A Gopala Krishna

In this article, Figure 1 was published with incorrect image. The corrected Figure 1 is shown here. (Figure presented.) a Representation of crystal structure of APJR-AMG3054 complex (PDB: 5VBL). The modified peptide ligand is depicted in green (sticks) and the receptor is shown in orange (cartoon). b Amino acid sequence of apelin peptide isoforms. Residues of Apelin-36, Apelin-17, Apelin-13, and Pyro-glutamate (Pyr) Apelin-13 are indicated. Grey arrows show the site of cleavage by PCSK3/furin enzyme in the larger isoforms, Apelin-36 and Apelin-17 c Comparison between Apelin-17 and AMG3054. The latter is a modified cyclic peptide (cyclised between Glu10 and Lys13, indicated by dotted lines). The modified residues of AMG3054 are coloured in pink. hARG, homoarginine; CHA, cyclohexylalanine; OIC, Octahydroindole-2-carboxylic Acid; NLE, Norleucine; 4-Cl-PHE, 4-choloro-Phenylalanine The original article has been corrected.

P21 Activated Kinase 1 (PAK1) is an oncogenic serine/threonine kinase known to play a significant role in the regulation of cytoskeleton and cell morphology. Runt-related transcription factor 3 (RUNX3) was initially known for its tumor suppressor function, but recent studies have reported the oncogenic role of RUNX3 in various cancers. Previous findings from our laboratory provided evidence that Threonine 209 phosphorylation of RUNX3 acts as a molecular switch in dictating the tissue-specific dualistic functions of RUNX3 for the first time. Based on these proofs and to explore the translational significance of these findings, we designed a small peptide (RMR) from the protein sequence of RUNX3 flanking the Threonine 209 phosphorylation site. The selection of this specific peptide from multiple possible peptides was based on their binding energies, hydrogen bonding, docking efficiency with the active site of PAK1 and their ability to displace PAK1–RUNX3 interaction in our prediction models. We found that this peptide is stable both in in vitro and in vivo conditions, not toxic to normal cells and inhibits the Threonine 209 phosphorylation in RUNX3 by PAK1. We also tested the efficacy of this peptide to block the RUNX3 Threonine 209 phosphorylation mediated tumorigenic functions in in vitro cell culture models, patient-derived explant (PDE) models and in in vivo tumor xenograft models. These results proved that this peptide has the potential to be developed as an efficient therapeutic molecule for targeting RUNX3 Threonine 209 phosphorylation-dependent tumor phenotypes.

Apelin receptor (APJR) is a class A peptide (apelin) binding G protein-coupled receptor (GPCR) that plays a significant role in regulating blood pressure, cardiac output, and maintenance of fluid homeostasis. It is activated by a wide range of endogenous peptide isoforms of apelin and elabela. The apelin peptide isoforms contain distinct structural features that aid in ligand recognition and activation of the receptor. Site-directed mutagenesis and structure-based studies have revealed the involvement of extracellular and transmembrane regions of the receptor in binding to the peptide isoforms. The structural features of APJR activation of the receptor as well as mediating G-protein and β-arrestin-mediated signaling are delineated by multiple mutagenesis studies. There is increasing evidence that the structural requirements of APJR to activate G-proteins and β-arrestins are different, leading to biased signaling. APJR also responds to mechanical stimuli in a ligand-independent manner. A multitude of studies has focused on developing both peptide and non-peptide agonists and antagonists specific to APJR. Apelin/elabela-activated APJR orchestrates major signaling pathways such as extracellular signal-regulated kinase (ERKs), protein kinase B (PKB/Akt), and p70S. This review focuses on the structural and functional characteristics of apelin, elabela, APJR, and their interactions involved in the binding and activation of the downstream signaling cascade. We also focus on the diverse signaling profile of APJR and its ligands and their involvement in various physiological systems.

The characteristic features of cancer cells are aberrant (acidic) intracellular pH and elevated levels of phosphatidylserine. The primary focus of cancer research is concentrated on the discovery of biomarkers directed towards early diagnosis and therapy. It has been observed that azoxymethane-treated mice demonstrate an increased expression of calnuc (a multi-domain, Ca2+- and DNA-binding protein) in their colon, suggesting it to be a good biomarker of carcinogenesis. We show that culture supernatants from tumor cells have significantly higher amounts of secreted calnuc compared to non-tumor cells, selectively packaged into exosomes. Exosomal calnuc is causal for epithelial-mesenchymal transition and atypical migration in non-tumor cells, which are key events in tumorigenesis and metastasis. In vitro studies reveal a significant affinity for calnuc towards phosphatidylserine, specifically to its C-terminal region, leading to the formation of 'molten globule' conformation. Similar structural changes are observed at acidic pH (pH 4), which demonstrates the role of the acidic microenvironment in causing the molten globule conformation and membrane interaction. On a precise note, we propose that the molten globule structure of calnuc caused by aberrant conditions in cancer cells to be the causative mechanism underlying its exosome-mediated secretion, thereby driving metastasis.

