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1.
Anticancer Res ; 44(2): 561-565, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38307547

RESUMO

BACKGROUND/AIM: Cytoglobin (Cygb), a protein involved in cellular oxygen metabolism and protection, has garnered attention owing to its potential role in the initiation and progression of cancer, particularly colon cancer (CC). This study investigated the expression and significance of Cygb in CC. PATIENTS AND METHODS: This study included 145 patients who underwent R0 surgery for CC (clinical stage II/III) at our institution between January 2007 and December 2014. Immunohistochemical analysis was performed to evaluate the Cygb expression patterns in CC tissues. Additionally, the correlation between Cygb expression levels and the clinicopathological characteristics of patients with CC was investigated. RESULTS: Colon cancer tissues were categorized into high-expression (95 cases) and low-expression (50 cases) groups. Cygb was highly expressed in well-differentiated cases, whereas its expression decreased in poorly differentiated cases. No significant differences in other clinicopathological factors were observed between the two groups. Cygb expression had no significant effect on recurrence-free survival or overall survival. CONCLUSION: This study contributes to the growing understanding of Cygb expression and its significance in CC. The expression of Cygb in CC was found to be unrelated to the recurrence rate and prognosis, but showed a correlation with differentiation status.


Assuntos
Neoplasias do Colo , Globinas , Humanos , Citoglobina , Globinas/metabolismo
2.
PeerJ ; 12: e16898, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38332807

RESUMO

Agrobacterium tumefaciens is a soil-borne pathogenic bacterium that causes crown gall disease in many plants. Chemotaxis offers A. tumefaciens the ability to find its host and establish infection. Being an aerobic bacterium, A. tumefaciens possesses one chemotaxis system with multiple potential chemoreceptors. Chemoreceptors play an important role in perceiving and responding to environmental signals. However, the studies of chemoreceptors in A. tumefaciens remain relatively restricted. Here, we characterized a cytoplasmic chemoreceptor of A. tumefaciens C58 that contains an N-terminal globin domain. The chemoreceptor was designated as Atu1027. The deletion of Atu1027 not only eliminated the aerotactic response of A. tumefaciens to atmospheric air but also resulted in a weakened chemotactic response to multiple carbon sources. Subsequent site-directed mutagenesis and phenotypic analysis showed that the conserved residue His100 in Atu1027 is essential for the globin domain's function in both chemotaxis and aerotaxis. Furthermore, deleting Atu1027 impaired the biofilm formation and pathogenicity of A. tumefaciens. Collectively, our findings demonstrated that Atu1027 functions as an aerotaxis receptor that affects agrobacterial chemotaxis and the invasion of A. tumefaciens into its host.


Assuntos
Agrobacterium tumefaciens , Quimiotaxia , Agrobacterium tumefaciens/genética , Quimiotaxia/genética , Tumores de Planta/microbiologia , Plantas , Globinas
3.
J Inorg Biochem ; 252: 112482, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38218138

RESUMO

Bacteria utilize heme proteins, such as globin coupled sensors (GCSs), to sense and respond to oxygen levels. GCSs are predicted in almost 2000 bacterial species and consist of a globin domain linked by a central domain to a variety of output domains, including diguanylate cyclase domains that synthesize c-di-GMP, a major regulator of biofilm formation. To investigate the effects of middle domain length and heme edge residues on GCS diguanylate cyclase activity and cellular function, a putative diguanylate cyclase-containing GCS from Shewanella sp. ANA-3 (SA3GCS) was characterized. Binding of O2 to the heme resulted in activation of diguanylate cyclase activity, while NO and CO binding had minimal effects on catalysis, demonstrating that SA3GCS exhibits greater ligand selectivity for cyclase activation than many other diguanylate cyclase-containing GCSs. Small angle X-ray scattering analysis of dimeric SA3GCS identified movement of the cyclase domains away from each other, while maintaining the globin dimer interface, as a potential mechanism for regulating cyclase activity. Comparison of the Shewanella ANA-3 wild type and SA3GCS deletion (ΔSA3GCS) strains identified changes in biofilm formation, demonstrating that SA3GCS diguanylate cyclase activity modulates Shewanella phenotypes.


