Structural Plasticity Allows Calumenin-1 to Moonlight as a Ca2+-Independent Chaperone: Pb2+ Enables Probing Alternate Inhibitory Conformation.

Human calumenin-1 (HsCalu-1) is an endoplasmic reticulum (ER) and Golgi-resident Ca2+-binding protein of the hepta-EF-hand superfamily that plays a vital role in maintaining the cytoplasmic Ca2+ concentration below toxic levels by interacting with Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and ryanodine receptors (RyR), indicating its role in Ca2+ homeostasis in the ER. HsCalu-1 seems to be able to exhibit structural plasticity to achieve its plethora of functions. In this study, we demonstrate that HsCalu-1 acts as a chaperone in both its intrinsically disordered state (apo form) and the structured state (Ca2+-bound form). HsCalu-1 chaperone activity is independent of Ca2+ and Pb2+ binding attenuating its chaperone-like activity. Incidentally, Pb2+ binds to HsCalu-1 with lower affinity (KD = 38.46 µM) (compared to Ca2+-binding), leading to the formation of a less-stable conformation as observed by a sharp drop in its melting temperature Tm from 67 °C in the Ca2+-bound form to 43 °C in the presence of Pb2+. The binding site for Pb2+ was mapped as being in the EF-Hand-234 domain of HsCalu-1, a region that overlaps with the Ca2+-dependent initiator of its functional fold. A change in the secondary and tertiary structure, leading to a less-stable but compact conformation upon Pb2+ binding, is the mechanism by which the chaperone-like activity of HsCalu-1 is diminished. Our results not only demonstrate the chaperone activity by a protein in its disordered state but also explain, using Pb2+ as a probe, that the multiple functions of calumenin are due to its ability to adopt a quasi-stable conformation.

Calnuc-derived nesfatin-1-like peptide is an activator of tumor cell proliferation and migration.

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.

Correction to: Structure-function relationship and physiological role of apelin and its G protein coupled receptor.

[This corrects the article DOI: 10.1007/s12551-023-01044-x.].

Gobind: an inspiring enigma.

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.

Structure-function relationship and physiological role of apelin and its G protein coupled receptor.

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 ß-arrestin-mediated signaling are delineated by multiple mutagenesis studies. There is increasing evidence that the structural requirements of APJR to activate G-proteins and ß-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.

Small peptide inhibitor from the sequence of RUNX3 disrupts PAK1-RUNX3 interaction and abrogates its phosphorylation-dependent oncogenic function.

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.

Critical APJ receptor residues in extracellular domains that influence effector selectivity.

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 ß-arrestin-mediated signaling. Mutation-dependent variation in ligand binding and signaling was observed. W197 A in ECL2 and L276 L277 W279 -AAA in ECL3 were deficient in Gi and ß-arrestin signaling pathways with relatively preserved Gq -mediated signaling. T169 T170 -AA, Y182 A, and T190 A mutants in ECL2 showed impaired ß-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.

Aberrant environment and PS-binding to calnuc C-terminal tail drives exosomal packaging and its metastatic ability.

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.

A Change in Domain Cooperativity Drives the Function of Calnuc.

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.

Molecular evolution guided functional analyses reveals Nucleobindin-1 as a canonical E-box binding protein promoting Epithelial-to-Mesenchymal transition (EMT).

Calcium binding proteins (CBPs) function in response to changes in intracellular calcium (Ca2+) levels by modulating intracellular signaling pathways. Calcium sensors, including Nucleobindins (Nucb1/2) undergo Ca2+-binding induced conformational changes and bind to target proteins. Nucleobindins possess additional uncharacterized domains including partly characterized EF-hands. We study the molecular evolution of Nucleobindins in eukaryotes emphasizing on the N-terminal DNA binding domain (DBD) that emerged as a result of domain insertion event in Nucb1/2 domain-scaffold in an ancestor to the opisthokonts. Our results from in silico analyses and functional assays revealed that DBD of Nucb1 binds to canonical E-box sequences and triggers cell epithelial-mesenchymal transition (EMT). Thus, post gene duplication, Nucb1 has emerged as unconventional Ca2+-binding transcriptional regulators that can induce EMT.

Molecular determinants on extracellular loop domains that dictate interaction between ß-arrestin and human APJ receptor.

