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A regulação genética é o processo usado para controlar o momento, a localização e a quantidade em que os genes são expressos. O processo pode ser complicado e é realizado por uma variedade de mecanismos, inclusive através de proteínas reguladoras e modificação química do DNA. A regulação genética é fundamental para a capacidade de um organismo responder às mudanças ambientais.

Fonte: https://www.genome.gov/genetics-glossary/Gene-Regulation

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Sequências homólogas que resultaram do Blast e foram colocadas no Clustal Omega com a finalidade de se obter a fologenia da proteína HLA-DRB1.

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O gene CD14 codifica uma proteína chamada receptor de lipopolissacarídeo (LPS), que está envolvida na resposta imunológica do organismo a bactérias. A regulação do gene CD14 é crucial para garantir que a expressão dessa proteína seja mantida em níveis apropriados para as necessidades do sistema imunológico.

Concluindo, este gene é um antígeno de diferenciação monocítica que regula as respostas imunes inatas a patógenos.

Fonte: https://www.ncbi.nlm.nih.gov/gene/929

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Official Symbol

Official Full Name

CD14 moleculeprovided by HGNC

Primary source

HGNC:HGNC:1628

See related

Ensembl:ENSG00000170458 MIM:158120; AllianceGenome:HGNC:1628

Gene type

protein coding

RefSeq status

REVIEWED

Organism

Homo sapiens

Lineage

Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo

Summary

The protein encoded by this gene is a surface antigen that is preferentially expressed on monocytes/macrophages. It cooperates with other proteins to mediate the innate immune response to bacterial lipopolysaccharide, and to viruses. This gene has been identified as a target candidate in the treatment of SARS-CoV-2-infected patients to potentially lessen or inhibit a severe inflammatory response. Alternative splicing results in multiple transcript variants encoding the same protein. [provided by RefSeq, Aug 2020]

Annotation information

Note: This gene has been reviewed for its involvement in coronavirus biology, and is relevant for COVID-19 treatment.

Expression

Broad expression in placenta (RPKM 87.5), liver (RPKM 85.2) and 23 other tissues See more

Orthologs

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CD14 CD14 molecule [ Homo sapiens (human) ]

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Official Symbol Official Full Name CD14 moleculeprovided by HGNC Primary source HGNC:HGNC:1628 See related Ensembl:ENSG00000170458 MIM:158120; AllianceGenome:HGNC:1628 Gene type protein coding RefSeq status REVIEWED Organism Homo sapiens Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo Summary The protein encoded by this gene is a surface antigen that is preferentially expressed on monocytes/macrophages.

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Através da análise da árvore filogenética fornecida pelo Clustal Omega, é possível verificar que existe um ancestral comum e que a partir deste surgem dois grupos distintos. A proteína major histocompatibility complex class II DR beta 1 precursor, com o código NP_001346123, associada ao gene HLA-DRB1 no Homo sapiens (query) apresenta maior proximidade com outras proteínas da mesma espécie como AAH24269 HLA-DRB1 protein e UMZ08559 MHC class II antigen, o que se pode verificar pelo número de ramificações que têm em comum e pelo comprimento destas em relação ao ancestral comum.

Tal vai de encontro ao esperado através da análise dos resultados fornecidos pelo Blast.

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Clustal Omega is a multiple sequence alignment program. It produces biologically meaningful multiple sequence alignments of divergent sequences. Evolutionary relationships can be seen via viewing Cladograms or Phylograms.

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As modificações pós-tradução são alterações de resíduos de aminoácidos por interação da proteína com outras moléculas e correspondem a uma mutação pontual na proteína.

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O alinhamento múltiplo de sequências é uma ferramenta usada para estudar genes ou proteínas intimamente relacionados, a fim de encontrar as relações evolutivas entre estes e identificar padrões compartilhados entre genes funcional ou estruturalmente relacionados.

[1]https://www.sciencedirect.com/topics/medicine-and-dentistry/multiple-sequence-alignment

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A regulação do gene HLA-DRB1 refere-se aos mecanismos que controlam a expressão desse gene. A expressão do HLA-DRB1 é altamente regulada para garantir que as células do sistema imunológico possam reconhecer e responder a antígenos estranhos, enquanto evitam reações autoimunes contra os próprios tecidos do organismo.

Em termos clínicos, variações no gene HLA-DRB1 têm sido associadas a diferentes condições e doenças.

Fonte: HLA-DRB1 pode ser regulado de forma antagônica pelo promotor coordenado e 3′-UTR sob seleção estabilizadora - PMC (nih.gov⁾

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PLoS One. 2011; 6(10): e25794.

Abstract

HLA-DRB1 is the most polymorphic MHC (major histocompatibility complex) class II gene in human, and plays a crucial role in the development and function of the immune system. Extensive polymorphisms exist in the promoter and 3′-UTR of HLA-DRB1, especially a LTR (Long terminal repeat) element in the promoter, which may be involved in the expression regulation. However, it remains unknown how the polymorphisms in the whole promoter region and 3′-UTR to regulate the gene expression. In this study, we investigated the extensive polymorphisms in the HLA-DRB1 promoter and 3′-UTR, and how these polymorphisms affect the gene expression in both independent and jointly manners. It was observed that most of the haplotypes in the DRB1 promoter and 3′-UTR were clustered into 4 conserved lineages (H1, H2, H3 and H4), and showed high linkage disequilibrium. Compared with H1 and H2 lineage, a LTR element in the promoter of H3 and H4 lineage significantly suppressed the promoter activity, whereas the activity of the linked 3′-UTR increased, leading to no apparent difference in the final expression product between H1/H2 and H3/H4 lineage. Nevertheless, compared with the plasmid with a promoter and 3′-UTR from the same lineage, the recombinant plasmid with a promoter from H2 and a 3′-UTR from H3 showed about double fold increased luciferase activity, Conversely, the recombinant plasmid with a promoter from H3 and a 3′-UTR from H2 resulted in about 2-fold decreased luciferase activity. These results indicate that the promoter and 3′-UTR of HLA-DRB1 may antagonistically regulate the gene expression, which may be subjected to stabilizing selection. These findings may provide a novel insight into the mechanisms of the diseases associated with HLA-DRB1 genes.

Introduction

Major histocompatibility complex (MHC) plays a crucial role in the regulation of immune response. Since the landmark discovery of MHC restricted T-cell recognition by Zinkernagel and Doherty [1], Numerous studies have concentrated on how T-cells are trained to recognize self-peptide∶self-MHC complex (pMHC) by positive and negative selection in thymus [2]-[4]. Both the avidity of the T-cell receptor (TCR) to the pMHC and the density of pMHC on the thymic cortical epithelial cell contributes to the consequence of the positive and negative selection for T-cells [5]. Although previous studies have revealed that the concentration of self-peptide ligands and their affinity to TCR may influence the outcome of positive selection and negative selection [6]-[7], but very limited studies were reported on how the amount of pMHC affects the consequences of positive or negative selection for thymocytes. Patients lacking MHC class II, known as the bare lymphocyte syndrome (BLS), were subjected to severe immune deficiency, and they were devoid of peripheral CD4+ T-cells [8]. Additionally, it was shown that decreased expression of HLA-DR molecules in thymoma may affect on CD4+ T-cell development [9]. Analysis based on BLS patients suggested that abnormal MHC II expression level may impair CD4+ T-cell development. Apart from this, MHC-II molecules also play very important roles in the native immune response. Deficiency of MHC-II attenuates the Toll-like receptors (TLRs) triggered inflammation [10].

