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1.
Int J Mol Sci ; 25(12)2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38928146

RESUMO

Mitochondrial quality control is essential in mitochondrial function. To examine the importance of Parkin-dependent mechanisms in mitochondrial quality control, we assessed the impact of modulating Parkin on proteome flux and mitochondrial function in a context of reduced mtDNA fidelity. To accomplish this, we crossed either the Parkin knockout mouse or ParkinW402A knock-in mouse lines to the Polg mitochondrial mutator line to generate homozygous double mutants. In vivo longitudinal isotopic metabolic labeling was followed by isolation of liver mitochondria and synaptic terminals from the brain, which are rich in mitochondria. Mass spectrometry and bioenergetics analysis were assessed. We demonstrate that slower mitochondrial protein turnover is associated with loss of mtDNA fidelity in liver mitochondria but not synaptic terminals, and bioenergetic function in both tissues is impaired. Pathway analysis revealed loss of mtDNA fidelity is associated with disturbances of key metabolic pathways, consistent with its association with metabolic disorders and neurodegeneration. Furthermore, we find that loss of Parkin leads to exacerbation of Polg-driven proteomic consequences, though it may be bioenergetically protective in tissues exhibiting rapid mitochondrial turnover. Finally, we provide evidence that, surprisingly, dis-autoinhibition of Parkin (ParkinW402A) functionally resembles Parkin knockout and fails to rescue deleterious Polg-driven effects. Our study accomplishes three main outcomes: (1) it supports recent studies suggesting that Parkin dependence is low in response to an increased mtDNA mutational load, (2) it provides evidence of a potential protective role of Parkin insufficiency, and (3) it draws into question the therapeutic attractiveness of enhancing Parkin function.


Assuntos
DNA Polimerase gama , DNA Mitocondrial , Camundongos Knockout , Mutação , Ubiquitina-Proteína Ligases , Animais , DNA Polimerase gama/genética , DNA Polimerase gama/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Camundongos , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteômica/métodos , Proteoma/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genética , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética
2.
J Mol Cell Cardiol ; 149: 27-40, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32956670

RESUMO

Identification of proteins that interact with Cx43 has been instrumental in the understanding of gap junction (GJ) regulation. An in vitro phosphorylation screen identified that Protein tyrosine kinase 2 beta (Pyk2) phosphorylated purified Cx43CT and this led us to characterize the impact of this phosphorylation on Cx43 function. Mass spectrometry identified Pyk2 phosphorylates Cx43 residues Y247, Y265, Y267, and Y313. Western blot and immunofluorescence staining using HeLaCx43 cells, HEK 293 T cells, and neonatal rat ventricular myocytes (NRVMs) revealed Pyk2 can be activated by Src and active Pyk2 interacts with Cx43 at the plasma membrane. Overexpression of Pyk2 increases Cx43 phosphorylation and knock-down of Pyk2 decreases Cx43 phosphorylation, without affecting the level of active Src. In HeLaCx43 cells treated with PMA to activate Pyk2, a decrease in Cx43 GJ intercellular communication (GJIC) was observed when assayed by dye transfer. Moreover, PMA activation of Pyk2 could be inhibited by the small molecule PF4618433. This partially restored GJIC, and when paired with a Src inhibitor, returned GJIC to the no PMA control-level. The ability of Pyk2 and Src inhibitors to restore Cx43 function in the presence of PMA was also observed in NRVMs. Additionally, an animal model of myocardial infarction induced heart failure showed a higher level of active Pyk2 activity and increased interaction with Cx43 in ventricular myocytes. Src inhibitors have been used to reverse Cx43 remodeling and improve heart function after myocardial infarction; however, they alone could not fully restore proper Cx43 function. Our data suggest that Pyk2 may need to be inhibited, in addition to Src, to further (if not completely) reverse Cx43 remodeling and improve intercellular communication.


