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Membrane-bound proteins that change protonation during function use specific protein groups to bind and transfer protons. Knowledge of the identity of the proton-binding groups is of paramount importance to decipher the reaction mechanism of the protein, and protonation states of prominent are studied extensively using experimental and computational approaches. Analyses of model transporters and receptors from different organisms, and with widely different biological functions, indicate common structure-sequence motifs at internal proton-binding sites. Proton-binding dynamic hydrogen-bond networks that are exposed to the bulk might provide alternative proton-binding sites and proton-binding pathways. In this perspective article I discuss protonation coupling and proton binding at internal and external carboxylate sites of proteins that use proton transfer for function. An inter-helical carboxylate-hydroxyl hydrogen-bond motif is present at functionally important sites of membrane proteins from archaea to the brain. External carboxylate-containing H-bond clusters are observed at putative proton-binding sites of protonation-coupled model proteins, raising the question of similar functionality in spike protein S.

Intra- and extracellular pH regulation is a pivotal function of all cells and tissues. Net outward transport of H+ is a prerequisite for normal physiological function, since a number of intracellular processes, such as metabolism and energy supply, produce acid. In tumor tissues, distorted pH regulation results in extracellular acidification and the formation of a hostile environment in which cancer cells can outcompete healthy local host cells. Cancer cells employ a variety of H+/HCO3−-coupled transporters in combination with intra- and extracellular carbonic anhydrase (CA) isoforms, to alter intra- and extracellular pH to values that promote tumor progression. Many of the transporters could closely associate to CAs, to form a protein complex coined “transport metabolon”. While transport metabolons built with HCO3−-coupled transporters require CA catalytic activity, transport metabolons with monocarboxylate transporters (MCTs) operate independently from CA catalytic function. In this article, we assess some of the processes and functions of CAs for tumor pH regulation and discuss the role of intra- and extracellular pH regulation for cancer pathogenesis and therapeutic intervention.

Many tumor cells produce vast amounts of lactate and acid, which have to be removed from the cell to prevent intracellular lactacidosis and suffocation of metabolism. In the present study, we show that proton-driven lactate flux is enhanced by the intracellular carbonic anhydrase CAII, which is colocalized with the monocarboxylate transporter MCT1 in MCF-7 breast cancer cells. Co-expression of MCTs with various CAII mutants in Xenopus oocytes demonstrated that CAII facilitates MCT transport activity in a process involving CAII-Glu69 and CAII-Asp72, which could function as surface proton antennae for the enzyme. CAII-Glu69 and CAII-Asp72 seem to mediate proton transfer between enzyme and transporter, but CAII-His64, the central residue of the enzyme’s intramolecular proton shuttle, is not involved in proton shuttling between the two proteins. Instead, this residue mediates binding between MCT and CAII. Taken together, the results suggest that CAII features a moiety that exclusively mediates proton exchange with the MCT to facilitate transport activity.

Proteins that bind protons at cell membrane interfaces often expose to the bulk clusters of carboxylate and histidine sidechains that capture protons transiently and, in proton transporters, deliver protons to an internal site. The protonation-coupled dynamics of bulk-exposed carboxylate clusters, also known as proton antennas, is poorly described. An essential open question is how water-mediated bridges between sidechains of the cluster respond to protonation change and facilitate transient proton storage. To address this question, here I studied the protonation-coupled dynamics at the proton-binding antenna of PsbO, a small extrinsinc subunit of the photosystem II complex, with atomistic molecular dynamics simulations and systematic graph-based analyses of dynamic protein and protein-water hydrogen-bond networks. The protonation of specific carboxylate groups is found to impact the dynamics of their local protein-water hydrogen-bond clusters. Regardless of the protonation state considered for PsbO, carboxylate pairs that can sample direct hydrogen bonding, or bridge via short hydrogen-bonded water chains, anchor to nearby basic or polar protein sidechains. As a result, carboxylic sidechains of the hypothesized antenna cluster are part of dynamic hydrogen bond networks that may rearrange rapidly when the protonation changes. [Display omitted] • Graph analyses reveal the protonation-coupled dynamics of a proton antenna cluster. • Local protein interactions and water bridging shape dynamics of carboxylic clusters. • Dynamic protein-water H-bond clusters couple to protonation of carboxylic groups. • Protocol developed for model antenna protein applicable to proton-binding proteins. [ABSTRACT FROM AUTHOR]

Membrane-bound proteins that change protonation during function use specific protein groups to bind and transfer protons. Knowledge of the identity of the proton-binding groups is of paramount importance to decipher the reaction mechanism of the protein, and protonation states of prominent are studied extensively using experimental and computational approaches. Analyses of model transporters and receptors from different organisms, and with widely different biological functions, indicate common structure-sequence motifs at internal proton-binding sites. Proton-binding dynamic hydrogen-bond networks that are exposed to the bulk might provide alternative proton-binding sites and proton-binding pathways. In this perspective article I discuss protonation coupling and proton binding at internal and external carboxylate sites of proteins that use proton transfer for function. An inter-helical carboxylate-hydroxyl hydrogen-bond motif is present at functionally important sites of membrane proteins from archaea to the brain. External carboxylate-containing H-bond clusters are observed at putative proton-binding sites of protonation-coupled model proteins, raising the question of similar functionality in spike protein S., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bondar.)