Assuntos
GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli , Shewanella , Globinas/química , Oxigênio/metabolismo , Proteínas de Escherichia coli/química , Fósforo-Oxigênio Liases/química , Biofilmes , Heme/química , Proteínas de Bactérias/química
4.
Int J Mol Sci ; 25(2)2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38279321

RESUMO

Specific sequences within RNA encoded by human genes essential for survival possess the ability to activate the RNA-dependent stress kinase PKR, resulting in phosphorylation of its substrate, eukaryotic translation initiation factor-2α (eIF2α), either to curb their mRNA translation or to enhance mRNA splicing. Thus, interferon-γ (IFNG) mRNA activates PKR through a 5'-terminal 203-nucleotide pseudoknot structure, thereby strongly downregulating its own translation and preventing a harmful hyper-inflammatory response. Tumor necrosis factor-α (TNF) pre-mRNA encodes within the 3'-untranslated region (3'-UTR) a 104-nucleotide RNA pseudoknot that activates PKR to enhance its splicing by an order of magnitude while leaving mRNA translation intact, thereby promoting effective TNF protein expression. Adult and fetal globin genes encode pre-mRNA structures that strongly activate PKR, leading to eIF2α phosphorylation that greatly enhances spliceosome assembly and splicing, yet also structures that silence PKR activation upon splicing to allow for unabated globin mRNA translation essential for life. Regulatory circuits resulting in each case from PKR activation were reviewed previously. Here, we analyze mutations within these genes created to delineate the RNA structures that activate PKR and to deconvolute their folding. Given the critical role of intragenic RNA activators of PKR in gene regulation, such mutations reveal novel potential RNA targets for human disease.


Assuntos
Precursores de RNA , RNA , Humanos , RNA/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Biossíntese de Proteínas , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , RNA Mensageiro/genética , Fosforilação , Fator de Necrose Tumoral alfa/metabolismo , Nucleotídeos/metabolismo , Globinas/genética , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo
5.
Biochemistry ; 63(4): 523-532, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38264987

RESUMO

Globin-coupled sensors constitute an important family of heme-based gas sensors, an emerging class of heme proteins. In this study, we have identified and characterized a globin-coupled sensor phosphodiesterase containing an HD-GYP domain (GCS-HD-GYP) from the human pathogen Vibrio fluvialis, which is an emerging foodborne pathogen of increasing public health concern. The amino acid sequence encoded by the AL536_01530 gene from V. fluvialis indicated the presence of an N-terminal globin domain and a C-terminal HD-GYP domain, with HD-GYP domains shown previously to display phosphodiesterase activity toward bis(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates numerous important physiological functions in bacteria, including in bacterial pathogens. Optical absorption spectral properties of GCS-HD-GYP were found to be similar to those of myoglobin and hemoglobin and of other bacterial globin-coupled sensors. The binding of O2 to the Fe(II) heme iron complex of GCS-HD-GYP promoted the catalysis of the hydrolysis of c-di-GMP to its linearized product, 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), whereas CO and NO binding did not enhance the catalysis, indicating a strict discrimination of these gaseous ligands. These results shed new light on the molecular mechanism of gas-selective catalytic regulation by globin-coupled sensors, with these advances apt to lead to a better understanding of the family of globin-coupled sensors, a still growing family of heme-based gas sensors. In addition, given the importance of c-di-GMP in infection and virulence, our results suggested that GCS-HD-GYP could play an important role in the ability of V. fluvialis to sense O2 and NO in the context of host-pathogen interactions.