Assuntos
Comunicação Celular , Conexina 43/metabolismo , Quinase 2 de Adesão Focal/antagonistas & inibidores , Junções Comunicantes/metabolismo , Quinases da Família src/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Linhagem Celular , Conexina 43/química , Modelos Animais de Doenças , Quinase 2 de Adesão Focal/metabolismo , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/patologia , Ventrículos do Coração/patologia , Humanos , Mutação/genética , Fosforilação , Ligação Proteica , Domínios Proteicos , Ratos , Acetato de Tetradecanoilforbol/farmacologia , Quinases da Família src/genética , Quinases da Família src/metabolismo
3.
J Biol Chem ; 294(1): 341-350, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30401746

RESUMO

Gap junctions are intercellular conduits that permit the passage of ions, small metabolites, and signaling molecules between cells. Connexin32 (Cx32) is a major gap junction protein in the liver and brain. Phosphorylation is integral to regulating connexin assembly, degradation, and electrical and metabolic coupling, as well as to interactions with molecular partners. Cx32 contains two intracellular tyrosine residues, and tyrosine phosphorylation of Cx32 has been detected after activation of the epidermal growth factor receptor; however, the specific tyrosine residue and the functional implication of this phosphorylation remain unknown. To address the limited available information on Cx32 regulation by tyrosine kinases, here we used the Cx32 C-terminal (CT) domain in an in vitro kinase-screening assay, which identified ephrin (Eph) receptor family members as tyrosine kinases that phosphorylate Cx32. We found that EphB1 and EphA1 phosphorylate the Cx32CT domain residue Tyr243 Unlike for Cx43, the tyrosine phosphorylation of the Cx32CT increased gap junction intercellular communication. We also demonstrated that T-cell protein-tyrosine phosphatase dephosphorylates pTyr243 The data presented above along with additional examples throughout the literature of gap junction regulation by kinases, indicate that one cannot extrapolate the effect of a kinase on one connexin to another.


Assuntos
Conexinas/metabolismo , Junções Comunicantes/metabolismo , Receptor EphA1/metabolismo , Receptor EphB1/metabolismo , Células CACO-2 , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Junções Comunicantes/genética , Células HeLa , Humanos , Proteína Tirosina Fosfatase não Receptora Tipo 2 , Receptor EphA1/genética , Receptor EphB1/genética , Proteína beta-1 de Junções Comunicantes
4.
J Mol Cell Cardiol ; 126: 36-49, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30448479

RESUMO

Phosphorylation regulates connexin43 (Cx43) function from assembly/disassembly to coupling at the plaque. Src is a tyrosine kinase known to both phosphorylate Cx43 (residues Y247 and Y265) and affect gap junction intercellular communication. However, the Cx43 carboxyl-terminal (CT) domain contains additional tyrosine residues and proteomic discovery mass spectrometry data identified Y313 as a potential phosphorylation target. Based upon the study of Lin et al. (2001) J. Cell Biol., which still observed tyrosine phosphorylation by Src when using a Cx43 Y247/Y265F mutant, we addressed the possibility of Y313 phosphorylation (pY313) by Src. In vitro Src phosphorylation of purified Cx43CT followed by mass spectroscopy revealed that Src also phosphorylates Y313. This observation was confirmed by repeating the in vitro phosphorylation using different combinations of Cx43CT Y → F mutants and a general anti-pTyr antibody. Next, a phospho-specific antibody was generated to help characterize the importance of pY313. We established an in cyto experimental system by stably expressing Cx43 WT and mutants (Y247F, Y265F, Y313F, Y247/265F, Y247/313F, Y265/313F, or Y247/265/313F) in Cx43-deficient HeLa cells. Cx43 WT and mutants, in the absence of v-Src, localized to the plasma membrane and formed gap junctions. When v-Src was over-expressed, Cx43 WT localized intracellularly, while all of the single and double mutants remained able to form plaques and transfer dye, albeit variable in number and amount, respectively. Complete Src-resistance was only achieved with the Cx43 Y247/265/313F mutant. Furthermore, Cx43 Y265F inhibited the ability of v-Src to phosphorylate Y247 and Y313 as well as phosphorylation at both Y265 and Y313 was necessary to inhibit the Cx43 interaction with Drebrin. Finally, we observed in diseased cardiac tissue, in which Src is active, an increase in intercalated disc and intracellular localized Cx43 pY313.


Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Neuropeptídeos/metabolismo , Fosfotirosina/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Especificidade de Anticorpos , Conexina 43/química , Células HeLa , Humanos , Miocárdio/metabolismo , Miocárdio/patologia , Fosforilação , Ligação Proteica , Ratos
5.
FASEB J ; 31(12): 5217-5233, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28778975

RESUMO

The Eps15-homology domain-containing (EHD) protein family comprises 4 members that regulate endocytic recycling. Although the kidney expresses all 4 EHD proteins, their physiologic roles are largely unknown. This study focused on EHD4, which we found to be expressed differentially across nephron segments with the highest expression in the inner medullary collecting duct. Under baseline conditions, Ehd4-/- [EHD4-knockout (KO)] mice on a C57Bl/6 background excreted a higher volume of more dilute urine than control C57Bl/6 wild-type (WT) mice while maintaining a similar plasma osmolality. Urine excretion after an acute intraperitoneal water load was significantly increased in EHD4-KO mice compared to WT mice, and although EHD4-KO mice concentrated their urine during 24-h water restriction, urinary osmolality remained significantly lower than in WT mice, suggesting that EHD4 plays a role in renal water handling. Total aquaporin 2 (AQP2) and phospho-S256-AQP2 (pAQP2) protein expression in the inner medulla was similar in the two groups in baseline conditions. However, localization of both AQP2 and pAQP2 in the renal inner medullary principal cells appeared more dispersed, and the intensity of apical membrane staining for AQP2 was reduced significantly (by ∼20%) in EHD4-KO mice compared to WT mice in baseline conditions, suggesting an important role of EHD4 in trafficking of AQP2. Together, these data indicate that EHD4 play important roles in the regulation of water homeostasis.-Rahman, S. S., Moffitt, A. E. J., Trease, A. J., Foster, K. W., Storck, M. D., Band, H., Boesen, E. I. EHD4 is a novel regulator of urinary water homeostasis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Urina/química , Água/metabolismo , Animais , Aquaporina 2/metabolismo , Aquaporina 4/metabolismo , Arginina Vasopressina/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Feminino , Homeostase/genética , Homeostase/fisiologia , Medula Renal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética
6.
Int J Mol Sci ; 19(5)2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29748463

RESUMO

Connexins are integral membrane building blocks that form gap junctions, enabling direct cytoplasmic exchange of ions and low-molecular-mass metabolites between adjacent cells. In the heart, gap junctions mediate the propagation of cardiac action potentials and the maintenance of a regular beating rhythm. A number of connexin interacting proteins have been described and are known gap junction regulators either through direct effects (e.g., kinases) or the formation of larger multifunctional complexes (e.g., cytoskeleton scaffold proteins). Most connexin partners can be categorized as either proteins promoting coupling by stimulating forward trafficking and channel opening or inhibiting coupling by inducing channel closure, internalization, and degradation. While some interactions have only been implied through co-localization using immunohistochemistry, others have been confirmed by biophysical methods that allow detection of a direct interaction. Our understanding of these interactions is, by far, most well developed for connexin 43 (Cx43) and the scope of this review is to summarize our current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by demonstrating their importance at the intercalated disc, a major hub for Cx43 regulation and Cx43 mediated effects.


Assuntos
Conexina 43/genética , Citoesqueleto/genética , Junções Comunicantes/genética , Mapas de Interação de Proteínas/genética , Fenômenos Biofísicos , Comunicação Celular/genética , Conexina 43/química , Citoesqueleto/química , Junções Comunicantes/química , Humanos , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética
7.
Int J Mol Sci ; 19(6)2018 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-29882937