HLA-DR molecules are constitutively expressed on the cell surface of B lymphocytes, thymic cortical epithelial cells, dendritic cells (DCs), monocytes and other antigen presenting cells (APC), with the function of presenting antigens T-cells [11]-[12]. DR molecules function as the heterodimer consisted of DR alpha (encoded by DRA locus) and DR beta chains (encoded by DRB loci, including DRB1, DRB3, DRB4 and DRB5), HLA-DRA is monomorphic, whereas HLA-DRB, especially DRB1, is highly polymorphic [13]-[14]. Among the MHC II members, HLA-DR plays the most crucial role in the human immune system since it accounts for more than 90% of the HLA class II isotypes expressed on antigen-presenting cells [14]. Because the HLA-DRB1 genes located on different DR haplotypes are vastly diversified, and some DR haplotypes also comprise another DRB encoding gene except for DRB1, so 2-4 very distinct HLA-DR heterodimers can be formed in an individual depending on the DR haplotypes [15]. The expression level of DRB alleles determines the amount of a variety of functional DR heterodimers on the surface of APCs, which may in turn influence the quantity and ratio of mature T-cells with different specificity. In other words, the expression regulation of HLA-DR molecules is very important for CD4+ T-cells selection and activation during the adaptive immune response. Since the expression level of HLA-DRB1 loci was demonstrated to be much higher than that of other HLA-DRB coding loci ( DRB3, DRB4 and DRB5) [16]-[17], and HLA-DRB1 was also the most reported MHC gene in association with different diseases [18]-[26]. The regulation of HLA-DRB1 expression, the dominant locus which expresses HLA-DR, must be under delicate control. Previous studies revealed that the polymorphisms in the core promoter of HLA-DRB1 gene caused a significant change of the promoter activity [17], [27]-[31]. However, the mechanism underlying this effect is still unknown. Polymorphisms in regulatory cis-elements may impact on the phenotypic outcomes in a highly variable and unpredictable fashion [32]. Many retro-elements, such as LINEs (long interspersed nuclear elements), SINEs (short interspersed nuclear elements) and LTRs (long terminal repeats), could play important roles in the regulation of gene expression [33]. In the past decades, 3′-UTR was also demonstrated to contain important cis-elements for gene expression regulation. It is very popular that distinct length of 3′-UTRs are produced by alternative polyadenylation (APA) in different cell types and cell stages, regulating the final product of gene expression [34]. To understand the evolution of the regulatory region of HLA-DRB1 and assess the functional consequence of the polymorphisms in these regions, we sequenced about 4.6 kb of HLA-DRB1 promoter region including 5′-UTR and 2.6 kb of 3′ non-coding region (3′-NCR) containing entire 3′-UTR from 26 normal Han Chinese, and used the dual luciferase reporter gene assay system to experimentally determine the effect of the polymorphisms in HLA-DRB1 promoter and 3′-UTR on gene expression. This study first explored how the polymorphisms in the promoter and 3′-UTR of HLA-DRB1 to regulate the gene expression.

Results

Most of the Haplotypes in HLA-DRB1 Promoter and 3′-UTR were Grouped into Four Distinct Lineages

A total of 51 promoter sequences and 50 3′-UTR sequences were obtained, and the new sequences were submitted to GenBank ( http://www.nlm.nih.ncbi.org) and assigned a GenBank accession number of FJ464430 to FJ464461. Phylogenetic analysis showed that the sequences from both promoter and 3′-UTR were clustered apparently into 4 deep clades, which were designated as H1, H2, H3 and H4 for promoter clades ( Figure 1A ), and UTH1, UTH2, UTH3 and UTH4 for 3′-UTR ( Figure 1B ), respectively. One unique 3′-UTR haplotype denoted as UTHx was derived from HLA-DRB1*10:01:01 ( Figure 1B ). Linkage analysis between promoters, exon2 and 3′-UTRs revealed four tightly linked lineages, namely H1-UTH1-exon2 (DR01, DR15 and DR16), H2-UTH2-exon2 (DR03, DR08, DR11, DR12, DR13 and DR14), H3-UTH3-exon2 (DR07 and DR09) and H4-UTH4-exon2 (DR04) ( Figure 2 ). The evolutionary relationship among all haplotypes except for UTHx is very similar on the 3′-UTR tree and the 3′-UTR downstream (3′-UTR-DS) tree. The UTHx was clustered with UTH2 lineage on the 3′-UTR tree, but it is more ancient on the 3′-UTR-DS tree ( Figure S1).

There are extreme differences among lineages but low diversity within lineages in both promoter and 3′-UTR. The frequency spectrum analysis showed that the most moderate frequency alleles exist in the DRB1 promoters ( Figure S2) and the similar distribution of a frequency spectrum is observed for 3′-UTR (data not shown). When we performed Tajima's D test and Fu and Li's test by taking account for all lineages together or only one lineage, a notable difference was observed ( Table S1). When we considered all lineages together, the nucleotide diversity (π) are 0.05053 and 0.07263 in the promoter region and 3′-UTR, respectively. In promoter region, Tajima's D is 2.43186 ( P<0.05 ). Fu and Li's D* test statistic is 2.09095 ( P<0.02) and Fu and Li's F* test statistic is 2.73971 ( P<0.02). In 3′-UTR, Tajima's D is 2.3799 with a statistical significance P<0.05, Fu and Li's D* and F* is 1.8129 and 2.4305, respectively, and both with a statistical significance ( P<0.02; Table S1). The above results suggested that the promoter and 3′-UTR may have been subjected to balancing selection. However, when we only selected the sequences grouping in certain lineage or sub-lineage for analysis, low nucleotide diversity was observed. For H3 lineage, the nucleotide diversity (π) is 0.00083. Tajima's D is −2.37453 ( P<0.001 ). Fu and Li's D* test statistic is −3.26961 ( P<0.02) and Fu and Li's F* test statistic is −3.47765 ( P<0.02). Tajima's D, Fu and Li's D* and F* test for UTH1 are −1.84699, −2.50331 and −2.65713, respectively ( P<0.05) ( Table S1). These results indicated that the polymorphism patterns within H3 and UTH1 lineage may have been shaped by purifying or positive selection. The nucleotide diversity between H12 (H1 and H2) and H34 (H3 and H4) in the 3′-UTR region is significantly higher than that in the promoter region. However, the nucleotide diversity between H1 and H2, or H3 and H4, in the 3′-UTR region is slightly lower than that in the promoter region ( Figure 3 ). The above indicated that the 3′-UTR diverged faster between H12 and H34 than the promoter, suggesting the 3′-UTR was subjected to strong positive selection after the separation of the DRB1 lineage of H12 and H34.

At least one SNP was observed in each of the cis-elements such as TATA box, CAAT box, Y box, X box and W box ( Figure S3). We compared the SNP density in these cis-elements, their flanking sequences from −1 to −200 bp, as well as 5′-UTR among 4 DRB1 promoter lineages. SNP density is 72.5 SNPs/kb in the regulatory boxes, 110.0 SNPs/ kb in flanking sequences and 45.0 SNPs/ kb in 5′-UTR, respectively. The SNP density in flanking sequences is higher than that in the regulatory boxes and 5′-UTR, suggesting the regulatory boxes and 5′-UTR were subjected to purifying selection.

The Promoter Activity of HLA-DRB1 Decreased in the Absence of Its Linked 5′-UTR

Dual luciferase reporter system was used to analyze the expression activity of different promoter constructs. Whereas no significant variation in luciferase expression level was observed when the reporter gene was placed under the control of different promoters together with the same 5′-UTR from H2 lineage ( Figure 4A ). We found a remarkable effect of different 5′-UTRs on the luciferase level. The expression of luciferase (P1-5′U1, P1-5′U2, P1-5′U3 and P1-5′U4) driven by the same promoter sequence derived from H1 lineage and different 5′-UTRs obtained from H1, H2, H3 and H4 lineages dropped increasingly, and statistic significant differences were found between P1-5′U1 and P1-5′U4, and between P1-5′U2 and P1-5′U4 ( P<0.05), suggesting the polymorphisms in the 5′-UTR could affect the promoter activity ( Figure 4B ). We also investigated the influence of the DRB1 promoter with or without 5′-UTR on the expression levels. Analysis for the four paired corresponding promoter constructs showed that the DRB1 promoter with its linked 5′-UTR had much higher transcription activity than that without 5′-UTR in Raji cells ( Figure 4C ). However, no significant difference was observed among the constructs (P1-5′U1H, P2-5′U2H, P3-5′U3H and P4-5′U4H) that include constitutive promoter and 5′-UTR or among the constructs (P1-W, P2-W, P3-W and P4-W) that include only DRB1 promoter sequence without 5′-UTR sequence ( Figure 4C ). These results indicate that the polymorphisms in the proximal promoter (−1 to −535 bp) of HLA-DRB1 gene by themselves do not impact on the promoter activity. However, the polymorphisms in the 5′-UTR may play an important role through coordinately regulating the gene expression with those in the promoter region.

