RESUMEN
Chronic liver diseases, fibrosis, cirrhosis, and HCC are often a consequence of persistent inflammation. However, the transition mechanisms from a normal liver to fibrosis, then cirrhosis, and further to HCC are not well understood. This study focused on the role of the tumor stem cell protein doublecortin-like kinase 1 (DCLK1) in the modulation of molecular factors in fibrosis, cirrhosis, or HCC. Serum samples from patients with hepatic fibrosis, cirrhosis, and HCC were analyzed via ELISA or NextGen sequencing and were compared with control samples. Differentially expressed (DE) microRNAs (miRNA) identified from these patient sera were correlated with DCLK1 expression. We observed elevated serum DCLK1 levels in fibrosis, cirrhosis, and HCC patients; however, TGF-ß levels were only elevated in fibrosis and cirrhosis. While DE miRNAs were identified for all three disease states, miR-12136 was elevated in fibrosis but was significantly increased further in cirrhosis. Additionally, miR-1246 and miR-184 were upregulated when DCLK1 was high, while miR-206 was downregulated. This work distinguishes DCLK1 and miRNAs' potential role in different axes promoting inflammation to tumor progression and may serve to identify biomarkers for tracking the progression from pre-neoplastic states to HCC in chronic liver disease patients as well as provide targets for treatment.
Asunto(s)
Quinasas Similares a Doblecortina , Inflamación , Péptidos y Proteínas de Señalización Intracelular , Cirrosis Hepática , Neoplasias Hepáticas , MicroARNs , Proteínas Serina-Treonina Quinasas , Humanos , MicroARNs/sangre , MicroARNs/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/sangre , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/sangre , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/sangre , Cirrosis Hepática/genética , Cirrosis Hepática/sangre , Inflamación/genética , Inflamación/sangre , Masculino , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/sangre , Femenino , Enfermedad Crónica , Hepatopatías/sangre , Hepatopatías/genética , Persona de Mediana Edad , Carcinogénesis/genética , Anciano , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genéticaRESUMEN
While significant strides have been made in understanding cancer biology, the enhancement in patient survival is limited, underscoring the urgency for innovative strategies. Epigenetic modifications characterized by hereditary shifts in gene expression without changes to the DNA sequence play a critical role in producing alternative gene isoforms. When these processes go awry, they influence cancer onset, growth, spread, and cancer stemness. In this review, we delve into the epigenetic and isoform nuances of the protein kinase, doublecortin-like kinase 1 (DCLK1). Recognized as a hallmark of tumor stemness, DCLK1 plays a pivotal role in tumorigenesis, and DCLK1 isoforms, shaped by alternative promoter usage and splicing, can reveal potential therapeutic touchpoints. Our discussion centers on recent findings pertaining to the specific functions of DCLK1 isoforms and the prevailing understanding of its epigenetic regulation via its two distinct promoters. It is noteworthy that all DCLK1 isoforms retain their kinase domain, suggesting that their unique functionalities arise from non-kinase mechanisms. Consequently, our research has pivoted to drugs that specifically influence the epigenetic generation of these DCLK1 isoforms. We posit that a combined therapeutic approach, harnessing both the epigenetic regulators of specific DCLK1 isoforms and DCLK1-targeted drugs, may prove more effective than therapies that solely target DCLK1.
