During the past three decades, mice, zebrafish, fruit flies, and have been the primary model organisms used for the study of various biological phenomena. These models have also been adopted and developed to investigate the physiological roles of carbonic anhydrases (CAs) and carbonic anhydrase-related proteins (CARPs). These proteins belong to eight CA families and are identified by Greek letters: α, β, γ, δ, ζ, η, θ, and ι. Studies using model organisms have focused on two CA families, α-CAs and β-CAs, which are expressed in both prokaryotic and eukaryotic organisms with species-specific distribution patterns and unique functions. This review covers the biological roles of CAs and CARPs in light of investigations performed in model organisms. Functional studies demonstrate that CAs are not only linked to the regulation of pH homeostasis, the classical role of CAs, but also contribute to a plethora of previously undescribed functions.

Lysine glutarylation (Kglu) of mitochondrial proteins is associated with glutaryl-CoA dehydrogenase (GCDH) deficiency, which impairs lysine/tryptophan degradation and causes destruction of striatal neurons during catabolic crisis with subsequent movement disability. By investigating the role of Kglu modifications in this disease, we compared the brain and liver glutarylomes of Gcdh-deficient mice. In the brain, we identified 73 Kglu sites on 37 mitochondrial proteins involved in various metabolic degradation pathways. Ultrastructural immunogold studies indicated that glutarylated proteins are heterogeneously distributed in mitochondria, which are exclusively localized in glial cells. In liver cells, all mitochondria contain Kglu-modified proteins. Glutarylation reduces the catalytic activities of the most abundant glutamate dehydrogenase (GDH) and the brain-specific carbonic anhydrase 5b and interferes with GDH-protein interactions. We propose that Kglu contributes to the functional heterogeneity of mitochondria and may metabolically adapt glial cells to the activity and metabolic demands of neighboring GCDH-deficient neurons.

Membrane-associated carbonic anhydrase (CA) isoform IV participates in carbon metabolism and pH homeostasis and is implicated in the development of eye diseases such as retinitis pigmentosa and glaucoma. A series of substituted benzenesulfonamides were designed and their binding affinity to CA IV was determined by fluorescent thermal shift assay and isothermal titration calorimetry (ITC). Compound [(4-chloro-2-phenylsulfanyl-5-sulfamoyl-benzoyl)amino]propyl acetate (19) bound CA IV with the K of 1.0 nM and exhibited significant selectivity over the remaining 11 human CA isoforms. The compound could be developed as a drug targeting CA IV. Various forms of recombinant CA IV were produced in Escherichia coli and mammalian cell cultures. Comparison of their temperature stability in various buffers and salt solutions demonstrated that CA IV is most stable at slightly alkaline conditions and at elevated sodium sulfate concentrations. High-resolution X-ray crystallographic structures of ortho-Cl and meta-thiazole-substituted benzene sulfonamide in complex with CA IV revealed the position of and interactions between the ligand and the protein. Sulfonamide inhibitor binding to CA IV is linked to several reactions-the deprotonation of the sulfonamide amino group, the protonation of CA-Zn(II)-bound hydroxide at the active site of CA IV, and the compensating reactions of the buffer. The dissection of binding-linked reactions yielded the intrinsic thermodynamic parameters, characterizing the interaction between CA IV and the sulfonamides in the binding-able protonation forms, including Gibbs energy, enthalpy, and entropy, that could be used for the characterization of binding to any CA in the process of drug design.

Human CAXII was initially identified as a cancer marker in different cancers and tumors. Expression of CAXII is regulated by hypoxia and estrogen receptors. CAXII expression has been also detected in several tissues, whereas in cancer and tumor tissues its expression is several fold higher. In brain tumors, an alternatively spliced form of CAXII is expressed. Higher expression of CAXII in breast cancer is indicative of lower grade disease. CAXII plays a key role in several physiological functions. Mutation in the CAXII gene causes cystic fibrosis-like syndrome and salt wasting disease. CAXII is also seen in nuclear pulposus cells of the vertebrae. Aging dependent stiffness or degeneration of backbone correlates with CAXII expression level. This finding suggests a possible implication of CAXII as a biomarker for chronic back pain and a pharmacological target for possible treatment of chronic back pain.

Skin wound closure occurs when keratinocytes migrate from the edge of the wound and re-epithelialize the epidermis. Their migration takes place primarily before any vascularization is established, that is, under hypoxia, but relatively little is known regarding the factors that stimulate this migration. Hypoxia and an acidic environment are well-established stimuli for cancer cell migration. The carbonic anhydrases (CAs) contribute to tumor cell migration by generating an acidic environment through the conversion of carbon dioxide to bicarbonate and a proton. On this basis, we explored the possible role of CAs in tissue regeneration using mouse skin wound models. We show that the expression of mRNAs encoding CA isoforms IV and IX are increased (~25 × and 4 ×, respectively) during the wound hypoxic period (days 2-5) and that cells expressing CAs form a band-like structure beneath the migrating epidermis. RNA-Seq analysis suggested that the CA IV-specific signal in the wound is mainly derived from neutrophils. Due to the high level of induction of CA IV in the wound, we treated skin wounds locally with recombinant human CA IV enzyme. Recombinant CA IV significantly accelerated wound re-epithelialization. Thus, CA IV could contribute to wound healing by providing an acidic environment in which the migrating epidermis and neutrophils can survive and may offer novel opportunities to accelerate wound healing under compromised conditions.