition, and a powerful family impact driven by the unrelenting nature of disease progression.


Assuntos
Lipofuscinoses Ceroides Neuronais , Humanos , Criança , Lipofuscinoses Ceroides Neuronais/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/uso terapêutico , Glicoproteínas de Membrana/genética , Pais , Progressão da Doença , Inquéritos e Questionários
11.
J Cell Biol ; 223(6)2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38478018

RESUMO

The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway.


Assuntos
Vesículas Citoplasmáticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animais , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Mamíferos/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Munc18/análise , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vesículas Citoplasmáticas/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo
12.
Naturwissenschaften ; 111(2): 16, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38483597

RESUMO

Heat shock proteins are constitutively expressed chaperones induced by cellular stress, such as changes in temperature, pH, and osmolarity. These proteins, present in all organisms, are highly conserved and are recruited for the assembly of protein complexes, transport, and compartmentalization of molecules. In fungi, these proteins are related to their adaptation to the environment, their evolutionary success in acquiring new hosts, and regulation of virulence and resistance factors. These characteristics are interesting for assessment of the host adaptability and ecological transitions, given the emergence of infections by these microorganisms. Based on phylogenetic inferences, we compared the sequences of HSP9, HSP12, HSP30, HSP40, HSP70, HSP90, and HSP110 to elucidate the evolutionary relationships of different fungal organisms to suggest evolutionary patterns employing the maximum likelihood method. By the different reconstructions, our inference supports the hypothesis that these classes of proteins are associated with pathogenic gains against endothermic hosts, as well as adaptations for phytopathogenic fungi.


Assuntos
Proteínas de Choque Térmico , Chaperonas Moleculares , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Filogenia , Sequência de Aminoácidos , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo
13.
Proc Natl Acad Sci U S A ; 121(12): e2309326121, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38483986

RESUMO

Hsp90s are ATP-dependent chaperones that collaborate with co-chaperones and Hsp70s to remodel client proteins. Grp94 is the ER Hsp90 homolog essential for folding multiple secretory and membrane proteins. Grp94 interacts with the ER Hsp70, BiP, although the collaboration of the ER chaperones in protein remodeling is not well understood. Grp94 undergoes large-scale conformational changes that are coupled to chaperone activity. Within Grp94, a region called the pre-N domain suppresses ATP hydrolysis and conformational transitions to the active chaperone conformation. In this work, we combined in vivo and in vitro functional assays and structural studies to characterize the chaperone mechanism of Grp94. We show that Grp94 directly collaborates with the BiP chaperone system to fold clients. Grp94's pre-N domain is not necessary for Grp94-client interactions. The folding of some Grp94 clients does not require direct interactions between Grp94 and BiP in vivo, suggesting that the canonical collaboration may not be a general chaperone mechanism for Grp94. The BiP co-chaperone DnaJB11 promotes the interaction between Grp94 and BiP, relieving the pre-N domain suppression of Grp94's ATP hydrolysis activity. In structural studies, we find that ATP binding by Grp94 alters the ATP lid conformation, while BiP binding stabilizes a partially closed Grp94 intermediate. Together, BiP and ATP push Grp94 into the active closed conformation for client folding. We also find that nucleotide binding reduces Grp94's affinity for clients, which is important for productive client folding. Alteration of client affinity by nucleotide binding may be a conserved chaperone mechanism for a subset of ER chaperones.


Assuntos
Proteínas de Choque Térmico HSP70 , Dobramento de Proteína , Humanos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Nucleotídeos , Trifosfato de Adenosina/metabolismo
14.
Nat Commun ; 15(1): 2299, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38485940

RESUMO

Designing complex synthetic materials for enzyme immobilization could unlock the utility of biocatalysis in extreme environments. Inspired by biology, we investigate the use of random copolymer brushes as dynamic immobilization supports that enable supra-biological catalytic performance of immobilized enzymes. This is demonstrated by immobilizing Bacillus subtilis Lipase A on brushes doped with aromatic moieties, which can interact with the lipase through multiple non-covalent interactions. Incorporation of aromatic groups leads to a 50 °C increase in the optimal temperature of lipase, as well as a 50-fold enhancement in enzyme activity. Single-molecule FRET studies reveal that these supports act as biomimetic chaperones by promoting enzyme refolding and stabilizing the enzyme's folded and catalytically active state. This effect is diminished when aromatic residues are mutated out, suggesting the importance of π-stacking and π-cation interactions for stabilization. Our results underscore how unexplored enzyme-support interactions may enable uncharted opportunities for using enzymes in industrial biotransformations.