Assuntos
Globinas , Diester Fosfórico Hidrolases , Vibrio , Humanos , Diester Fosfórico Hidrolases/genética , Globinas/genética , Proteínas de Bactérias/química , Catálise , GMP Cíclico/metabolismo , Heme/química
6.
Br J Haematol ; 204(2): 399-401, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37985143

RESUMO

The genetic underpinnings of beta-thalassaemia encompass a myriad of molecular mechanisms. The ability of synonymous mutations, an often-overlooked category of variants, to influence ß-globin expression and phenotypic disease is highlighted by this report by Gorivale et al. Commentary on: Gorivale et al. When a synonymous mutation breaks the silence in a thalassaemia patient. Br J Haematol 2024;204:677-682.


Assuntos
Talassemia , Talassemia beta , Humanos , Mutação Silenciosa , Mutação , Talassemia beta/genética , Globinas beta/genética , Globinas/genética
7.
Blood Cells Mol Dis ; 104: 102761, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37271682

RESUMO

ß-Thalassemia is a genetic form of anemia due to mutations in the ß-globin gene, that leads to ineffective and extramedullary erythropoiesis, abnormal red blood cells and secondary iron-overload. The severity of the disease ranges from mild to lethal anemia based on the residual levels of globins production. Despite being a monogenic disorder, the pathophysiology of ß-thalassemia is multifactorial, with different players contributing to the severity of anemia and secondary complications. As a result, the identification of effective therapeutic strategies is complex, and the treatment of patients is still suboptimal. For these reasons, several models have been developed in the last decades to provide experimental tools for the study of the disease, including erythroid cell lines, cultures of primary erythroid cells and transgenic animals. Years of research enabled the optimization of these models and led to decipher the mechanisms responsible for globins deregulation and ineffective erythropoiesis in thalassemia, to unravel the role of iron homeostasis in the disease and to identify and validate novel therapeutic targets and agents. Examples of successful outcomes of these analyses include iron restricting agents, currently tested in the clinics, several gene therapy vectors, one of which was recently approved for the treatment of most severe patients, and a promising gene editing strategy, that has been shown to be effective in a clinical trial. This review provides an overview of the available models, discusses pros and cons, and the key findings obtained from their study.


Assuntos
Talassemia beta , Animais , Humanos , Talassemia beta/genética , Talassemia beta/terapia , Eritropoese/genética , Ferro/metabolismo , Globinas/genética , Modelos Animais de Doenças
8.
J Inorg Biochem ; 250: 112387, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37914583

RESUMO

Most hemoproteins display an all-α-helical fold, showing the classical three on three (3/3) globin structural arrangement characterized by seven or eight α-helical segments that form a sandwich around the heme. Over the last decade, a completely distinct class of heme-proteins called nitrobindins (Nbs), which display an all-ß-barrel fold, has been identified and characterized from both structural and functional perspectives. Nbs are ten-stranded anti-parallel all-ß-barrel heme-proteins found across the evolutionary ladder, from bacteria to Homo sapiens. Myoglobin (Mb), commonly regarded as the prototype of monomeric all-α-helical globins, is involved along with the oligomeric hemoglobin (Hb) in diatomic gas transport, storage, and sensing, as well as in the detoxification of reactive nitrogen and oxygen species. On the other hand, the function(s) of Nbs is still obscure, even though it has been postulated that they might participate to O2/NO signaling and metabolism. This function might be of the utmost importance in poorly oxygenated tissues, such as the eye's retina, where a delicate balance between oxygenation and blood flow (regulated by NO) is crucial. Dysfunction in this balance is associated with several pathological conditions, such as glaucoma and diabetic retinopathy. Here a detailed comparison of the structural, spectroscopic, and functional properties of Mb and Nbs is reported to shed light on the similarities and differences between all-α-helical and all-ß-barrel heme-proteins.