Asunto(s)
Quinasas Similares a Doblecortina , Neoplasias , Humanos , Epigénesis Genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Isoformas de Proteínas/metabolismo , Neoplasias/genética , Neoplasias/terapia , Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismoRESUMEN
Type-II toxin-antitoxin (TA) systems are comprised of two tightly interacting proteins, and operons encoding these systems have been identified throughout the genomes of bacteria. In contrast to secretion system effector-immunity pairs, TA systems must remain paired to protect the host cell from toxicity. Continual depletion of the antitoxin results in a shorter half-life than that of the toxin, though it is unclear if antitoxins can be effectively degraded when complexed with toxins. The current work probed the protein-protein interface of the PaParDE1 TA system, guided by an X-ray crystal structure, to determine contributions of antitoxin amino acids to interaction kinetics and affinity. These studies identified a "hotspot" position that alters the binding mode and resulting affinity (KD) from 152 pM for a 1:1 model for wild type to 25.5 and 626 nM for a 2:1 model with mutated antitoxin. This correlates with an altered induced secondary structure upon complexation with PaParE1 and increased kinetics of Lon protease digestion of the antitoxin despite the toxin presence. However, the decreased affinity at this hotspot was essentially reversed when the antitoxin dimerization region was deleted, yielding insights into complex interactions involved in the tight association. Removal of the antitoxin C-terminal seven amino acids, corresponding to the site of a disorder-to-order transition, completely prevents association. These studies combine to provide a model for the initiation of the TA interaction and highlight how manipulation of the sequence can impact the antitoxin disorder-to-order transition, weakening the affinity and resulting in increased antitoxin susceptibility to degradation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteasa La/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/química , Toxinas Bacterianas/química , Cristalografía por Rayos X , Escherichia coli/química , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/química , Humanos , Cinética , Proteasa La/química , Unión Proteica , Mapas de Interacción de Proteínas , Proteolisis , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/químicaRESUMEN
The sea lamprey (Petromyzon marinus) undergoes substantial genomic alterations during embryogenesis in which specific sequences are deleted from the genome of somatic cells yet retained in cells of the germ line. One element that undergoes diminution in P. marinus is Germ1, which consists of a somatically rare (SR) region and a fragment of 28S rDNA. Although the SR-region has been used as a marker for genomic alterations in lampreys, the evolutionary significance of its diminution is unknown. We examined the Germ1 element in five additional species of lamprey to better understand its evolutionary significance. Each representative species contained sequences similar enough to the Germ1 element of P. marinus to be detected via PCR and Southern hybridizations, although the SR-regions of Lampetra aepyptera and Lethenteron appendix are quite divergent from the homologous sequences of Petromyzon and three species of Ichthyomyzon. Lamprey Germ1 sequences have a number of features characteristic of the R2 retrotransposon, a mobile element that specifically targets 28S rDNA. Phylogenetic analyses of the SR-regions revealed patterns generally consistent with relationships among the species included in our study, although the 28S-fragments of each species/genus were most closely related to its own functional rDNA, suggesting that the two components of Germ1 were assembled independently in each lineage. Southern hybridizations showed evidence of genomic alterations involving Germ1 in each species. Our results suggest that Germ1 is a R2 retroelement that occurs in the genome of P. marinus and other petromyzontid lampreys, and that its diminution is incidental to the reduction in rDNA copies during embryogenesis.
Asunto(s)
Tamaño del Genoma/fisiología , Lampreas/genética , ARN Ribosómico 28S/genética , Animales , Evolución Biológica , Deleción Cromosómica , Diploidia , Evolución Molecular , Peces/genética , Genoma/genética , Tamaño del Genoma/genética , Células Germinativas/fisiología , Lampreas/metabolismo , Filogenia , Retroelementos/genética , Especificidad de la EspecieRESUMEN
In most sexually reproducing organisms, the fundamental process of meiosis is implemented concurrently with two differentiation programs that occur at different rates and generate distinct cell types, sperm and oocytes. However, little is known about how the meiotic program is influenced by such contrasting developmental programs. Here we present a detailed timeline of late meiotic prophase during spermatogenesis in Caenorhabditis elegans using cytological and molecular landmarks to interrelate changes in chromosome dynamics with germ cell cellularization, spindle formation, and cell cycle transitions. This analysis expands our understanding C. elegans spermatogenesis, as it identifies multiple spermatogenesis-specific features of the meiotic program and provides a framework for comparative studies. Post-pachytene chromatin of spermatocytes is distinct from that of oocytes in both composition and morphology. Strikingly, C. elegans spermatogenesis includes a previously undescribed karyosome stage, a common but poorly understood feature of meiosis in many organisms. We find that karyosome formation, in which chromosomes form a constricted mass within an intact nuclear envelope, follows desynapsis, involves a global down-regulation of transcription, and may support the sequential activation of multiple kinases that prepare spermatocytes for meiotic divisions. In spermatocytes, the presence of centrioles alters both the relative timing of meiotic spindle assembly and its ultimate structure. These microtubule differences are accompanied by differences in kinetochores, which connect microtubules to chromosomes. The sperm-specific features of meiosis revealed here illuminate how the underlying molecular machinery required for meiosis is differentially regulated in each sex.