Assuntos
Bacillus subtilis , Enzimas Imobilizadas , Enzimas Imobilizadas/química , Estabilidade Enzimática , Bacillus subtilis/metabolismo , Lipase/metabolismo , Temperatura , Biocatálise , Chaperonas Moleculares/metabolismo
15.
BMC Neurol ; 24(1): 96, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38491364

RESUMO

BACKGROUND: The Limb Girdle Muscular Dystrophies (LGMDs) are characterized by progressive weakness of the shoulder and hip girdle muscles as a result of over 30 different genetic mutations. This study is designed to develop clinical outcome assessments across the group of disorders. METHODS/DESIGN: The primary goal of this study is to evaluate the utility of a set of outcome measures on a wide range of LGMD phenotypes and ability levels to determine if it would be possible to use similar outcomes between individuals with different phenotypes. We will perform a multi-center, 12-month study of 188 LGMD patients within the established Genetic Resolution and Assessments Solving Phenotypes in LGMD (GRASP-LGMD) Research Consortium, which is comprised of 11 sites in the United States and 2 sites in Europe. Enrolled patients will be clinically affected and have mutations in CAPN3 (LGMDR1), ANO5 (LGMDR12), DYSF (LGMDR2), DNAJB6 (LGMDD1), SGCA (LGMDR3), SGCB (LGMDR4), SGCD (LGMDR6), or SGCG (LGMDR5, or FKRP-related (LGMDR9). DISCUSSION: To the best of our knowledge, this will be the largest consortium organized to prospectively validate clinical outcome assessments (COAs) in LGMD at its completion. These assessments will help clinical trial readiness by identifying reliable, valid, and responsive outcome measures as well as providing data driven clinical trial decision making for future clinical trials on therapeutic agents for LGMD. The results of this study will permit more efficient clinical trial design. All relevant data will be made available for investigators or companies involved in LGMD therapeutic development upon conclusion of this study as applicable. TRIAL REGISTRATION: Clinicaltrials.gov NCT03981289; Date of registration: 6/10/2019.


Assuntos
Distrofia Muscular do Cíngulo dos Membros , Sarcoglicanopatias , Humanos , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , Distrofia Muscular do Cíngulo dos Membros/genética , Fenótipo , Músculo Esquelético , Mutação/genética , Proteínas do Tecido Nervoso/genética , Chaperonas Moleculares/genética , Proteínas de Choque Térmico HSP40/genética , Pentosiltransferases/genética , Anoctaminas/genética
16.
Mol Cell ; 84(6): 1101-1119.e9, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38428433

RESUMO

Mitochondrial outer membrane ⍺-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse proteins remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse ⍺-helical substrates reveals that these components are organized into distinct targeting pathways that act on substrates based on their topology. NAC is required for the efficient targeting of polytopic proteins, whereas signal-anchored proteins require TTC1, a cytosolic chaperone that physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, the targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems.


Assuntos
Membranas Mitocondriais , Proteínas de Saccharomyces cerevisiae , Animais , Membranas Mitocondriais/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismo , Mamíferos/metabolismo
17.
ACS Appl Mater Interfaces ; 16(11): 13411-13421, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38456838

RESUMO

The development of sustainable biomaterials and surfaces to prevent the accumulation and proliferation of viruses and bacteria is highly demanded in healthcare areas. This study describes the assembly and full characterization of two new bioactive silver(I) coordination polymers (CPs) formulated as [Ag(aca)(µ-PTA)]n·5nH2O (1) and [Ag2(µ-ada)(µ3-PTA)2]n·4nH2O (2). These products were generated by exploiting a heteroleptic approach based on the use of two different adamantoid building blocks, namely 1,3,5-triaza-7-phosphaadamantane (PTA) and 1-adamantanecarboxylic (Haca) or 1,3-adamantanedicarboxylic (H2ada) acids, resulting in the assembly of 1D (1) and 3D (2). Antiviral, antibacterial, and antifungal properties of the obtained compounds were investigated in detail, followed by their incorporation as bioactive dopants (1 wt %) into hybrid biopolymers based on acid-hydrolyzed starch polymer (AHSP). The resulting materials, formulated as 1@AHSP and 2@AHSP, also featured (i) an exceptional antiviral activity against herpes simplex virus type 1 and human adenovirus (HAd-5) and (ii) a remarkable antibacterial activity against Gram-negative bacteria. Docking experiments, interaction with human serum albumin, mass spectrometry, and antioxidation studies provided insights into the mechanism of antimicrobial action. By reporting these new silver CPs driven by adamantoid building blocks and the derived starch-based materials, this study endows a facile approach to access biopolymers and interfaces capable of preventing and reducing the proliferation of a broad spectrum of different microorganisms, including bacteria, fungi, and viruses.