Solid tumors are metabolically highly active tissues, which produce large amounts of acid. The acid/base balance in tumor cells is regulated by the concerted interplay between a variety of membrane transporters and carbonic anhydrases (CAs), which cooperate to produce an alkaline intracellular, and an acidic extracellular, environment, in which cancer cells can outcompete their adjacent host cells. Many acid/base transporters form a structural and functional complex with CAs, coined "transport metabolon". Transport metabolons with bicarbonate transporters require the binding of CA to the transporter and CA enzymatic activity. In cancer cells, these bicarbonate transport metabolons have been attributed a role in pH regulation and cell migration. Another type of transport metabolon is formed between CAs and monocarboxylate transporters, which mediate proton-coupled lactate transport across the cell membrane. In this complex, CAs function as "proton antenna" for the transporter, which mediate the rapid exchange of protons between the transporter and the surroundings. These transport metabolons do not require CA catalytic activity, and support the rapid efflux of lactate and protons from hypoxic cancer cells to allow sustained glycolytic activity and cell proliferation. Due to their prominent role in tumor acid/base regulation and metabolism, transport metabolons might be promising drug targets for new approaches in cancer therapy. [ABSTRACT FROM AUTHOR]

Intra- and extracellular pH regulation is a pivotal function of all cells and tissues. Net outward transport of H + is a prerequisite for normal physiological function, since a number of intracellular processes, such as metabolism and energy supply, produce acid. In tumor tissues, distorted pH regulation results in extracellular acidification and the formation of a hostile environment in which cancer cells can outcompete healthy local host cells. Cancer cells employ a variety of H + /HCO 3 - -coupled transporters in combination with intra- and extracellular carbonic anhydrase (CA) isoforms, to alter intra- and extracellular pH to values that promote tumor progression. Many of the transporters could closely associate to CAs, to form a protein complex coined "transport metabolon". While transport metabolons built with HCO 3 - -coupled transporters require CA catalytic activity, transport metabolons with monocarboxylate transporters (MCTs) operate independently from CA catalytic function. In this article, we assess some of the processes and functions of CAs for tumor pH regulation and discuss the role of intra- and extracellular pH regulation for cancer pathogenesis and therapeutic intervention.

Highly glycolytic tumor cells release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbate extracellular acidification and support the formation of a hostile environment. Transport activity of MCTs can be facilitated by non-catalytic interaction with carbonic anhydrase IX (CAIX), the expression of which has been shown to be upregulated under hypoxia. We have now studied the mechanisms that enable CAIX-mediated facilitation of proton-coupled lactate transport in breast cancer cells and Xenopus oocytes. Our results indicate that the proteoglycan like (PG) domain of CAIX could function as 'proton antenna' to facilitate MCT transport activity. Truncation of the PG domain and application of a PG-binding antibody significantly reduced proton-coupled lactate transport in MCT-expressing oocytes and hypoxic breast cancer cells, respectively. Furthermore, application of the PG-binding antibody reduced proliferation and migration of hypoxic cancer cells, suggesting that facilitation of proton-coupled lactate flux by the CAIX PG domain contributes to cancer cell survival under hypoxic conditions., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Many tumor cells produce vast amounts of lactate and acid, which have to be removed from the cell to prevent intracellular lactacidosis and suffocation of metabolism. In the present study, we show that proton-driven lactate flux is enhanced by the intracellular carbonic anhydrase CAII, which is colocalized with the monocarboxylate transporter MCT1 in MCF-7 breast cancer cells. Co-expression of MCTs with various CAII mutants in Xenopus oocytes demonstrated that CAII facilitates MCT transport activity in a process involving CAII-Glu69 and CAII-Asp72, which could function as surface proton antennae for the enzyme. CAII-Glu69 and CAII-Asp72 seem to mediate proton transfer between enzyme and transporter, but CAII-His64, the central residue of the enzyme's intramolecular proton shuttle, is not involved in proton shuttling between the two proteins. Instead, this residue mediates binding between MCT and CAII. Taken together, the results suggest that CAII features a moiety that exclusively mediates proton exchange with the MCT to facilitate transport activity., Competing Interests: SN, SJ, SA, SL, JD, HB No competing interests declared, (© 2018, Noor et al.)