A LTR Element Located at the Distal Promoter of H3 and H4 Lineage Led to the Repression of Promoter Activity

The insertion of repetitive elements into the gene promoter region usually affects the promoter activity. In order to explore the potential influence of the LTR element in the H3 and H4 lineages on activity of their promoters, we created constructs by cloning the long promoter region (from +66 bp to −3200 bp) from each DRB1 lineage into pGL3.0 basic vector, and simultaneously constructed several recombinants by inserting the LTR into the promoter sequence from H2 lineage. Compared with the constructs of H3AIC (from H3 lineage) and H4AIC (from H4 lineage) which comprise a LTR element, the long promoter constructs H1AC (from H1 lineage) and H2AC (from H2 lineage) which contain no LTR element showed significantly increased expression activity ( P<0.01) ( Figure 5 ). Furthermore, the expression activity of a construct (H2AIC) by inserting a LTR element derived from H3 lineage greatly decreased ( P<0.001) ( Figure 5 ). The above results suggest the insertion of LTR element significantly impact on the promoter activity. However, deletion of the LTR element in a construct H3AC could not apparently increase the promoter activity, and yet the transcription activity of a construct (IH3C) that excluding the upstream part of the LTR was significantly decreased ( P<0.05) ( Figure 5 ). Another construct IH2C was recombined with the proximal promoter from H2 lineage and a LTR element, unexpectedly, whose transcriptional activity was not decreased compared with that of H2AC ( Figure 5 ). These findings indicate that the influence of the LTR on promoter activity was depended on the sequence environment, and may act through altering the secondary or tertiary structures required for the binding of promoter complex.

Polymorphisms in the 3′-UTR of HLA-DRB1 Counteract the Influence of that in Its Linked Promoter on Expression

In order to find out whether the polymorphisms in 3′-UTR of HLA-DRB1 are involved in the regulation of gene expression, the luciferase activities with various 3′-UTR constructs were determined in 293T and Raji cells. A significant higher luciferase activity was observed for the 3′-UTR from DRB1 lineages of UTH3 and UTH4 than that from DRB1 lineages of UTH1, UTH2 and UTHx ( Figure 6A ). Similar results were obtained when different DRB1 3′-UTR sequences from various lineages were placed in the constructs containing the same HLA-DRB1 promoter and 5′-UTR sequence originated from lineage H2, ( Figure 6B ). The above results showed that the effect of the polymorphisms in 3′-UTR on the expression regulation is reverse to that in the promoter from same lineages. That being the case, then similar luciferase activity should be observed among the constructs in which the luciferase gene was placed under the control of both 3′-UTR and LP from the same lineage. This point was experimentally demonstrated in Raji cells ( Figure 6C ). Moreover, apparently increased luciferase activity was observed with the recombinant plasmid (H2AC-3′U3) in which the luciferase gene was placed under the control of 3′-UTR from H3 lineage and LP from H2 lineage, and opposite changes was obtained for the recombinant plasmid H3AIC-3′U2 in which the luciferase gene was placed under the control of 3′-UTR from H2 lineage and LP from H3 lineage ( Figure 6C ). These results indicate that the changes in expression of the HLA-DRB1 gene caused by the polymorphisms in promoters can be counteracted by that in their linked 3′-UTRs, it is strongly suggested that HLA-DRB1 gene have been subjected to stabilizing selection in the evolutionary history.

Discussion

In previous studies, it was reported that polymorphisms in cis-elements located in the core promoter of HLA-DRB1 genes led to apparent expression difference [17], [27]-[31]. However, in this study, no significant difference in promoter activity was observed between 4 distinct promoter lineages within a region of about 600 bp, though in which polymorphisms almost reside in every cis-element. Moreover, the promoter activity was influenced by the polymorphism in 5′-UTR only as they linked with the same promoter sequence ( Figure 4B ). The above phenomenon suggested that the sequence out of the core promoter (from TIS to about −300 bp) also contributes much to the promoter activity, and the promoter activity of different HLA-DRB1 lineages may be affected by the antagonistic action among polymorphisms in the whole promoter region. Inhibition of the promoter activity by the LTR element in upstream of −950 indicated that it is hard to suppose the promoter activity for different DRB1 lineage by investigating its core promoter region.

It is very interesting that the impact of the polymorphisms in the 3′-UTR on the gene expression is just reverse to that in the promoter, suggesting antagonistic regulation between the promoter and 3′-UTR of HLA-DRB1 gene. This regulatory mechanism ensures the 4 distinct HLA-DRB1 lineages, although with different promoter activity, may have a similar final expression product. So we proposed that it may be very important to maintain an identical expression level for two distinct HLA-DRB1 alleles in an individual, and HLA-DRB1 gene was subjected to stabilizing selection in the evolutionary history.

Stabilizing selection between the alleles from distinct DRB1 lineages suggested that the maintenance of equal expression of two distinct DRB1 alleles in an individual must have a very important biological significance. Although the expression of most genes in primate and model organisms was demonstrated to be subjected to stabilizing selection [35]-[38], yet it was poorly understood for the evolutionary mechanism. In this study, we experimentally demonstrated a typical mechanism for stabilizing selection, which was dependent on the antagonistic coevolution between the promoter and 3′-UTR. However, what's the biological significance for maintaining the relatively stable expression among the distinct DRB1 lineages? In previous studies, it was revealed that the ratio variation of MHC II I-A g7 gene product to I-A k product in mixed MHC mice determined the difference in autoproliferative response, and the higher ratio would cause more autoreactive T-cells to evade negative selection into the periphery and readily elicit autoimmune response [39]. Inefficient presentation of Self-peptide to T-cells in the thymus causes a part of T-cells evading from the negative selection and entering into the periphery, which is one of the hypothetic mechanisms for autoimmune disease demonstrated experimentally [39]-[40]. Lessons from the bare lymphocyte syndrome (BLS) told us that the quantity of MHC II molecules play key roles in thymocytes selection [41]. HLA-DRB1 is the most extensively studied MHC II genes, which are constitutively expressed in B lymphocytes, dendritic cells and macrophage cells, and is induced to express in other cells [42]. Many previous studies concluded that the polymorphisms in the cis-elements of HLA-DRB1 promoters could greatly affect the promoter activity [17], [27]-[28], which led to a puzzling question of what would have happened if the various promoters lead to differential expression of two distinct HLA-DRB1 alleles in an individual. Not only does the positive selection need interaction with the MHC-peptide complex (pMHC), but also does the negative selection [7]. Therefore, the types of MHC and its quantities on the cell surface could affect the selection results for mature T-cells. Maintaining the appropriate ratio of gene products of two heterozygous alleles may be critical for T-cells selection and activation, which could interpret why the polymorphisms in the DRB1 3′-UTR is implicated in the expression regulation on the opposite effect of the polymorphisms in the DRB1 promoter. Therefore, balancing the mature T-cell types may be the fundamental biological significance for the stabilizing selection acting on the expression of HLA-DRB1 genes, which may provide new insight for the research on why DRB1 genes were associated with so many diseases.