Assuntos
Prata , Vírus , Humanos , Prata/farmacologia , Prata/química , Polímeros/farmacologia , Polímeros/química , Antibacterianos/farmacologia , Antibacterianos/química , Bactérias , Antivirais/farmacologia , Amido , Proteínas Sanguíneas , Chaperonas Moleculares
18.
Sci Adv ; 10(12): eadk9884, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38507480

RESUMO

Molecular chaperones are protective in neurodegenerative diseases by preventing protein misfolding and aggregation, such as extracellular amyloid plaques and intracellular tau neurofibrillary tangles in Alzheimer's disease (AD). In addition, AD is characterized by an increase in astrocyte reactivity. The chaperone HSPB1 has been proposed as a marker for reactive astrocytes; however, its astrocytic functions in neurodegeneration remain to be elucidated. Here, we identify that HSPB1 is secreted from astrocytes to exert non-cell-autonomous protective functions. We show that in human AD brain, HSPB1 levels increase in astrocytes that cluster around amyloid plaques, as well as in the adjacent extracellular space. Moreover, in conditions that mimic an inflammatory reactive response, astrocytes increase HSPB1 secretion. Concomitantly, astrocytes and neurons can uptake astrocyte-secreted HSPB1, which is accompanied by an attenuation of the inflammatory response in reactive astrocytes and reduced pathological tau inclusions. Our findings highlight a protective mechanism in disease conditions that encompasses the secretion of a chaperone typically regarded as intracellular.


Assuntos
Doença de Alzheimer , Astrócitos , Humanos , Astrócitos/metabolismo , Proteínas tau/metabolismo , Placa Amiloide/patologia , Neuroproteção , Chaperonas Moleculares/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas de Choque Térmico/metabolismo
19.
Mol Biol Cell ; 35(4): ar59, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38446639

RESUMO

GRP170 (Hyou1) is required for mouse embryonic development, and its ablation in kidney nephrons leads to renal failure. Unlike most chaperones, GRP170 is the lone member of its chaperone family in the ER lumen. However, the cellular requirement for GRP170, which both binds nonnative proteins and acts as nucleotide exchange factor for BiP, is poorly understood. Here, we report on the isolation of mouse embryonic fibroblasts obtained from mice in which LoxP sites were engineered in the Hyou1 loci (Hyou1LoxP/LoxP). A doxycycline-regulated Cre recombinase was stably introduced into these cells. Induction of Cre resulted in depletion of Grp170 protein which culminated in cell death. As Grp170 levels fell we observed a portion of BiP fractionating with insoluble material, increased binding of BiP to a client with a concomitant reduction in its turnover, and reduced solubility of an aggregation-prone BiP substrate. Consistent with disrupted BiP functions, we observed reactivation of BiP and induction of the unfolded protein response (UPR) in futile attempts to provide compensatory increases in ER chaperones and folding enzymes. Together, these results provide insights into the cellular consequences of controlled Grp170 loss and provide hypotheses as to why mutations in the Hyou1 locus are linked to human disease.


Assuntos
Desenvolvimento Embrionário , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico HSP70 , Animais , Humanos , Camundongos , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Chaperonas Moleculares/metabolismo
20.
Mol Plant Pathol ; 25(3): e13442, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38476100

RESUMO

The type VI secretion system (T6SS) of many gram-negative bacteria injects toxic effectors into adjacent cells to manipulate host cells during pathogenesis or to kill competing bacteria. However, the identification and function of the T6SS effectors remains only partly known. Pantoea ananatis, a gram-negative bacterium, is commonly found in various plants and natural environments, including water and soil. In the current study, genomic analysis of P. ananatis DZ-12 causing brown stalk rot on maize demonstrated that it carries three T6SS gene clusters, namely, T6SS-1, T6SS-2, and T6SS-3. Interestingly, only T6SS-1 secretion systems are involved in pathogenicity and bacterial competition. The study also investigated the T6SS-1 system in detail and identified an unknown T6SS-1-secreted effector TseG by using the upstream T6SS effector chaperone TecG containing a conserved domain of DUF2169. TseG can directly interact with the chaperone TecG for delivery and with a downstream immunity protein TsiG for protection from its toxicity. TseG, highly conserved in the Pantoea genus, is involved in virulence in maize, potato, and onion. Additionally, P. ananatis uses TseG to target Escherichia coli, gaining a competitive advantage. This study provides the first report on the T6SS-1-secreted effector from P. ananatis, thereby enriching our understanding of the various types and functions of type VI effector proteins.


Assuntos
Pantoea , Sistemas de Secreção Tipo VI , Sistemas de Secreção Tipo VI/metabolismo , Pantoea/genética , Sistemas de Secreção Bacterianos/genética , Virulência/genética , Antibacterianos , Chaperonas Moleculares , Proteínas de Bactérias/metabolismo
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