Assuntos
Globinas , Mioglobina , Humanos , Globinas/química , Heme/química , Hemoglobinas/química , Mioglobina/química , Análise Espectral
9.
J Inorg Biochem ; 250: 112405, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37977965

RESUMO

The vertebrate respiratory protein cytoglobin (Cygb) is thought to exert multiple cellular functions. Here we studied the phenotypic effects of a Cygb knockout (KO) in mouse on the transcriptome level. RNA sequencing (RNA-Seq) was performed for the first time on sites of major endogenous Cygb expression, i.e. quiescent and activated hepatic stellate cells (HSCs) and two brain regions, hippocampus and hypothalamus. The data recapitulated the up-regulation of Cygb during HSC activation and its expression in the brain. Differential gene expression analyses suggested a role of Cygb in the response to inflammation in HSCs and its involvement in retinoid metabolism, retinoid X receptor (RXR) activation-induced xenobiotics metabolism, and RXR activation-induced lipid metabolism and signaling in activated cells. Unexpectedly, only minor effects of the Cygb KO were detected in the transcriptional profiles in hippocampus and hypothalamus, precluding any enrichment analyses. Furthermore, the transcriptome data pointed at a previously undescribed potential of the Cygb- knockout allele to produce cis-acting effects, necessitating future verification studies.


Assuntos
Globinas , Células Estreladas do Fígado , Animais , Camundongos , Citoglobina/genética , Citoglobina/metabolismo , Citoglobina/farmacologia , Perfilação da Expressão Gênica , Globinas/genética , Globinas/metabolismo , Células Estreladas do Fígado/metabolismo , Hipocampo/metabolismo , Camundongos Knockout , Transcriptoma
10.
Int J Mol Sci ; 24(21)2023 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-37958992

RESUMO

Globins have been studied as model proteins to elucidate the principles of protein evolution. This was achieved by understanding the relationship between amino acid sequence, three-dimensional structure, physicochemical properties, and physiological function. Previous molecular phylogenies of chordate globin genes revealed the monophyletic evolution of urochordate globins and suggested convergent evolution. However, to provide evidence of convergent evolution, it is necessary to determine the physicochemical and functional similarities between vertebrates and urochordate globins. In this study, we determined the expression patterns of Ciona globin genes using real-time RT-PCR. Two genes (Gb-1 and Gb-2) were predominantly expressed in the branchial sac, heart, and hemocytes and were induced under hypoxia. Combined with the sequence analysis, our findings suggest that Gb-1/-2 correspond to vertebrate hemoglobin-α/-ß. However, we did not find a robust similarity between Gb-3, Gb-4, and vertebrate globins. These results suggested that, even though Ciona globins obtained their unique functions differently from vertebrate globins, the two of them shared some physicochemical features and physiological functions. Our findings offer a good example for understanding the molecular mechanisms underlying gene co-option and convergence, which could lead to evolutionary innovations.


Assuntos
Ciona intestinalis , Anfioxos , Animais , Humanos , Globinas/genética , Ciona intestinalis/genética , Anfioxos/genética , Vertebrados/genética , Sequência de Aminoácidos , Família Multigênica , Filogenia , Evolução Molecular
11.
Biochim Biophys Acta Mol Cell Res ; 1870(8): 119558, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37549740

RESUMO

Cytoglobin (Cygb) is an evolutionary ancient heme protein with yet unclear physiological function(s). Mammalian Cygb is ubiquitously expressed in all tissues and is proposed to be involved in reactive oxygen species (ROS) detoxification, nitric oxide (NO) metabolism and lipid-based signaling processes. Loss-of-function studies in mouse associate Cygb with apoptosis, inflammation, fibrosis, cardiovascular dysfunction or oncogenesis. In zebrafish (Danio rerio), two cygb genes exist, cytoglobin 1 (cygb1) and cytoglobin 2 (cygb2). Both have different coordination states and distinct expression sites within zebrafish tissues. The biological roles of the cygb paralogs are largely uncharacterized. We used a CRISPR/Cas9 genome editing approach and generated a knockout of the penta-coordinated cygb1 for in vivo analysis. Adult male cygb1 knockouts develop phenotypic abnormalities, including weight loss. To identify the molecular mechanisms underlying the occurrence of these phenotypes and differentiate between function and effect of the knockout we compared the transcriptomes of cygb1 knockout at different ages to age-matched wild-type zebrafish. We found that immune regulatory and cell cycle regulatory transcripts (e.g. tp53) were up-regulated in the cygb1 knockout liver. Additionally, the expression of transcripts involved in lipid metabolism and transport, the antioxidative defense and iron homeostasis was affected in the cygb1 knockout. Cygb1 may function as an anti-inflammatory and cytoprotective factor in zebrafish liver, and may be involved in lipid-, iron-, and ROS-dependent signaling.