The promoter regions of MHC II genes were subjected to Balancing selection [43]-[44]. In this study, extreme diversity among lineages observed in HLA-DRB1 regulatory region suggested a balance selection. However, very limited polymorphism within a lineage could not be explained by balance selection. Moreover, each promoter and 3′-UTR lineage are linked with many diversified exons, implicating that the regulatory region and the exons of HLA-DRB1 gene were subjected to different natural selection. The limited polymorphisms in the promoter regions within a lineage might partly be attributed to the reduction of diversity by recombination. Nevertheless, no recombination signal was observed between the lineages. It was well accepted that the exon 2 of HLA-DRB1 gene have been subjected to balancing selection in its evolutionary history [45]. The exon 2 of HLA-DRB1 gene encodes antigen recognition site (ARS) and peptide binding site (PBS), so high diversity in this region implies that it may present more extensive exogenous antigens, and the populations could be protected against more pathogens [46]-[47]. Moreover, it is beneficial for populations maintaining the diversity in exon 2 of DRB1 by balanced selection [48]-[49]. When plague was prevalent in certain periods, some special alleles which could present the antigen in the most efficient way would be selectively survived. And in this circumstance, positive selection played a major role. Because of a microorganism evolving very fast, the ARS and PBR were necessarily driven to evolve faster than any other part of the gene [50]. As it was proposed in previous works, the pathogens may be the main force which drives and maintains the remarkable polymorphism in the MHC [51]-[52]. Anyway, the polymorphic pattern in exon 2 of DRB1 gene is the consequence of ongoing balanced selection and infrequently positive selection. Since our results showed that the corresponding promoter lineage and 3′-UTR lineage were linked tightly, and they cooperatively worked to keep the similar expression among different DRB1 lineages, suggesting that most mutation occurred in the two regions would be deleterious. The fact that SNP density in cis-elements and 5′-UTR is lower than in their flanking regions also indicates that purifying selection have acted on DRB1 upstream regulatory region. Therefore, the complicated polymorphism pattern in the promoter and 3′-UTR of HLA-DRB1 must have been attributed to the hitch-hiking by the exons which are subjected to balancing selection, positive selection or other natural selection.

The nucleotide diversities between lineage 12 (lineage 1 and lineage 2) and lineage 34 (lineage 3 and lineage 4) in the 3′-UTR region is much higher than that in the promoter region, suggesting that the 3′-UTR and the promoter were subjected to different natural selection. The 3′-UTR from lineage 3 and 4 might be subjected to positive selection so as to evade the suppression to counteract the inhibition of its promoter activity. UTHx belongs to DRB1*10:01:01 which was recombined with the allele on the DR52 haplotype in intron 1 and has the most recent common ancestor with the alleles on DR51 and DR52 haplotype [53]. The position of UTHx haplotype on the 3′-UTR tree and 3′-UTR-DS tree suggested that the 3′-UTR have been subjected to stronger natural selection than the downstream sequence. Most part of the above may be contributed by the selective pressure of maintaining the stable expression of the different HLA-DRB1 gene.

In conclusion, the expression of HLA-DRB1 may be antagonistically regulated by its coordinately evolved promoter and 3′-UTR under stabilizing selection. The polymorphic pattern in the DRB1 promoter and 3′-UTR was shaped by multiple natural selection, such as purifying selection, positive selection, stabilizing selection, and hitch-hiking by the polymorphic sites in the exons. The findings in this study implicated that balancing the expression of two different MHC II alleles in an individual may be very important, which provide a novel insight into the pathogenic mechanism of MHC involving in many diseases.

Materials and Methods

Samples

Whole blood samples were collected from 26 Han Chinese volunteers from the different provinces. A written informed consent was obtained from each participant, and this study was conducted with the approval of the Institute Research Medical Ethics Committee of Sun Yat-Sen University. Genome DNA was extracted with Blood DNA kit (D3392-02) purchased from OMEGA Bio-tek, Inc., Guangzhou, China.

Primer design

We downloaded 3 HLA-DRB1 reference sequences which located at different DR haplotypes, namely DR51 ( DRB1*15:01:01, GenBank accession no. NG_002432), DR52 ( DRB1*03:01:01, GenBank accession no. NG_002392), and DR53 ( DRB1*04:01:01, GenBank accession no. NG_002433). Sequence alignment was performed with ClustalX 1.81, and all primers were designed with primer premier 5.0. The primer pairs and their relative positions were shown in Figure S4. Also, the sequencing primers were designed and most of which were lineage specific. All the primers were synthesized by Invitrogen (Shanghai, China) and the primer sequences were shown in Table S2.

Long fragment amplification and investigation of linkage relationships

The amplification was performed in a 50 µl of reaction system containing 5 µl of 10×LA buffer, 0.25 mM of dNTP, 0.24 µM of each primer, 20 ng of genome DNA and 2.5 U of TakaRa LA Taq polymerase in which pfu enzyme was supplemented according to 1∶19. A total of 35 cycles run in the PCR after preheating at 92°C for 3 minutes, and for each cycle with denaturing at 95°C for 15 seconds, annealing at x °C (the "x" denotes the annealing temperature that varies according to different primer pairs) for 30 seconds and extension at 68°C for y (the "y" means the extension time for amplifying different length of fragments) minutes, then elongating at 68°C for 10 minutes. The PCR products were cleaned with Gel extraction Kit (OMEGA Bio-tek, Inc., Guangzhou, China), then sequenced directly and finally cloned into T-easy vector (Promega Corporation, Madison, WI).

To investigate linkage relationship among promoter, exon 2, and 3′-UTR, we designed 2 universal primer pairs (and) to amplify the fragment containing promoter region and exon 2, as well as the fragment containing 3′-UTR and exon 2 ( Figure S4). Then the fragments were cloned into T-easy vector, and one positive clone was sequenced from two ends with sequencing primers T7 and SP6 for each segment. The end sequences were aligned with the known promoter sequences, exon 2 sequences and 3′-UTR sequences, respectively. Genotyping for exon 2 of HLA-DRB1 gene was according to the description of Kotsch [54]. We could get the linkage relationship between promoter regions and 3′-UTRs easily through analyzing the overlapping exon 2.

Sequencing

The allele specific PCR product was purified by gel extraction with DNA Gel Extraction Kit (OMEGA bio-tek, Inc., Guangzhou, China) followed by sequencing with haplotype specific primers ( Table S2) on an ABI 3730 sequencer using BigDye Terminator V3.0 cycle sequencing kit. The plasmid was extracted with Plasmid Mini kit (OMEGA bio-tek, Inc., Guangzhou, China) followed by sequencing. All different sequences were submitted to NCBI and the GenBank accession numbers were given as FJ464430 to FJ464461.

Construction of expression vectors

The promoter lineages H1, H2, H3 and H4 were defined according to the clusters of the promoter sequences ( Figure 1A ). H1 represents the promoter sequences derived from DRB1*01, DRB1*15 and DRB1*16; H2 indicates the promoter sequences from DRB1*03, DRB1*08, DRB1*10, DRB1*11, DRB1*12, DRB1*13 and DRB1*14; H3 denotes the promoter sequences from DRB1*09 and DRB1*07, and H4 indicates the promoter sequences from DRB1*04. In order to compare the influence of different promoter lineages of HLA-DRB1 and their 5′-UTR on the gene expression level, we constructed 3 kinds of vectors by inserting DRB1 promoter derived fragments with or without 5′-UTR into pGL3.0 Basic vectors. The first kind of promoter constructs is that the luciferase gene was placed under the control of different promoters from distinct DRB1 lineages and fused with the 5′-UTR from lineages H2. We denominated these vectors as P1-5′U2, P2-5′U2, P3-5′U2 and P4-5′U2 (P1-4 represent the promoter derived from promoter lineages H1-4, and the "5′U2" indicates a 5′-UTR from promoter lineage H2). The fragment, including the promoter and 5′-UTR sequence, were amplified from lineage H4 was cloned into pGL3.0 basic vector as a control (P4-5′U4) ( Figure S5A). The second kind of promoter constructs contains the same promoter sequence amplified from H1 lineage and various 5′-UTRs from distinct DRB1 lineage, which were denoted as P1-5′U1, P1-5′U2, P1-5′U3 and P1-5′U4, respectively. In these two constructs, the fusion fragment of promoter sequence and 5′-UTR were inserted into the Hind III and Nco I site, so they do not contain the 5′-UTR of luciferase reporter gene from the vector itself ( Figure S5B). The third kind of promoter vectors was constructed by inserting the PCR segments from different DRB1 lineages containing with or without the 5′-UTR into the Kpn I and Sma I sites, which were designated as P1-5′U1H, P2-5′U2H, P3-5′U3H, P4-5′U4H, P1-W, P2-W, P3-W and P4-W, respectively. The former 4 vectors contain the 5′-UTRs, and the later 4 vectors are absent from the DRB1 derived 5′-UTR. ( Figure S5C).