Asunto(s)
Caenorhabditis elegans/citología , Meiosis , Espermatogénesis , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Regulación del Desarrollo de la Expresión Génica , Masculino , Espermatozoides/citología , Espermatozoides/crecimiento & desarrollo , Espermatozoides/metabolismoRESUMEN
Giardia lamblia is present in the intestinal lumen as a binucleate, flagellated trophozoite or a quadranucleate, immotile cyst. Here we used the plant lectin wheat germ agglutinin (WGA), which binds to the disaccharide di-N-acetyl-chitobiose (GlcNAc(2)), which is the truncated Asn-linked glycan (N-glycan) of Giardia, to affinity purify the N-glycomes (glycoproteins with N-glycans) of trophozoites and cysts. Fluorescent WGA bound to the perinuclear membranes, peripheral acidified vesicles, and plasma membranes of trophozoites. In contrast, WGA bound strongly to membranes adjacent to the wall of Giardia cysts and less strongly to the endoplasmic reticulum and acidified vesicles. WGA lectin-affinity chromatography dramatically enriched secreted and membrane proteins of Giardia, including proteases and acid phosphatases that retain their activities. With mass spectroscopy, we identified 91 glycopeptides with N-glycans and 194 trophozoite-secreted and membrane proteins, including 42 unique proteins. The Giardia oligosaccharyltransferase, which contains a single catalytic subunit, preferred N glycosylation sites with Thr to those with Ser in vivo but had no preference for flanking amino acids. The most-abundant glycoproteins in the N-glycome of trophozoites were lysosomal enzymes, folding-associated proteins, and unique transmembrane proteins with Cys-, Leu-, or Gly-rich repeats. We identified 157 secreted and membrane proteins in the Giardia cysts, including 20 unique proteins. Compared to trophozoites, cysts were enriched in Gly-rich repeat transmembrane proteins, cyst wall proteins, and unique membrane proteins but had relatively fewer Leu-rich repeat proteins, folding-associated proteins, and unique secreted proteins. In summary, there are major changes in the Giardia N-glycome with the differentiation from trophozoites to cysts.
Asunto(s)
Asparagina/metabolismo , Giardia lamblia/crecimiento & desarrollo , Giardia lamblia/metabolismo , Glicoproteínas/metabolismo , Polisacáridos/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Cromatografía de Afinidad , Giardia lamblia/química , Giardia lamblia/genética , Glicoproteínas/química , Glicoproteínas/genética , Datos de Secuencia Molecular , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Trofozoítos/química , Trofozoítos/crecimiento & desarrollo , Trofozoítos/metabolismo , Aglutininas del Germen de Trigo/metabolismoRESUMEN
BACKGROUND: The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role san-1, the MAD3 homologue, has in Caenorhabditis elegans embryos we used RNA interference (RNAi) to identify genes synthetic lethal with the viable san-1(ok1580) deletion mutant. RESULTS: The san-1(ok1580) animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of san-1(ok1580) embryos is significantly reduced when HCP-1 (CENP-F homologue), MDF-1 (MAD-1 homologue), MDF-2 (MAD-2 homologue) or BUB-3 (predicted BUB-3 homologue) are reduced by RNAi. Interestingly, the viability of san-1(ok1580) embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of san-1(ok1580);hcp-1(RNAi) embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging). Several of the san-1(ok1580);hcp-1(RNAi) animals displayed abnormal kinetochore (detected by MPM-2) and microtubule structure. The survival of mdf-2(RNAi);hcp-1(RNAi) embryos but not bub-3(RNAi);hcp-1(RNAi) embryos was also compromised. Finally, we found that san-1(ok1580) and bub-3(RNAi), but not hcp-1(RNAi) embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein. CONCLUSION: Together, these data suggest that in the C. elegans embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that san-1(ok1580);hcp-1(RNAi) animals had a severe viability defect whereas in the san-1(ok1580);hcp-2(RNAi) and san-1(ok1580);hcp-2(ok1757) animals the viability defect was not as severe suggesting that hcp-1 and hcp-2 are not completely redundant.