Assuntos
Globinas , Peixe-Zebra , Masculino , Camundongos , Animais , Citoglobina/genética , Citoglobina/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Globinas/genética , Globinas/metabolismo , Metabolismo dos Lipídeos/genética , Espécies Reativas de Oxigênio , Estresse Oxidativo/genética , Homeostase/genética , Lipídeos , Mamíferos/metabolismo
12.
Redox Biol ; 65: 102838, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37573836

RESUMO

Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.


Assuntos
Globinas , Proteína HMGB2 , Animais , Camundongos , Citoglobina/genética , Dano ao DNA , Globinas/genética , Globinas/metabolismo , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Fatores de Transcrição/genética
13.
Biochemistry ; 62(18): 2727-2737, 2023 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-37647623

RESUMO

Proteins have undergone evolutionary processes to achieve optimal stability, increased functionality, and novel functions. Comparative analysis of existent and ancestral proteins provides insights into the factors that influence protein stability and function. Ancestral sequence reconstruction allows us to deduce the amino acid sequences of ancestral proteins. Here, we present the structural and functional characteristics of an ancestral protein, AncMH, reconstructed to be the last common ancestor of hemoglobins and myoglobins. Our findings reveal that AncMH harbors heme and that the heme binds oxygen. Furthermore, we demonstrate that the ferrous heme in AncMH is pentacoordinated, similar to that of human adult hemoglobin and horse myoglobin. A detailed comparison of the heme pocket structure indicates that the heme pocket in AncMH is more similar to that of hemoglobin than that of myoglobin. However, the autoxidation of AncMH is faster than that of both hemoglobin and myoglobin. Collectively, our results suggest that ancestral proteins of hemoglobins and myoglobins evolved in steps, including the hexa- to pentacoordination transition, followed by stabilization of the oxygen-bound form.


Assuntos
Globinas , Heme , Adulto , Humanos , Animais , Cavalos , Globinas/genética , Mioglobina/genética , Sequência de Aminoácidos , Oxigênio
14.
Biol Pharm Bull ; 46(7): 1027-1030, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37394635

RESUMO

Globin digest (GD) inhibits dietary hypertriglyceridemia; however, its effects on physical fatigue remain unknown. Therefore, this study aimed to investigate the potential anti-fatigue effects of GD. Repeated administration of GD and valine (Val)-Val-tyrosine (Tyr)-proline (Pro), a component of GD, for five days prevented the forced walking-induced decrease in locomotion. Furthermore, GD treatment reversed the forced walking-induced increase in blood lactate levels in mice and increased phosphorylated AMP-activated protein kinase (p-AMPK) in the soleus muscle, suggesting that the anti-fatigue effect of GD involves AMPK activation in the soleus muscle through reduced blood lactate.


Assuntos
Globinas , Hiperlipidemias , Camundongos , Animais , Globinas/metabolismo , Globinas/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Músculo Esquelético/metabolismo , Lactatos
15.
J Clin Immunol ; 43(8): 1873-1880, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37505322

RESUMO

PURPOSE: This study aimed to investigate the correlation between calculated globulin (CG, total protein level minus albumin level) and the gamma globulin fraction (Gamma), obtained from serum protein electrophoresis with serum IgG levels in adults (≥ 18 years). METHODS: Using linear regression models, analyses of CG and Gamma levels correlation with IgG levels in adults were performed. Receiver-operator curves were created to determine cutoff values and the respective sensitivity and specificity measures. RESULTS: A total of 886 samples were analyzed. CG and Gamma were positively and statistically correlated with IgG levels (r2 = 0.4628 for CG, and = 0.7941 for Gamma, p < 0.0001 for both analyses). For the detection of hypogammaglobulinemia, i.e., IgG level below the reference value (6 g/L), a CG cutoff value of 24 g/L showed a sensitivity of 86.2% (95% CI 69.4-94.5) and a specificity of 92% (90.0-93.6). A Gamma cutoff value of 7.15 g/L yielded a sensitivity of 100% (88.3-100) and a specificity of 96.8 (95.3-97.8). CONCLUSION: Both CG and Gamma levels determined by protein electrophoresis analysis may be used to screen for antibody deficiencies in adults, enabling earlier diagnosis of antibody deficiencies in a routine clinical setting.


