Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc-dependent metalloenzymes that catalyze the reversible hydration of carbon dioxide, a reaction essential for acid-base regulation, respiration, and metabolic integration in vertebrates. In animals, CA activity is exclusively encoded by the α-CA family, which has undergone extensive expansion during vertebrate evolution. This chapter presents a phylogenetic synthesis of vertebrate α-CAs, integrating sequence-based analyses with comparative genomics, structural constraints, and patterns of subcellular localization. Phylogenetic evidence indicates that the modern vertebrate CA repertoire originated early in the evolution of life, and was shaped by ancient whole-genome duplication events, followed by differential retention, divergence, and lineage-specific gene loss. Cytosolic, mitochondrial, membrane-associated, and secreted CAs form well-supported evolutionary lineages that primarily reflect diversification in regulation, cellular targeting and physiological roles, rather than changes in catalytic mechanism. Catalytically inactive CA-related proteins (CARPs) constitute a distinct evolutionary branch derived from gene duplication and functional divergence of catalytically active CAs. Molecular analyses reveal strong evolutionary constraints on residues essential for zinc coordination and proton transfer, whereas surface-exposed and regulatory regions show greater evolutionary flexibility. Comparative analyses across vertebrates highlight the broad conservation of major CA clades, together with lineage-specific differences in isoform repertoires. By embedding functional interpretation within a phylogenetic framework, this chapter provides an evolutionary basis for comparative and biomedical studies of vertebrate CAs.
Telomeric sequences are hotspots for ultraviolet light (UV) induced cyclobutane pyrimidine dimers (CPD) and pyrimidine(6-4)pyrimidone photoproducts (6-4 PP), due to pyrimidine runs on both the TTAGGG and CCCTAA containing strands. Photoproducts are repaired by global genome nucleotide excision repair (GG-NER) or by transcription-coupled (TC-NER) in regions of active transcription. Since telomeres are transcribed into long telomeric repeat-containing RNA (TERRA) molecules, here we tested roles for both TC-NER and GG-NER in telomere stability following UVC irradiation. XPC-deficient cells, incapable of GG-NER, failed to exhibit significant reductions in 6-4 PPs and CPDs at telomeres during recovery times, indicating that TC-NER cannot compensate for detectable photoproduct removal at telomeres when GG-NER is absent. TERRA analysis confirmed active telomere transcription in these cell lines. Loss of total NER or specifically GG-NER in XPA-deficient or XPC-deficient cells, respectively, increased telomere losses and telomere fragility following UV irradiation. These data provide direct evidence that NER is required to prevent UV damage-induced telomere aberrations and that transcription at telomeres is likely insufficient to drive substantial TC-NER-mediated photoproduct removal.
BACKGROUND/AIM:This study aimed to evaluate the anticancer efficacy of 5-Fluorouracil (5-FU) in Caco-2 human colorectal adenocarcinoma cells treated with siRNA-mediated MLH1 gene inhibition, in terms of cell viability, apoptosis, and related gene/protein expression. MATERIALS AND METHODS:Caco-2 cells were treated with a dose of 10 µM 5-FU. Cell viability was assessed by CVDK-8 analysis, and apoptosis was determined by flow cytometry using Annexin V-FITC/PI staining. Expression levels of MLH1, CDK2, CDK4, CDK5, CDK6, PTEN, EGFR, mTOR, PI3K, AKT3, ERK, PARP1, and GAPDH genes were examined by RT-qPCR analysis. Protein expression levels were analyzed using Western blot for PI3K, p-AKT, AKT1, mTOR, MAPK-p38, Caspase-3, Bcl-2, STAT3, JAK2, MLH1, MDR1, p53, and proteins; β-Actin was used as an internal control. RESULTS:CVDK-8 analysis showed that application of 10 µM 5-FU significantly reduced cell viability in Caco-2 cells. Flow cytometry results revealed a significant increase in both early- and late-apoptotic cell populations following 5-FU treatment. DISCUSSION:Gene and protein expression analyses showed significant changes in signaling pathways associated with apoptosis and cell proliferation. In this study, the effects of combining MLH1 gene silencing with 5-FU on colorectal cancer cells were comprehensively evaluated using Cell viability, RT-qPCR, flow cytometry, and Western blot analyses. It was determined that the combined application significantly reduced cell viability and increased apoptosis. Molecular-level findings on gene and protein expression support this effect. The results suggest that MLH1 gene silencing may increase sensitivity to 5-FU, offering a potential combination therapy approach in colorectal cancer.