One LTR (MER11C) element is about 1.1 kb, which was inserted into the promoter region of H3 and H4 lineages upstream at the position −938 to −2065. Here, we defined the position of LTR elements as indel site, and use an "A" represent the distal promoter that located upstream of the indel site, an "I" indicates the LTR elements, a "C" denotes the proximal promoter that located between translation start site and indel site, and H1-4 represent the 4 distinct promoter lineage. To investigate whether the LTR element influences on the transcription activity of the promoter, we constructed 4 constitutive promoter vectors designated as H1AC, H2AC, H3AIC and H4AIC, respectively. Simultaneously, we constructed four recombinant plasmids namely H2AIC, IH2C, H3AC and IH3C, respectively (shown on the left of Figure 5 ).

We amplified the fragment containing the complete 3′-UTR and about 300 bp long downstream sequence from the poly(A) signal site from each 3′-NCR lineage of HLA-DRB1, which is about 586-594 bp long. We denoted the fragments of 3′-UTR from UTH1, UTH2, UTH3, UTH4 and UTHx as 3′U1, 3′U2, 3′U3, 3′U4 and 3′Ux, respectively, and constructed them into pGL3.0 Basic vector or pGL3.0 control vector between Xba I and BamH I site. We successfully constructed four different kinds of vectors and confirmed its fidelity by sequencing. 1) The vector in which the 3′-UTR and 5′-UTR of the luciferase gene were simultaneously substituted for that from different DRB1 lineages ( Figure S6A). 2) The luciferase gene was placed under the control of the promoter from H2 lineage and the 3′-UTR from different DRB1 lineages ( Figure S6B). 3) The luciferase gene was placed under the control of the long promoter and 3′-UTR from the same DRB1 lineage, and simultaneously created two recombinant plasmids in which the luciferase gene was placed under the control of the long promoter from H2 lineage (H2AC) and the 3′-UTR from UTH3 lineage (3′U3), as well as from H3 lineage (H3AIC) and the 3′-UTR from UTH2 lineage (3′U2).( Figure S6C).

Transfection and luciferase assay

All plasmids for transfection were extracted with Qiagen Midi Kit-25 (Valencia CA, USA) according to manufacturer's instruction. The concentration of each construct was measured with Spectrophotometer ND-1000. Burkitt's lymphoma B cell line Raji was propagated with RPMI 1640 culture medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. All promoter constructs and an internal control (pRL-TK vector) were co-transfected into Raji cells by electroporation, and into 293T cells with lipofactamine2000 (Invitrogen). The electroporation was performed as following: the mix of 200-400 µl cells and plasmids were transferred into a 4 mm cuvette pre-cooled on ice and discharged for 25 ms at 160 V on a Genepulser Xcell (Bio-Rad). Then, the cells were transferred to wells containing 2 ml RPMI 1640 complete culture medium and incubated at 37°C in a CO2 incubator with moist ambient air for 36-48 hours. The procedure of transfection with lipofactamine2000 was according to the manufacturer's instruction.

Transfected cells were harvested and schizolysed with passive lysis buffer attached with the Dual-Luciferase reporter assay system (Promega). Then, the enzyme activity of the reporter gene including the internal control was assayed with the Dual-Luciferase reporter assay system on a chemoluminescence meter (Monolight 3010C, Pharmingen). The fluorescence intensity representing promoter activity of each lineage was normalized by internal control and comparison was performed among all lineages.

Data analysis

Sequence assembly was performed manually with BioEdit version 7.0 and the sequence alignment was carried out with clustalX 1.81. The polymorphism level, π (the average number of pairwise nucleotide differences per site) and θw (the number of segregating sites per nucleotide), was calculated with DnaSP version 4.50.3 [55]. Phylogenetic trees were constructed by MEGA3.1 [56] using the distance matrix calculated by Kimura's two-parameter method and the neighbor-joining method. The reliability of the NJ trees was assessed by bootstrapping with 1,000 pseudosamples.

Data for analysis of promoter activity were expressed as the mean ± standard error of mean (s.e.m.) from at least 3 separate experiments. Unless otherwise noted, the differences between groups were analyzed using Student t test when only 2 groups, or assessed by one-way ANOVA when more than 2 groups were compared. All tests performed were two-sided. Differences were considered statistically significant at P<0.05.

Supporting Information

Figure S3

Polymorphism in the Core Promoter Region of Partial DRB Alleles from human, chimpanzee, rhesus and marmoset. The numbering is relative to the transcription start site. The cis-element box was encircled with a pane and the core sequence in a cis-element box was marked with underline.

(JPG)

Acknowledgments

We thank Shangwu Chen and Shengfeng Huang for helpful discussions.

Footnotes

Competing Interests: The authors have declared that no competing interests exist.

Funding: This work was supported by Key Project (No 30730089) from National Natural Science Foundation of China ( http://www.nsfc.gov.cn), Project 2007CB815800 of the National Basic Research Program (973), projects 2006AA09Z433 and 2008AA092603 of the State High-Tech Development Project (863), project 2007DFA30840 of International S&T Cooperation Program from the Ministry of Science and Technology of China ( http://www.most.gov.cn). Dr. Xu is the recipent of "Outstanding Young Scientist Award" from National Natural Science Foundation of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Articles from PLOS ONE are provided here courtesy of PLOS

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HLA-DRB1 May Be Antagonistically Regulated by the Coordinately Evolved Promoter and 3′-UTR under Stabilizing Selection

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HLA-DRB1 is the most polymorphic MHC (major histocompatibility complex) class II gene in human, and plays a crucial role in the development and function of the immune system. Extensive polymorphisms exist in the promoter and 3′-UTR of HLA-DRB1, ...

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A rede de interação proteína-proteína (rede IPP) codifica as interações entre as proteínas e auxilia no mapeamento do interatoma de um organismo. Para compreender um sistema complexo, é preciso entender como seus componentes moleculares interagem entre si, e as redes são excelentes modelos para codificar e interpretar essas interações.

A ferramenta String permite visualizar essas interações. As interações incluem associações diretas (físicas) e indiretas (funcionais). Estas decorrem na previsão computacional, da transferência de conhecimento entre organismos e das interações agregadas de outros bancos de dados (primários).

Através da análise da rede de interações obtidas associa as relações entre a proteína alvo de estudo e outras proteínas de outros genes. Pela sua análise as proteínas associadas/ligadas são provenientes do mesmo organismo, Homo sapiens.

Exemplo de proteína interativa:

TLR2 (Receptor Tipo Toll 2): a sua função principal é iniciar respostas imunológicas, incluindo a produção de citocinas e a ativação de células imunes, para combater infecções. O TLR2 desempenha um papel crucial na detecção de patógenos e na regulação da resposta imunológica. Desregulações em sua regulação foram associadas a diversas doenças inflamatórias e autoimunes.