Assuntos
Agamaglobulinemia , Doenças da Imunodeficiência Primária , Humanos , Adulto , Eletroforese , Globinas , Imunoglobulina G
16.
Biophys J ; 122(15): 3117-3132, 2023 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-37353934

RESUMO

Artificial proteins representing the consensus of a set of homologous sequences have attracted attention for their increased thermodynamic stability and conserved activity. Here, we applied the consensus approach to a b-type heme-binding protein to inspect the contribution of a dissociable cofactor to enhanced stability and the chemical consequences of creating a generic heme environment. We targeted the group 1 truncated hemoglobin (TrHb1) subfamily of proteins for their small size (∼120 residues) and ease of characterization. The primary structure, derived from a curated set of ∼300 representative sequences, yielded a highly soluble consensus globin (cGlbN) enriched in acidic residues. Optical and NMR spectroscopies revealed high-affinity heme binding in the expected site and in two orientations. At neutral pH, proximal and distal iron coordination was achieved with a pair of histidine residues, as observed in some natural TrHb1s, and with labile ligation on the distal side. As opposed to studied TrHb1s, which undergo additional folding upon heme binding, cGlbN displayed the same extent of secondary structure whether the heme was associated with the protein or not. Denaturation required guanidine hydrochloride and showed that apo- and holoprotein unfolded in two transitions-the first (occurring with a midpoint of ∼2 M) was shifted to higher denaturant concentration in the holoprotein (∼3.7 M) and reflected stabilization due to heme binding, while the second transition (∼6.2 M) was common to both forms. Thus, the consensus sequence stabilized the protein but exposed the existence of two separately cooperative subdomains within the globin architecture, masked as one single domain in TrHb1s with typical stabilities. The results suggested ways in which specific chemical or thermodynamic features may be controlled in artificial heme proteins.


Assuntos
Globinas , Hemeproteínas , Globinas/química , Dobramento de Proteína , Termodinâmica , Heme/metabolismo , Desnaturação Proteica
17.
PLoS Genet ; 19(5): e1010727, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37216374

RESUMO

We report three novel deletions involving the Multispecies Conserved Sequences (MCS) R2, also known as the Major Regulative Element (MRE), in patients showing the α-thalassemia phenotype. The three new rearrangements showed peculiar positions of the breakpoints. 1) The (αα)ES is a telomeric 110 kb deletion ending inside the MCS-R3 element. 2) The (αα)FG, 984 bp-long, ends 51 bp upstream to MCS-R2; both are associated with a severe α-thalassemia phenotype. 3) The (αα)CT, 5058 bp-long starts at position +93 of MCS-R2 and is the only one associated to a mild α-thalassemia phenotype. To understand the specific role of different segments of the MCS-R2 element and of its boundary regions we carried out transcriptional and expression analysis. Transcriptional analysis of patients' reticulocytes showed that (αα)ES was unable to produce α2-globin mRNA, while a high level of expression of the α2-globin genes (56%) was detected in (αα)CT deletion, characterized by the presence of the first 93 bp of MCS-R2. Expression analysis of constructs containing breakpoints and boundary regions of the deletions (αα)CT and (αα)FG, showed comparable activity both for MCS-R2 and the boundary region (-682/-8). Considering that the (αα)CT deletion, almost entirely removing MCS-R2, has a less severe phenotype than the (αα)FG α0thalassemia deletion, removing both MCS-R2 almost entirely and an upstream 679 bp, we infer for the first time that an enhancer element must exist in this region that helps to increase the expression of the α-globin genes. The genotype-phenotype relationship of other previously published MCS-R2 deletions strengthened our hypothesis.