RESUMO

Activation of Wnt signaling induces Connexin43 (Cx43) expression via the transcriptional activity of ß-catenin, and results in the enhanced accumulation of the Cx43 protein and the formation of gap junction channels. In response to Wnt signaling, ß-catenin co-localizes with the Cx43 protein itself as part of a complex at the gap junction plaque. Work from several labs have also shown indirect evidence of this interaction via reciprocal co-immunoprecipitation. Our goal for the current study was to identify whether ß-catenin directly interacts with Cx43, and if so, the location of that direct interaction. Identifying residues involved in direct protein⁻protein interaction is of importance when they are correlated to the phosphorylation of Cx43, as phosphorylation can modify the binding affinities of Cx43 regulatory protein partners. Therefore, combining the location of a protein partner interaction on Cx43 along with the phosphorylation pattern under different homeostatic and pathological conditions will be crucial information for any potential therapeutic intervention. Here, we identified that ß-catenin directly interacts with the Cx43 carboxyl-terminal domain, and that this interaction would be inhibited by the Src phosphorylation of Cx43CT residues Y265 and Y313.


Assuntos
Conexina 43/química , Conexina 43/metabolismo , beta Catenina/metabolismo , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Humanos , Espectroscopia de Ressonância Magnética , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Ratos , Ressonância de Plasmônio de Superfície , beta Catenina/química
8.
J Mol Cell Cardiol ; 111: 69-80, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28754342

RESUMO

Gap junctions, composed of connexins, mediate electrical coupling and impulse propagation in the working myocardium. In the human heart, the spatio-temporal regulation and distinct functional properties of the three dominant connexins (Cx43, Cx45, and Cx40) suggests non-redundant physiological roles for each isoform. There are substantial differences in gating properties, expression, and trafficking among these isoforms, however, little is known about the determinants of these different phenotypes. To gain insight regarding these determinants, we focused on the carboxyl-terminal (CT) domain because of its importance in channel regulation and large degree of sequence divergence among connexin family members. Using in vitro biophysical experiments, we identified a structural feature unique to Cx45: high affinity (KD~100nM) dimerization between CT domains. In this study, we sought to determine if this dimerization occurs in cells and to identify the biological significance of the dimerization. Using a bimolecular fluorescence complementation assay, we demonstrate that the CT domains dimerize at the plasma membrane. By inhibiting CT dimerization with a mutant construct, we show that CT dimerization is necessary for proper Cx45 membrane localization, turnover, phosphorylation status, and binding to protein partners. Furthermore, CT dimerization is needed for normal intercellular communication and hemichannel activity. Altogether, our results demonstrate that CT dimerization is a structural feature important for correct Cx45 function. This study is significant because discovery of how interactions mediated by the CT domains can be modulated would open the door to strategies to ameliorate the pathological effects of altered connexin regulation in the failing heart.


Assuntos
Conexinas/química , Conexinas/metabolismo , Citoplasma/metabolismo , Miocárdio/metabolismo , Multimerização Proteica , Transdução de Sinais , Sequência de Aminoácidos , Animais , Comunicação Celular , Membrana Celular/metabolismo , Cães , Junções Comunicantes/metabolismo , Células HeLa , Humanos , Ativação do Canal Iônico , Células Madin Darby de Rim Canino , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Domínios Proteicos , Proteólise
9.
Biopolymers ; 105(3): 143-62, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26542351

RESUMO

The connexin carboxyl-terminal (CxCT) domain plays a role in the trafficking, localization, and turnover of gap junction channels, as well as the level of gap junction intercellular communication via numerous post-translational modifications and protein-protein interactions. As a key player in the regulation of gap junctions, the CT presents itself as a target for manipulation intended to modify function. Specific to intrinsically disordered proteins, identifying residues whose secondary structure can be manipulated will be critical toward unlocking the therapeutic potential of the CxCT domain. To accomplish this goal, we used biophysical methods to characterize CxCT domains attached to their fourth transmembrane domain (TM4). Circular dichroism and nuclear magnetic resonance were complementary in demonstrating the connexin isoforms that form the greatest amount of α-helical structure in their CT domain (Cx45 > Cx43 > Cx32 > Cx50 > Cx37 ≈ Cx40 ≈ Cx26). Studies compared the influence of 2,2,2-trifluoroethanol, pH, phosphorylation, and mutations (Cx32, X-linked Charcot-Marie Tooth disease; Cx26, hearing loss) on the TM4-CxCT structure. While pH modestly influences the CT structure, a major structural change was associated with phosphomimetic substitutions. Since most connexin CT domains are phosphorylated throughout their life cycle, studies of phospho-TM4-CxCT isoforms will be critical toward understanding the role that structure plays in regulating gap junction function.