Fonte: http://revodonto.bvsalud.org/scielo.php?script=sci_arttext&pid=S1981-86372011000400019

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A fosforilação é a adição de um grupo fosfato (PO₄) a uma proteína ou outra molécula. Este mecanismo é um dos principais participantes nos mecanismos de regulação das proteínas.

A energia obtida na respiração ou na fotossíntese é utilizada para adicionar o grupo fosfato ao ADP (difosfato de adenosina) e convertê-lo em ATP. Esta molécula armazena essa energia, que fica à disposição da célula.

Fonte: https://pt.khanacademy.org/science/biology/cellular-respiration-and-fermentation/oxidative-phosphorylation/a/oxidative-phosphorylation-etc

A ubiquitinação é um importante processo biológico, que tem como finalidade regular as proteínas através da marcação daquelas que são indesejadas e, que posteriormente, são degradadas por organelos conhecidos como proteassoma.

Fonte: https://pt.wikipedia.org/wiki/Ubiquitina

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Uma árvore filogenética, também conhecida como filogenia, é um diagrama que representa as linhas de descendência evolutiva de diferentes espécies, organismos ou genes a partir de um antepassado comum.

[2]https://www.nature.com/scitable/topicpage/reading-a-phylogenetic-tree-the-meaning-of-41956/

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Analisando os resultados fornecidos pelo Cobalt, é possível verificar que as zonas mais conservadas das proteínas se apresentam a vermelho. Deste modo, a região mais conservada em todas as sequências encontra-se entre os aminoácidos 10 e 255.

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Uma superfamília homóloga é um grupo de proteínas que compartilham uma origem evolutiva comum.

Fonte: https://proteinswebteam.github.io/interpro-blog/2017/10/03/Homologous-superfamily/

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Beatriz Gomes | PG53482

Duarte Fonseca | PG54728

Maria Pereira | PG53491

Renato Ribeiro | PG53495

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CD14

HLA-DRB1

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Este gene codifica um proteína, produzida principalmente por macrófagos como parte do sistema imunológico.

O CD14 atua como co-receptor para a detecção de lipopolissacarídeo bacteriano (LPS), encontrados em bactérias gram-negativas. Quando esses componentes são reconhecidos, o CD14 desencadeia uma resposta imunológica, ativando as células do sistema imunológico para combater a infecção.

Além disso, o CD14 pode existir em duas formas principais: a forma solúvel e a forma ancorada à membrana por uma cauda de glicosilfosfatidilinositol (GPI) (mCD14). A forma solúvel circula no sangue e pode ajudar na neutralização de toxinas bacterianas. A forma ligada à membrana está presente nas células do sistema imunológico, onde são detectados agentes patogénicos.

Portanto, o CD14 desempenha um papel crucial na resposta imunológica do corpo contra infecções bacterianas, desencadeando uma série de eventos que ajudam a eliminar os agentes patogénicos invasores.

Fonte: https://www.frontiersin.org/articles/10.3389/fcimb.2013.00032/full

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Com o intuito de analisar as sequências e as respetivas features presentes no NCBI foram desenvolvidas scripts, para ler os ficheiros guardados em formato GenBank.

Através da ferramenta Jupyter Notebook, foi possível retornar as anotações de cada gene, assim como número de features, o tipo e a localização. Para além disso, também se obtém o índice das sequências codificantes.

Por fim, procedeu-se à analise da lista de qualifiers do ficheiro de cada gene, tendo em conta que as respetivas scripts se encontram no GitHub.

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Recorrendo ao NCBI, através da ferramenta blastp, foi possível determinar que a sequência mais homóloga é a sequência com o acession number CAL38573.1, designada de hypothetical protein.

Apresenta um comprimento de 375 aminoácidos e está associado ao organismo synthetic construct.

Atendendo ao valor do query cover (100%), pode-se concluir que a sequência da base de dados abrange 100% da sequência alvo.

Analisando o valor de identities, verifica-se que a extensão em que as duas sequências têm os mesmos resíduos nas mesmas posições do alinhamento é de 99,73%.

Apresenta um total score e max score de 750, tendo dois bit a menos do que a sequência em estudo.

Atendendo ao valor do E-value, sabe-se que quanto menor o seu valor mais significativa é a correspondência. Como neste caso é 0, pode-se concluir que existe uma total compatibilidade e homologia entre as sequências.

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Contribute to G1Bioinf/Projeto_G1 development by creating an account on GitHub.

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Recorrendo ao NCBI, através da ferramenta blastp, foi possível determinar que a sequência mais homóloga é a sequência com o acession number CAD0297745.1, designada de MHC class II antigen.

Apresenta um comprimento de 266 aminoácidos e está associado ao organismo Homo sapiens.

Atendendo ao valor do query cover (100%), pode-se concluir que a sequência da base de dados abrange 100% da sequência alvo.

Analisando o valor de identities, verifica-se que a extensão em que as duas sequências têm os mesmos resíduos nas mesmas posições do alinhamento é de 99,62%.

Apresenta um total score e max score de 551, tendo apenas um bit a menos do que a sequência em estudo.

Atendendo ao valor do E-value, sabe-se que quanto menor o seu valor mais significativa é a correspondência. Como neste caso é 0, pode-se concluir que existe uma total compatibilidade e homologia entre as sequências.

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Contribute to G1Bioinf/Projeto_G1 development by creating an account on GitHub.

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Este gene está localizado no complexo principal de histocompatibilidade (MHC) humano, que é uma região genómica crucial para o sistema imunológico. O MHC é dividido em classes I e II, e o HLA-DRB1 faz parte da classe II.

O HLA-DRB1 codifica uma das subunidades da molécula de histocompatibilidade do tipo II chamada HLA-DR (Antígeno de Histocompatibilidade Leucocitária - Classe II, subtipo DR). As moléculas HLA-DR estão envolvidas na apresentação de antígenos para células T auxiliares CD4+.

Além disso, as variantes alélicas do gene HLA-DRB1 estão associadas a diferenças nas moléculas HLA-DRB1 e têm implicações em diversos contextos, como a susceptibilidade a certas doenças autoimunes. Por exemplo, certas variantes do HLA-DRB1 foram associadas a um maior risco de desenvolver artrite reumatoide.

Em resumo, o HLA-DRB1 é um gene do MHC de classe II que desempenha um papel crucial no sistema imunológico, especialmente na apresentação de antígenos e na regulação da resposta imune adaptativa.

Fonte: https://medlineplus.gov/genetics/gene/hla-drb1/

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Através da análise dos resultados é possível concluir que o ID relacionado com o gene CD14 é o 'NC_000005.10'.  Relativamente ao segundo gene, HLA-DRB1, o ID correspondente é o 'NG_029921.1'.

Para além disso, ambos pertencem ao mesmo organismo, à espécie Homo sapiens e ambas são moléculas de DNA.

A sua taxonomia completa começa em "Eucariota", e termina em "Homo". A descrição do gene CD14 é 'Homo sapiens chromosome 5, GRCh38.p14 Primary Assembly', estando o gene localizado no cromossoma 5.

Por sua vez, a descrição do gene HLA-DRB1 é 'Homo sapiens major histocompatibility complex, class II, DR beta 1 (HLA-DRB1), RefSeqGene on chromosome 6', estando este gene localizado no cromossoma 6.

Para o gene CD14 o número de features total é 11, constituída por: 1 do tipo source, 2 do tipo gene, 4 do tipo mRNA e 4 do tipo CDS.

Para o gene HLA-DRB1 o número de features total é 11, constituída por: 1 do tipo source, 1 do tipo gene, 1 do tipo mRNA, 6 do tipo exon, 1 do tipo CDS e 1 do tipo sig_peptide.

O gene CD14 codifica a proteína, monocyte differentiation antigen CD14 precursor, com ID 'NP_001167575.1'. Recorrendo ao Uniprot, esta proteína atua como co-receptor do LPS.