The human APJ receptor (APJR), activated by apelin isoforms, regulates cardiovascular functions and fluid homeostasis. Understanding its structure-function relationship is crucial for a comprehensive knowledge of signalling aberrations that cause several physiological disorders. Here, we demonstrate the influence of extracellular loop (ECL) domains in the mechanism of ß-arrestin-mediated signalling from human APJR: Apelin system. Alanine mutations of evolutionarily conserved residues were characterized using receptor internalization, ß-arrestin pull-down, Akt phosphorylation and cell migration assay. C281A and 268 KTL270 -AAA in ECL3 were deficient in all assays, whereas 183 MDYS186 -AAAA mutant in ECL2 showed impaired ß-arrestin-mediated signalling but demonstrated Gi -dependent cell migration. Our findings establish that conserved residues in the extracellular domain play a prominent role in modulating receptor interactions with the ß-arrestin signalling cascade.

Chaperoning Against Amyloid Aggregation: Monitoring In Vitro and In Vivo.

Protein aggregation and inclusion body formation have been a key causal phenomenon behind a majority of neurodegenerative disorders. Various approaches aimed at preventing the formation/elimination of protein aggregates are being developed to control these diseases. Molecular chaperones are a class of protein that not only direct the functionally relevant fold of the protein but also perform quality control against stress, misfolding/aggregation. Genes that encode molecular chaperones are induced and expressed in response to extreme stress conditions to "salvage" the cell by the "unfolded protein response" (UPR) signaling pathway. Here we describe in detail the various in vitro and in vivo assays involved in identifying the chaperone activity of proteins using human calnuc as a model protein. Calnuc is a Golgi resident, calcium-binding protein, identified as chaperone protein and is reported to protect the cells against the cytotoxicity caused by amyloidosis and ER stress. Calnuc is also reported to regulate Gαi activity and inflammation apart from the role of chaperoning against amyloid proteins.

Identification and characterization of signal peptide of Mitofusin1 (Mfn1).

Mitofusin1 (Mfn1) mediates outer mitochondrial membrane (OMM) fusion in Opisthokonts. The uncharacterized TM comprises to two helices (namely, the TM1 and TM2) connected by an intermembrane loop. Consistent with previous studies, our results from in silico analyses show that all mitofusins lack N terminal-MTS and the TM may act an internal MTS. We have identified a conserved region in TM domain that is responsible for mitochondrial localization of Mfn1/2. Thus, our results suggest the dual function of TM; in OMM anchoring and signaling Mfn1 to mitochondria. Our study illuminates the underlying role of TM for mitochondrial localization of Mfn1 on one hand and also paves a way for the development of tools for in silico prediction of cellular localization of proteins.

The Differential Response to Ca2+ from Vertebrate and Invertebrate Calumenin Is Governed by a Single Amino Acid Residue.

Calumenin (Calu) is a well-conserved multi-EF-hand-containing Ca2+-binding protein. In this work, we focused on the alterations that calumenin has undergone during evolution. We demonstrate that vertebrate calumenin is significantly different from its invertebrate homologues with respect to its response to Ca2+ binding. Human calumenin (HsCalu1) is intrinsically unstructured in the Ca2+ free form and responds to Ca2+ with a dramatic gain in structure. Calumenin from Caenorhabditis elegans (CeCalu) is structured even in the apo form, with no conformational change upon binding of Ca2+. We decode this structural and functional distinction by identifying a single "Leu" residue-based switch located in the fourth EF-hand of HsCalu1, occupied by "Gly" in the invertebrate homologues. We demonstrate that replacing Leu with Gly (L150G) in HsCalu1 enables the protein to adopt a structural fold even in the Ca2+ free form, similar to CeCalu, leading to ligand compensation (adoption of structure in the absence of Ca2+). The fourth (of seven) EF-hand of HsCalu1 nucleates the structural fold of the protein depending on the switch residue (Gly or Leu). Our analyses reveal that the Leu that replaced Gly from fishes onward is absolutely conserved in higher vertebrates, while lower organisms have Gly, not only enlarging the scope of Ca2+-dependent structural transitions but also drawing a boundary between the invertebrate and vertebrate calumenin. The evolutionary selection of the switch residue strongly corroborates the change in the structure of the protein and its pleiotropic functions and seems like it can be extended to the presence or absence of a heart in that organism.

The nucleotide-free state of heterotrimeric G proteins α-subunit adopts a highly stable conformation.