Assuntos
Conexinas/química , Isoformas de Proteínas/química , Dicroísmo Circular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína
10.
Biophys J ; 106(10): 2184-95, 2014 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-24853747

RESUMO

Mechanisms underlying the initiation and persistence of lethal cardiac rhythms are of significant clinical and scientific interests. Gap junctions are principally involved in forming the electrical connections between myocytes, and changes in distribution, density, and properties are consistent characteristics in arrhythmic heart disease. Therefore, understanding the structure and function of gap junctions during normal and abnormal impulse propagation are essential in the control of arrhythmias. For example, Cx45 is predominately expressed in the specialized myocytes of the impulse generation and conduction system. In both ventricular and atrial human working myocytes, Cx45 is present in very low quantities. However, a reduction in Cx43 coupled with an increased Cx45 protein levels within the ventricles have been observed after myocardial infarction and end-stage heart failure. Cx45 may influence electrical and/or metabolic coupling as a result of pathophysiological overexpression. Our goal was to identify mechanisms that could cause cellular coupling to be different between the cardiac connexins. Based upon the conserved transmembrane and extracellular loop segments, our focus was on identifying features within the divergent cytoplasmic portions. Here, we biophysically characterize the carboxyl-terminal domain of Cx45 (Cx45CT). Purification revealed the possibility of oligomeric species, which was confirmed by analytical ultracentrifugation experiments. Sedimentation equilibrium and circular dichroism studies of different Cx45CT constructs identified one region of α-helical structure (A333-N361) that mediates CT dimerization through hydrophobic contacts. Interestingly, the binding affinity of Cx45CT dimerization is 1000-fold stronger than Cx43CT dimerization. Cx45CT resonance assignments were also used to identify the binding sites and affinities of molecular partners involved in the Cx45 regulation; although none disrupted dimerization, many of these proteins interacted within one intrinsically disordered region (P278-P285). This domain has similarities with other cardiac connexins, and we propose they constitute a master regulatory domain, which contains overlapping molecular partner binding, cis-trans proline isomerization, and phosphorylation sites.


Assuntos
Conexinas/química , Conexinas/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Proteína alfa-5 de Junções Comunicantes
11.
Fla Entomol ; 97(2): 362-366, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25382863

RESUMO

The last 2 decades have produced a better understanding of insect-microbial associations and yielded some important opportunities for insect control. However, most of our knowledge comes from model systems. Thrips (Thysanoptera: Thripidae) have been understudied despite their global importance as invasive species, plant pests and disease vectors. Using a culture and primer independent next-generation sequencing and metagenomics pipeline, we surveyed the bacteria of the globally important pest, Scirtothrips dorsalis Hood. The most abundant bacterial phyla identified were Actinobacteria and Proteobacteria and the most abundant genera were Propionibacterium, Stenotrophomonas, and Pseudomonas. A total of 189 genera of bacteria were identified. The absence of any vertically transferred symbiont taxa commonly found in insects is consistent with other studies suggesting that thrips primarilly acquire resident microbes from their environment. This does not preclude a possible beneficial/intimate association between S. dorsalis and the dominant taxa identified and future work should determine the nature of these associations.