Assuntos
Talassemia alfa , Humanos , Talassemia alfa/genética , Globinas/genética , Fenótipo , Sequência Conservada , Elementos Facilitadores Genéticos/genética , Genótipo
18.
Methods Mol Biol ; 2648: 167-185, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37039991

RESUMO

Androglobin (ADGB), the most recently identified member of the mammalian globin family, is a chimeric protein with an unusual, embedded globin domain that is circularly permutated and exhibits hallmarks of a hexacoordinated heme-binding scheme. Whereas abundant expression of ADGB was initially found to be mainly restricted to cells in the postmeiotic stages of spermatogenesis, more recent RNA-Seq-based expression analysis data revealed that ADGB is detectable in cells carrying motile cilia or flagella. This very tight regulation of ADGB gene expression urges the need for alternative techniques to study endogenous expression in classical mammalian cell models, which do not express ADGB. We describe here the use of CRISPR activation (CRISPRa) technology to induce endogenous ADGB gene expression in HEK293T, MCF-7, and HeLa cells from its promoter and illustrate how this method can be employed to validate putative regulatory DNA elements of ADGB in promoter and enhancer regions.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Regulação da Expressão Gênica , Masculino , Humanos , Células HeLa , Células HEK293 , Globinas/genética , Globinas/metabolismo
19.
Biochim Biophys Acta Proteins Proteom ; 1871(4): 140913, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37004900

RESUMO

Out of the 34 globins in Caenorhabditis elegans, GLB-33 is a putative globin-coupled transmembrane receptor with a yet unknown function. The globin domain (GD) contains a particularly hydrophobic haem pocket, that rapidly oxidizes to a low-spin hydroxide-ligated haem state at physiological pH. Moreover, the GD has one of the fastest nitrite reductase activity ever reported for globins. Here, we use a combination of electronic circular dichroism, resonance Raman and electron paramagnetic resonance (EPR) spectroscopy with mass spectrometry to study the pH dependence of the ferric form of the recombinantly over-expressed GD in the presence and absence of nitrite. The competitive binding of nitrite and hydroxide is examined as well as nitrite-induced haem modifications at acidic pH. Comparison of the spectroscopic results with data from other haem proteins allows to deduce the important effect of Arg at position E10 in stabilization of exogenous ligands. Furthermore, continuous-wave and pulsed EPR indicate that ligation of nitrite occurs in a nitrito mode at pH 5.0 and above. At pH 4.0, an additional formation of a nitro-bound haem form is observed along with fast formation of a nitri-globin.


Assuntos
Caenorhabditis elegans , Globinas , Animais , Caenorhabditis elegans/metabolismo , Nitritos/metabolismo , Heme/metabolismo , Concentração de Íons de Hidrogênio
20.
Nat Commun ; 14(1): 2222, 2023 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-37076519

RESUMO

Variational autoencoders are unsupervised learning models with generative capabilities, when applied to protein data, they classify sequences by phylogeny and generate de novo sequences which preserve statistical properties of protein composition. While previous studies focus on clustering and generative features, here, we evaluate the underlying latent manifold in which sequence information is embedded. To investigate properties of the latent manifold, we utilize direct coupling analysis and a Potts Hamiltonian model to construct a latent generative landscape. We showcase how this landscape captures phylogenetic groupings, functional and fitness properties of several systems including Globins, ß-lactamases, ion channels, and transcription factors. We provide support on how the landscape helps us understand the effects of sequence variability observed in experimental data and provides insights on directed and natural protein evolution. We propose that combining generative properties and functional predictive power of variational autoencoders and coevolutionary analysis could be beneficial in applications for protein engineering and design.


Assuntos
Globinas , Fatores de Transcrição , Filogenia , Sequência de Aminoácidos , beta-Lactamases/genética
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