With the increasing incidence of neurodegenerative disorders, there is an urgent need to understand the protein folding process. Examining the folding process of multidomain proteins remains a prime challenge, as their complex conformational dynamics make them highly susceptible to misfolding and/or aggregation. The presence of multiple domains in a protein can lead to interaction between the partially folded domains, thereby driving misfolding and/or aggregation. Calnuc is one such multidomain protein for which Ca2+ binding plays a pivotal role in governing its structural dynamics and stability and, presumably, in directing its interactions with other proteins. We demonstrate differential structural dynamics between the Ca2+-free and Ca2+-bound forms of calnuc. In the absence of Ca2+, full-length calnuc displays equilibrium structural transitions with four intermediate states, reporting a sum of the behavioral properties of its individual domains. Fragment-based studies illustrate the sequential events of structure adoption proceeding in the following order: EF domain followed by the NT and LZ domains in the apo state. On the other hand, Ca2+ binding increases domain cooperativity and enables the protein to fold as a single unit. Single-tryptophan mutant proteins, designed in a domain-dependent manner, confirm an increase in the number of interdomain interactions in the Ca2+-bound form as compared to the Ca2+-free state of the protein, thereby providing insight into its folding process. The attenuated domain crosstalk in apo-calnuc is likely to influence and regulate its physiologically important intermolecular interactions.

Human APJ receptor/apelin receptor (APJR), activated by apelin peptide isoforms, regulates a wide range of physiological processes. The role of extracellular loop (ECL) domain residues of APJR in ligand binding and receptor activation has not been established yet. Based on multiple sequence alignment of APJ receptor from various organisms, we identified conserved residues in the extracellular domains. Alanine substitutions of specific residues were characterized to evaluate their ligand binding efficiency and Gq-, Gi-, and β-arrestin-mediated signaling. Mutation-dependent variation in ligand binding and signaling was observed. W197A in ECL2 and L276L277W279-AAA in ECL3 were deficient in Gi and β-arrestin signaling pathways with relatively preserved Gq-mediated signaling. T169T170-AA, Y182A, and T190A mutants in ECL2 showed impaired β-arrestin-dependent cell signaling while maintaining G protein-mediated signaling. Structural comparison with angiotensin II type I receptor revealed the importance of ECL2 and ECL3 residues in APJR ligand binding and signaling. Our results unequivocally confirm the specific role of these ECL residues in ligand binding and in orchestrating receptor conformations that are involved in preferential/biased signaling functions.

Gobind Khorana’s distinguished career spanned nearly six decades (1952–2011). His work resulted in remarkable achievements starting with the complicated synthesis of coenzyme A. He then pioneered the synthesis of DNA oligonucleotides, which enabled him to crack the genetic code. Using this experience, he ventured to accomplish the first complete synthesis of a gene. Not satisfied with elucidating the function of bacteriorhodopsin, Gobind took up another greater challenge, that of spearheading studies on visual rhodopsin, its mechanism of activation, and the consequent signal transduction pathway. This Editorial acts to introduce the articles appearing in this Issue Focus dedicated to celebrating the 100th anniversary of the year of his birth.

Calnuc (nucleobindin-1, nucb1) is a Ca2+-binding protein involved in the etiology of many human diseases. To understand the functions of calnuc, we have identified a nesfatin-1-like peptide (NLP) in its N terminus that is proteolyzed by a convertase enzyme in the secretory granules of cells. Mutational studies confirm the presence of a proteolytic cleavage site for proprotein convertase subtilisin/kexin type 1 (PCSK1). We demonstrate that NLP regulates Gαq-mediated intracellular Ca2+ dynamics, likely via a G-protein-coupled receptor. NLP treatment to carcinoma cell lines (SCC131 cells) promotes the expression of regulators of cell cycle, proliferation, and clonogenicity by the AKT/mTOR pathway. NLP is causative of augmented migration and epithelial–mesenchymal transition (EMT), illustrating its metastatic propensity and establishing its tumor promotion ability.