12.
bioRxiv ; 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38617282

RESUMO

Human Immunodeficiency Virus (HIV) is widely acknowledged for its profound impact on the immune system. Although HIV primarily affects peripheral CD4 T cells, its influence on the central nervous system (CNS) cannot be overlooked. Within the brain, microglia and CNS-associated macrophages (CAMs) serve as the primary targets for HIV, as well as for the simian immunodeficiency virus (SIV) in nonhuman primates. This infection can lead to neurological effects and the establishment of a viral reservoir. Given the gaps in our understanding of how these cells respond in vivo to acute CNS infection, we conducted single-cell RNA sequencing (scRNA-seq) on myeloid cells from the brains of three rhesus macaques 12-days after SIV infection, along with three uninfected controls. Our analysis revealed six distinct microglial clusters including homeostatic microglia, preactivated microglia, and activated microglia expressing high levels of inflammatory and disease-related molecules. In response to acute SIV infection, the population of homeostatic and preactivated microglia decreased, while the activated and disease-related microglia increased. All microglial clusters exhibited upregulation of MHC class I molecules and interferon-related genes, indicating their crucial roles in defending against SIV during the acute phase. All microglia clusters also upregulated genes linked to cellular senescence. Additionally, we identified two distinct CAM populations: CD14lowCD16hi and CD14hiCD16low CAMs. Interestingly, during acute SIV infection, the dominant CAM population changed to one with an inflammatory phenotype. Notably, specific upregulated genes within one microglia and one macrophage cluster were associated with neurodegenerative pathways, suggesting potential links to neurocognitive disorders. This research sheds light on the intricate interactions between viral infection, innate immune responses, and the CNS, providing valuable insights for future investigations.

13.
Biomolecules ; 13(4)2023 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-37189407

RESUMO

T and B cell receptor signaling involves the activation of Akt, MAPKs, and PKC as well as an increase in intracellular Ca2+ and calmodulin activation. While these coordinate the rapid turnover of gap junctions, also implicated in this process is Src, which is not activated as part of T and B cell receptor signaling. An in vitro kinase screen identified that Bruton's tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) phosphorylate Cx43. Mass spectroscopy revealed that BTK and ITK phosphorylate Cx43 residues Y247, Y265, and Y313, which are identical to the residues phosphorylated by Src. Overexpression of BTK or ITK in the HEK-293T cells led to increased Cx43 tyrosine phosphorylation as well as decreased gap junction intercellular communication (GJIC) and Cx43 membrane localization. In the lymphocytes, activation of the B cell receptor (Daudi cells) or T cell receptor (Jurkat cells) increased the BTK and ITK activity, respectively. While this led to increased tyrosine phosphorylation of Cx43 and decreased GJIC, the cellular localization of Cx43 changed little. We have previously identified that Pyk2 and Tyk2 also phosphorylate Cx43 at residues Y247, Y265, and Y313 with a similar cellular fate to that of Src. With phosphorylation critical to Cx43 assembly and turnover, and kinase expression varying between different cell types, there would be a need for different kinases to achieve the same regulation of Cx43. The work presented herein suggests that in the immune system, ITK and BTK have the capacity for the tyrosine phosphorylation of Cx43 to alter the gap junction function in a similar manner as Pyk2, Tyk2, and Src.


Assuntos
Conexina 43 , Interleucina-2 , Humanos , Tirosina Quinase da Agamaglobulinemia/metabolismo , Interleucina-2/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Quinase 2 de Adesão Focal , Comunicação Celular/fisiologia , Fosforilação , Junções Comunicantes/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Tirosina/metabolismo , Linfócitos T/metabolismo
14.
Adv Biol (Weinh) ; 7(8): e2300154, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37376822

RESUMO

Mitochondrial dysfunction has been implicated in neurodegenerative diseases like Parkinson's disease (PD). This study investigates the role of Parkin, a protein involved in mitochondrial quality control, and strongly linked to PD, in the context of mitochondrial DNA (mtDNA) mutations. Mitochondrial mutator mice (PolgD257A/D257A ) (Polg) are used and bred with Parkin knockout (PKO) mice or mice with disinhibited Parkin (W402A). In the brain, mtDNA mutations are analyzed in synaptosomes, presynaptic neuronal terminals, which are far from neuronal soma, which likely renders mitochondria there more vulnerable compared with brain homogenate. Surprisingly, PKO results in reduced mtDNA mutations in the brain but increased control region multimer (CRM) in synaptosomes. In the heart, both PKO and W402A lead to increased mutations, with W402A showing more mutations in the heart than PKO. Computational analysis reveals many of these mutations are deleterious. These findings suggest that Parkin plays a tissue-dependent role in regulating mtDNA damage response, with differential effects in the brain and heart. Understanding the specific role of Parkin in different tissues may provide insights into the underlying mechanisms of PD and potential therapeutic strategies. Further investigation into these pathways can enhance the understanding of neurodegenerative diseases associated with mitochondrial dysfunction.