O gene HLA-DRB1 codifica a proteína, major histocompatibility complex, class II, DR beta 1 precursor, com ID 'NP_001230894.1'. Através da Uniprot, esta proteína está envolvida no processo de eliminação de agentes infeciosos.

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Projeto_G1/Analise de seq e features final.ipynb at main · G1Bioinf/Projeto_G1

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Contribute to G1Bioinf/Projeto_G1 development by creating an account on GitHub.

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Contribute to G1Bioinf/Projeto_G1 development by creating an account on GitHub.

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A proteína codificada pelo gene CD14 é a Monocyte differentiation antigen CD14. Pertence ao organismo Homo sapiens (Human) e possui 375 aminoácidos.

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A proteína codificada pelo gene HLA-DRB1 é a HLA class II histocompatibility antigen, DRB1 beta. Pertence ao organismo Homo sapiens (Human) e possui 266 aminoácidos.

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No que diz respeito à função, analisaram-se os resultados obtidos sobre a função molecular e a atividade biológica.

Relativamente à função molecular, salienta-se a fonte de ligação ao receptor HLA-DRB1, ao complexo proteico classe II do MHC, a atividade do receptor MHC classe II, a ligação ao antígeno peptídico, a combinação de polissacarídeos, constituinte estrutural do citoesqueleto C e a ligação ao receptor de células T.

No que toca à atividade biológica, destacam-se o processamento de antígeno e apresentação de antígeno peptídico endógeno e exógeno via MHC, a detecção de bactérias, o desenvolvimento da epiderme, a resposta imunológica humoral, a resposta imune, a resposta inflamatória ao antígeno, a diferenciação de macrófagos, processamento e apresentação de antígeno de células dendríticas mieloides, a regulação negativa da resposta inflamatória ao estímulo antigénico, da proliferação de células, da fonte de produção de interferon tipo II, montagem de antígeno peptídico com complexo proteico MHC classe II, a regulação positiva da transdução de sinal NF-kappa B canónica 2, da ativação de células T alfa-beta positivas para CD4, de células T reguladoras alfa-beta positivas para CD4, positivas para CD25, transcrição modelada por DNA, da cascata ERK1 e ERK2, da resposta imune Source, da secreção de insulina envolvida resposta celular ao estímulo da glicose, da atividade da quinase, da cascata MAPK, da diferenciação de células T de memória, da diferenciação de monócitos Source, da fosforilação de proteínas, da ativação de células T1, da citotoxicidade mediada por células T3, mediada por células T às células tumorais, da entrada viral na célula hospedeira, tetramerização de proteínas 2. A regulação da produção de interleucina-10 e interleucina-4 C, regulação da diferenciação de células T auxiliares, transdução de sinal, via de sinalização do receptor de células T e respostas imune tipo T-helper são outros exemplos de atividade biológica.

Fonte: https://www.uniprot.org/uniprotkb/P01911/entry

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Utilizando a ferramenta InterPro, determinou-se o domínio e a super família homologa da proteína em estudo, visto que esta não pertence a nenhuma família . Assim sendo, a proteína pertence a quatro super famílias homologas, a Ig-like_fold, a MHC_I/II-like_Ag-recog, a MHC_II_a/b_N e a Ig-like_dom_sf.
No que diz respeito ao domínio esta proteína pertence a três domínios:

MHC_II_b_N - Esta entrada representa o domínio N-terminal (também chamado de domínio beta-1) da cadeia beta das glicoproteínas do MHC de classe II .As glicoproteínas do Complexo Principal de Histocompatibilidade (MHC) são receptores heterodiméricos de superfície celular que funcionam para apresentar fragmentos de peptídeos antígenos às células T responsáveis ​​pelas respostas imunes mediadas por células.

Ig-like_dom - A estrutura básica das moléculas de imunoglobulina (Ig) é um tetrâmero de duas cadeias leves e duas cadeias pesadas ligadas por ligações dissulfeto. Este tipo de domínio está envolvido em uma variedade de funções, incluindo reconhecimento célula-célula, receptores de superfície celular, estrutura muscular e sistema imunológico

Ig_C1-set - Esta entrada representa domínios do conjunto C1, que são domínios clássicos semelhantes a Ig que se assemelham ao domínio constante do anticorpo. Os domínios do conjunto C1 são encontrados quase exclusivamente em moléculas envolvidas no sistema imunológico, como nas cadeias leves e pesadas de imunoglobulina, nas moléculas do complexo principal de histocompatibilidade (MHC) de classe I e II

e em vários receptores de células T.

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Utilizando a ferramenta The Human Protein Atlas foi possível inferir que a expressão génica é maior no pulmão, nas trompas de falópio, no gânglio linfático e nas amígdalas.

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Relação entre o HLA-DRB1 e os vários tipos de cancro

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Relação entre o HLA-DRB1 e os vários tipos de cancro

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No que toca à relação com os diferentes cancros, este gene tem maior incidência no cancro no sistema linfático, cancro da pele e no mieloma múltiplo.

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Com a ajuda da ferramenta UniProt e através da análise da localização subcelular, conclui-se que esta localiza-se na membrana celular, na membrana do reticulo endoplasmático, na membrana do endossomo tardio e no autofagossoma.

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A alergia alimentar é uma patologia causada por uma reação alérgica a alguma substância presente no alimento, bebida ou aditivo alimentar consumido.

Como envolve um mecanismo imunológico, esta patologia leva ao aparecimento de sintomas poucos minutos após a sua ingestão. Estas reações podem atingir a pele e mucosas (urticária, angioedema), as vias respiratórias (rinoconjuntivite, asma), o intestino (vómitos, diarreia) e o sistema cardiovascular (choque anafiláctico, menos frequente mas mais grave).

As causas mais comuns de alergia alimentar estão no consumo de frutos do mar, castanhas, leite de vaca, ovo e amendoim.

Fonte: https://www.msdmanuals.com/pt-pt/casa/doen%C3%A7as-imunol%C3%B3gicas/rea%C3%A7%C3%B5es-al%C3%A9rgicas-e-outras-doen%C3%A7as-relacionadas-%C3%A0-hipersensibilidade/alergia-alimentar

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No que diz respeito à função, analisaram-se os resultados obtidos sobre a função molecular e a atividade biológica.

Relativamente à função molecular, salienta-se a fonte de ligação ao lipopolissacarídeo, ao ácido lipoteicóico, a atividade do receptor imune lipopolissacarídeo, do receptor de opsonina 2 e do receptor imune pepticoglicano.

No que toca à atividade biológica, destacam-se o processo apoptótico, a via de sinalização do receptor de superfície celular, a resposta celular ao lipopeptídeo diacil bacteriano, ao lipopolissacarídeo, ao ácido lipoteicóico, à molécula de origem bacteriana e ao lipopeptídeo triacil bacteriano, a resposta inflamatória, a resposta imune inata 2, a fagocitose 2, a regulação positiva da produção de citocinas, da endocitose, da produção de interleucina-8, da via de sinalização mediada por lipopolissacarídeos, da via de sinalização do receptor toll-like4, da produção do fator de necrose tumoral, da produção de interferon tipo I e II, a endocitose mediada por receptores, a resposta ao estímulo elétrico, ao etanol, ao ião magnésio 2, ao fator de necrose tumoral e a via de sinalização do receptor toll-like4.

Fonte: https://www.uniprot.org/uniprotkb/P08571/entry

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Utilizando a ferramenta InterPro, determinou-se a família da proteína e as suas super famílias homologas. Assim sendo, a proteína pertence à família Monocyte_diff_Ag_CD14. Os membros desta família são co-receptores para lipopolissacarídeos bacterianos.
No que diz respeito ao domínio esta proteína não apresenta domínios.

A proteína pertence ainda a uma super família homologa, a LRR_dom_sf.