RESUMEN
The back-to-back geometry of sister kinetochores is essential in preventing loss or damage of chromosomes during mitosis. Kinetochore orientation is generated in part by a process of resolving kinetochores at the centromere (centromere resolution) prior to spindle interactions. Because few of the genes required for centromere resolution are known, we used Caenorhabditis elegans to screen for conditional mutants defective in orienting sister kinetochores during mitosis. C. elegans is ideal for such screens because its chromosomes are holocentric. Here we identified an essential gene, cin-4, required for centromere resolution and for removal of cohesin from sites near sister kinetochores during mitosis. Given that compromised cohesin function restores centromere resolution in the absence of cin-4, CIN-4 likely acts to remove cohesin from the CENP-A chromatin enabling centromere resolution. CIN-4 has a high amino acid identity to the catalytic domain of topoisomerase II, suggesting a partial gene duplication of the C. elegans topoisomerase II gene, top-2. Similar to CIN-4, TOP-2 is also required for centromere resolution; however, the loss of TOP-2 is phenotypically distinct from the loss of CIN-4, suggesting that CIN-4 and TOP-2 are topoisomerase II isoforms that perform separate essential functions in centromere structure and function.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/enzimología , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Cinetocoros/metabolismo , Mitosis , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Dominio Catalítico , Inestabilidad Cromosómica , Cromosomas/metabolismo , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/genética , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Duplicación de Gen , Genes de Helminto , Modelos Genéticos , Datos de Secuencia Molecular , Mutación/genética , Sistemas de Lectura Abierta/genética , Transporte de Proteínas , Interferencia de ARN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , CohesinasRESUMEN
Prior to microtubule capture, sister centromeres resolve from one another, coming to rest on opposite surfaces of the condensing chromosome. Subsequent assembly of sister kinetochores at each sister centromere generates a geometry favorable for equal levels of segregation of chromatids. The holocentric chromosomes of Caenorhabditis elegans are uniquely suited for the study of centromere resolution and subsequent kinetochore assembly. In C. elegans, only two proteins have been identified as being necessary for centromere resolution, the kinase AIR-2 (prophase only) and the centromere protein HCP-4/CENP-C. Here we found that the loss of proteins involved in chromosome cohesion bypassed the requirement for HCP-4/CENP-C but not for AIR-2. Interestingly, the loss of cohesin proteins also restored the localization of HCP-6 to the kinetochore. The loss of the condensin II protein HCP-6 or MIX-1/SMC2 impaired centromere resolution. Furthermore, the loss of HCP-6 or MIX-1/SMC2 resulted in no centromere resolution when either nocodazole or RNA interference (RNAi) of the kinetochore protein KNL-1 perturbed spindle-kinetochore interactions. This result suggests that normal prophase centromere resolution is mediated by condensin II proteins, which are actively recruited to sister centromeres to mediate the process of resolution.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Profase , Adenosina Trifosfatasas/metabolismo , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero , Mitosis , Complejos Multiproteicos , Unión Proteica , Transporte de Proteínas , Interferencia de ARNRESUMEN
The Leishmania mexicana PFR2 locus encodes a component of the paraflagellar rod (PFR), a flagellar structure found only in the insect stage of the life cycle. PFR2 mRNA levels are 10-fold lower in the mammalian stage than in the insect stage. Nuclear run-on experiments indicate that the change in PFR2 mRNA abundance is achieved posttranscriptionally. Deletion and block substitution analysis of the entire 1,400-nucleotide 3' untranslated region (UTR) of PFR2C led to the identification of a regulatory element contained within 10 nucleotides of the 3' UTR, termed the PFR regulatory element (PRE), that is necessary for the 10-fold regulation of PFR2 mRNA levels. Comparison of the half-lives of PFR2 transcripts, identical except for the presence or absence of the PRE, revealed that the PRE acts by destabilizing the PFR2 mRNA in amastigotes. The PRE was inserted into a construct which directs the constitutive expression of a chimeric PFR2 transcript. Insertion of the PRE resulted in regulated expression of this transcript, demonstrating that the regulatory element is sufficient for promastigote-specific expression. Since the PRE is present in the 3' UTR of all L. mexicana PFR genes examined so far, we propose that it serves a means of coordinating expression of PFR genes.