Assuntos
Encéfalo , DNA Polimerase gama , Genoma Mitocondrial , Coração , Ubiquitina-Proteína Ligases , Animais , Camundongos , Camundongos Endogâmicos C57BL , Encéfalo/metabolismo , Mitocôndrias , Heteroplasmia , Camundongos Knockout , DNA Polimerase gama/genética , Ubiquitina-Proteína Ligases/genética
15.
Brain Behav Immun Health ; 31: 100656, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37484197

RESUMO

Animal disease models are important for neuroscience experimentation and in the study of neurodegenerative disorders. The major neurodegenerative disorder leading to motor impairments is Parkinson's disease (PD). The identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in PTEN-induced putative kinase 1 (Pink1) and the E3 ubiquitin ligase Parkin, two proteins involved in mitochondrial quality control, that could be harnessed to create animal models. However, to date, such models have not reproducibly recapitulated major aspects of the disease. Here, we describe the generation and phenotypic characterization of a combined Pink1/Parkin double knockout (dKO) rat, which reproducibly exhibits PD-relevant abnormalities, particularly in male animals. Motor dysfunction in Pink1/Parkin dKO rats was characterized by gait abnormalities and decreased rearing frequency, the latter of which was responsive to levodopa treatment. Pink1/Parkin dKO rats exhibited elevated plasma levels of neurofilament light chain and significant loss of tyrosine hydroxylase expression in the substantia nigra pars compacta (SNpc). Glial cell activation was also observed in the SNpc. Pink1/Parkin dKO rats showed elevated plasma and reduced cerebrospinal levels of alpha-synuclein as well as the presence of alpha-synuclein aggregates in the striatum. Further, the profile of circulating lymphocytes was altered, as elevated CD3+CD4+ T cells and reduced CD3+CD8+ T cells in Pink1/Parkin dKO rats were found. This coincided with mitochondrial dysfunction and infiltration of CD3+ T cells in the striatum. Altogether, the Pink1/Parkin dKO rats exhibited phenotypes similar to what is seen with PD patients, thus highlighting the suitability of this model for mechanistic studies of the role of Pink1 and Parkin in PD pathogenesis and as therapeutic targets.

16.
J Leukoc Biol ; 112(5): 969-981, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35686500

RESUMO

Microglia and macrophages are essential for homeostatic maintenance and innate immune response in the brain. They are the first line of defense against infections such as HIV/SIV in the brain. However, they are susceptible to infection and function as viral reservoirs even under effective viral suppression. While current antiretroviral regimens successfully suppress viremia and improve quality of life and lifespan, neurologic complications persist and are in part attributed to activated microglia. We sought to test the hypothesis that brain microglia return to a more homeostatic-like state when viremia is suppressed by combination antiretroviral therapy. Using the SIV-rhesus macaque model, we combined single-cell RNA sequencing, bioinformatics, and pathway analysis to compare gene expression profiles of brain myeloid cells under 4 conditions: uninfected, SIV infected, SIV infected with cART suppression, and SIV encephalitis (SIVE). Our study reveals greater myeloid diversity and an elevated proinflammatory state are associated with untreated SIV infection compared with uninfected animals. The development of encephalitis and suppression of viremia both reduced myeloid diversity. However, they had converse effects on the activation state of microglia and inflammation. Notably, suggestive of a restoration of a homeostatic state in microglia, gene expression and activation of pathways related to inflammation and immune response in cART-suppressed monkeys were most similar to that in uninfected monkeys. Untreated SIV infection shared characteristics, especially in brain macrophages to SIVE, with SIVE showing dramatic inflammation. In support of our hypothesis, our study demonstrates that cART indeed restores this key component of the brain's homeostatic state. Summary: ScRNA-seq of rhesus monkey microglia reveals clusters of cells in activated states in the setting of SIV infection, which is primarily reversed by suppressing viremia with combination antiretroviral therapy.