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Utilizando a ferramenta The Human Protein Atlas foi possível inferir que a expressão génica é maior na nasofaringe, endométrio e nas trompas de falópio.

# data[].attributes.headline
Relação entre o CD14 e os vários tipos de cancro

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Relação entre o CD14 e os vários tipos de cancro

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No que toca à relação com os diferentes cancros, este gene tem maior incidência no cancro pulmonar, cancro da cabeça, do pescoço e cancro das vias biliares.

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Com a ajuda da ferramenta UniProt e através da análise da localização subcelular, conclui-se que esta localiza-se na membrana celular, na jangada da membrana e nos complexo de Golgi .

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Posto isto, verificamos 2 tipos de modificações pós tradução: fosforilação e outro. Somando os locais de fosforilação, obtém-se 2 locais. Em relação à outra modificação, obtém-se 4 locais.

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Posto isto, verificamos 2 tipos de modificações pós tradução: fosforilação e ubiquitinação. Somando os locais de fosforilação, obtém-se 4 locais. Em relação à modificação de ubiquitinação, obtém-se 8 locais.

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Não foi identificado nenhum domínio transmembranar.

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Foi identificado 1 domínio transmembranar, na posição 228-250.

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Esta estrutura apresenta uma forma de beta solenoide com 11 domínios repetidas de leucinas. O CD14 é formado por 5 estruturas alfa hélice ordenado para o lado convexo da molécula, bem como 11 folhas betas no lado côncavo do beta solenoide, essa estrutura sugere a formação de um túnel na molécula.

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A molécula de classe II é um dímero constituído por uma cadeia alfa (DRA) e uma cadeia beta (DRB), ambas ancoradas na membrana.

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Para realizar o alinhamento múltiplo e a filogenia, utilizamos o Blast e o ClustalOmega, de modo a avaliar a conservação de sequências e posteriormente obter a árvore filogenética.

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O código implementado na secção "Análise de homologias por Blast" e "Ferramentas de análise das propriedades das proteínas", que se encontra no GitHub, foi baseado no código desenvolvido pelo grupo 4 do ano passado.

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Sequências homólogas que resultaram do Blast e foram colocadas no Clustal Omega com a finalidade de se obter a fologenia da proteina CD14.

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Através da análise da árvore filogenética fornecida pelo Clustal Omega, é possível verificar que existe um ancestral comum e que a partir deste surgem dois grupos distintos. A proteína monocyte differentiation antigen CD14 precursor, associada ao gene CD14 no Homo sapiens (query) apresenta maior proximidade com outras proteínas da mesma espécie como monocyte differentiation antigen CD14 e unnamed protein product, o que se pode verificar pelo número de ramificações que têm em comum e pelo comprimento destas em relação ao ancestral comum.

Tal vai de encontro ao esperado através da análise dos resultados fornecidos pelo Blast.

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Clustal Omega is a multiple sequence alignment program. It produces biologically meaningful multiple sequence alignments of divergent sequences. Evolutionary relationships can be seen via viewing Cladograms or Phylograms.

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A rede de interação proteína-proteína (rede IPP) codifica as interações entre as proteínas e auxilia no mapeamento do interatoma de um organismo. Para compreender um sistema complexo, é preciso entender como seus componentes moleculares interagem entre si, e as redes são excelentes modelos para codificar e interpretar essas interações.

A ferramenta String permite visualizar essas interações. As interações incluem associações diretas (físicas) e indiretas (funcionais). Estas decorrem na previsão computacional, da transferência de conhecimento entre organismos e das interações agregadas de outros bancos de dados (primários).

Através da análise da rede de interações obtidas associa as relações entre a proteína alvo de estudo e outras proteínas de outros genes. Pela sua análise as proteínas associadas/ligadas são provenientes do mesmo organismo, Homo sapiens.

Exemplo de proteína interativa:

HLA-B (Antígeno Leucocitário Humano B): a sua função é apresentar fragmentos de proteínas aos linfócitos T, desempenhando um papel crucial no reconhecimento e na resposta imunológica contra agentes patogénicos.

Fonte: https://www.msdmanuals.com/pt-pt/profissional/imunologia-dist%C3%BArbios-al%C3%A9rgicos/biologia-do-sistema-imunit%C3%A1rio/sistema-de-ant%C3%ADgeno-leucocit%C3%A1rio-humano-hla

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Analisando os resultados fornecidos pelo Cobalt, é possível verificar que as zonas mais conservadas das proteínas se apresentam a vermelho. Deste modo, a região mais conservada em todas as sequências encontra-se entre os aminoácidos 55 e 340.

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O presente trabalho tem como principal objetivo a utilização de ferramentas computacionais da área da Bioinformática para a análise de dois genes (CD14 e HLA-DRB1), focando o estudo das sequências associadas e a sua estrutura.

Os presentes genes provocam reações alérgicas a vários alimentos no corpo humano, nomeadamente os amendoins.

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Recorrendo à ferramenta DisGeNet, localizado no UniProt, é possível inferir que a proteína em estudo está relacionada com várias doenças como é o caso da asma alérgica, hiperreatividade brônquica, bronquiolite, entre outras.

Fonte: https://www.disgenet.org/browser/0/1/1/C1827849/geneid__929-source__ALL

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Segundo as bases de dados UniProt e OMIM, não foi possível associar diretamente nenhuma doença ao gene CD14.

Fonte: https://www.omim.org/entry/158120?search=CD14&highlight=cd14

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Na ferramenta Phospositeplus foi possível constatar que mutações nesta proteína podem desencadear o desenvolvimento de cancro, sendo o mais predominante o do endométrio.

Fonte: https://www.phosphosite.org/proteinAction.action?id=22133&showAllSites=true

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As mutações neste gene estão associadas à doença SARS-CoV-2.

Fonte: Molécula CD14 CD14 [Homo sapiens (humano)] - Gene - NCBI (nih.gov⁾

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Através da ferramenta IntAct, averigua-se que o gene CD14 interage com outros genes, sendo estes Homo sapiens e Mus musculus ou de outros animais, mamíferos, bactérias ou vírus.
Concluiu-se ainda que uma das ligações mais forte é com o gene PIK3R1.

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Recorrendo à ferramenta DisGeNet, localizado no UniProt, é possível inferir que a proteína em estudo está relacionada com várias doenças como é o caso da febre reumática, artrite rematóide precoce, úlcera duodenal, entre outras.

Fonte: https://www.disgenet.org/browser/0/1/1/C0035439/geneid__3123-source__ALL/

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Pela ferramenta OMIM (Online Mendelian Inheritance in Man), foi possível concluir que as variantes deste gene passam pelos alelos codificantes da esclerose múltipla e da sarcoidose.

Fonte: https://www.omim.org/entry/142857?search=HLA-DRB1&highlight=hla-drb1

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Na ferramenta Phospositeplus foi possível constatar que mutações nesta proteína podem desencadear o desenvolvimento de cancro, sendo o mais predominante o dos rins.

Fonte: https://www.phosphosite.org/proteinAction.action?id=6141101&showAllSites=true

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Através da ferramenta IntAct, averigua-se que o gene HLA-DRB1 interage com outros genes, sendo estes Homo sapiens e Escherichia coli ou de outros mamíferos ou bactérias.
Concluiu-se ainda que a ligação mais forte é com o gene HLA-DRA.

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As mutações neste gene estão associadas ao vírus da hepatite B.

Fonte: Complexo principal de histocompatibilidade HLA-DRB1, classe II, DR beta 1 [Homo sapiens (humano)] - Gene - NCBI (nih.gov⁾

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A realização deste trabalho permitiu o uso de ferramentas de bioinformática e o desenvolvimento de scripts através do BioPython, o que gerou o acesso automatizado aos mesmos dados. A partir deste podemos tirar conclusões quanto às características e funções de cada um dos genes alvo de estudo.