Assuntos
Encefalite , Infecções por HIV , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Animais , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Microglia , Viremia/tratamento farmacológico , Qualidade de Vida , Encefalite/complicações , Carga Viral
17.
Front Mol Neurosci ; 15: 852368, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35359570

RESUMO

Neurogenerative disorders, such as Alzheimer's disease (AD), represent a growing public health challenge in aging societies. Tauopathies, a subset of neurodegenerative disorders that includes AD, are characterized by accumulation of fibrillar and hyperphosphorylated forms of microtubule-associated protein tau with coincident mitochondrial abnormalities and neuronal dysfunction. Although, in vitro, tau impairs axonal transport altering mitochondrial distribution, clear in vivo mechanisms associating tau and mitochondrial dysfunction remain obscure. Herein, we investigated the effects of human tau on brain mitochondria in vivo using transgenic htau mice at ages preceding and coinciding with onset of tauopathy. Subcellular proteomics combined with bioenergetic assessment revealed pathologic forms of tau preferentially associate with synaptic over non-synaptic mitochondria coinciding with changes in bioenergetics, reminiscent of an aged synaptic mitochondrial phenotype in wild-type mice. While mitochondrial content was unaltered, mitochondrial maximal respiration was impaired in synaptosomes from htau mice. Further, mitochondria-associated tau was determined to be outer membrane-associated using the trypsin protection assay and carbonate extraction. These findings reveal non-mutant human tau accumulation at the synapse has deleterious effects on mitochondria, which likely contributes to synaptic dysfunction observed in the context of tauopathy.

18.
Biomolecules ; 10(10)2020 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-33080786

RESUMO

The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects.


Assuntos
Calmodulina/genética , Conexina 43/genética , Anormalidades Craniofaciais/genética , Anormalidades do Olho/genética , Deformidades Congênitas do Pé/genética , Sindactilia/genética , Anormalidades Dentárias/genética , Calmodulina/ultraestrutura , Movimento Celular/genética , Conexina 43/ultraestrutura , Anormalidades Craniofaciais/patologia , Citoplasma/genética , Anormalidades do Olho/patologia , Quinase 2 de Adesão Focal/genética , Deformidades Congênitas do Pé/patologia , Junções Comunicantes/genética , Células HeLa , Humanos , Mutação com Perda de Função/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Transporte Proteico/genética , Sindactilia/patologia , Anormalidades Dentárias/patologia
19.
J Genomics ; 2: 141-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25371763

RESUMO

The northern leopard frog Rana (Lithobates) pipiens is an important animal model, being used extensively in cancer, neurology, physiology, and biomechanical studies. R. pipiens is a native North American frog whose range extends from northern Canada to southwest United States, but over the past few decades its populations have declined significantly and is now considered uncommon in large portions of the United States and Canada. To aid in the study and conservation of R. pipiens, this paper describes the first R. pipiens transcriptome. The R. pipiens transcriptome was annotated using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Eukaryotic Orthologous Groups (KOG). Differential expression analysis revealed universal and tissue specific genes, and endocrine-related genes were identified. Transcriptome assemblies and other sequence data are available for download.

20.
J Genomics ; 2: 54-58, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24511328

RESUMO

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is a vector for the causative agents of Huanglongbing, which threatens citrus production worldwide. This study reports and discusses the first D. citri transcriptomes, encompassing the three main life stages of D. citri, egg, nymph and adult. The transcriptomes were annotated using Gene Ontology (GO) and insecticide-related genes within each life stage were identified to aid the development of future D. citri insecticides. Transcriptome assemblies and other sequence data are available for download at the International Asian Citrus Psyllid Genome Consortium website [http://psyllid.org/download] and at NCBI [http://www.ncbi.nlm.nih.gov/bioproject/29447].

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