Deciphering the mechanism of activation of heterotrimeric G proteins by their cognate receptors continues to be an intriguing area of research. The recently solved crystal structure of the ternary complex captured the receptor-bound α-subunit in an open conformation, without bound nucleotide has improved our understanding of the activation process. Despite these advancements, the mechanism by which the receptor causes GDP release from the α-subunit remains elusive. To elucidate the mechanism of activation, we studied guanine nucleotide-induced structural stability of the α-subunit (in response to thermal/chaotrope-mediated stress). Inherent stabilities of the inactive (GDP-bound) and active (GTP-bound) forms contribute antagonistically to the difference in conformational stability whereas the GDP-bound protein is able to switch to a stable intermediate state, GTP-bound protein loses this ability. Partial perturbation of the protein fold reveals the underlying influence of the bound nucleotide providing an insight into the mechanism of activation. An extra stable, pretransition intermediate, 'empty pocket' state (conformationally active-state like) in the unfolding pathway of GDP-bound protein mimics a gating system - the activation process having to overcome this stable intermediate state. We demonstrate that a relatively more complex conformational fold of the GDP-bound protein is at the core of the gating system. We report capturing this threshold, 'metastable empty pocket' conformation (the gate) of α-subunit of G protein and hypothesize that the receptor activates the G protein by enabling it to achieve this structure through mild structural perturbation.

Chaperone-like Activity of Calnuc Prevents Amyloid Aggregation.

Calnuc is a ubiquitously expressed protein of the EF-hand Ca2+-binding superfamily. Previous studies have implicated it in Ca2+-sensitive physiological processes, whereas details of its function and involvement in human diseases are lacking. Drawing upon the sequence homology of calnuc with calreticulin, we propose it functions as a molecular chaperone-like protein. In cells under thermal, chemical [urea and guanidinium chloride (GdmCl)], and acidic stress, calnuc exhibits properties similar to those of established chaperone-like proteins (GRP78, spectrin, and α-crystallin), effectively demonstrated by its ability to suppress aggregation of malate dehydrogenase (MDH), alcohol dehydrogenase, and catalase. Calnuc aids in refolding of MDH with retention of 80% of its enzymatic activity. In HEK293 cells subjected to heat shock, calnuc chaperones luciferase, protecting its activity. Our in vitro and cell culture results establish the ability of calnuc to inhibit fibrillation of insulin and lysozyme and validate its neuroprotective role in cells treated with amyloid fibrils. Calnuc also rescues cells from fibrillar toxicity (caused by misfolded or aggregated proteins), providing a plausible explanation for the previous observation of its low level of expression in brains affected by Alzheimer's disease. We propose that calnuc is possibly involved in controlling protein unfolding diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), prion disease, and type II diabetes.

Left-right axis asymmetry determining human Cryptic gene is transcriptionally repressed by Snail.

BACKGROUND: Establishment of the left-right axis is important for positioning organs asymmetrically in the developing vertebrate-embryo. A number of factors like maternally deposited molecules have emerged essential in initiating the specification of the axis; the downstream events, however, are regulated by signal-transduction and gene-expression changes identifying which remains a crucial challenge. The EGF-CFC family member Cryptic, that functions as a co-receptor for some TGF-beta ligands, is developmentally expressed in higher mammals and mutations in the gene cause loss or change in left-right axis asymmetry. Despite the strong phenotype, no transcriptional-regulator of this gene is known till date. RESULTS: Using promoter-analyses tools, we found strong evidence that the developmentally essential transcription factor Snail binds to the human Cryptic-promoter. We cloned the promoter-region of human Cryptic in a reporter gene and observed decreased Cryptic-promoter activation upon increasing Snail expression. Further, the expression of Cryptic is down-regulated upon exogenous Snail expression, validating the reporter assays and the previously identified role of Snail as a transcriptional repressor. Finally, we demonstrate using gel-shift assay that Snail in nuclear extract of PANC1 cells interacts with the promoter-construct bearing putative Snail binding sites and confirm this finding using chromatin immunoprecipitation assay. CONCLUSIONS: Snail represses the expression of human Cryptic and therefore, might affect the signaling via Nodal that has previously been demonstrated to specify the left-right axis using the EGF-CFC co-receptors.

Apelin binding to human APJ receptor leads to biased signaling.

BACKGROUND: Human APJ receptor (APJR), a rhodopsin family G-Protein Coupled Receptor (GPCR), activated by isoforms of peptide ligand apelin causing potent inotropic effect, is involved in cardiac function, angiogenesis and maintenance of fluid homeostasis. APJR is expressed in various organs e.g., heart, brain, kidney, muscles, etc. Hence, problems in APJR signaling lead to severe dysregulation in the pathophysiology of an organism. METHODS: Based on multiple sequence alignment of receptors from various organisms, we observe a large number of conserved residues in the extracellular side. Mutational studies including calcium mobilization, receptor internalization and ERK1/2 phosphorylation assays were performed. RESULTS: Stimulation of APJR and its mutants with apelin-13 led to mutation-dependent variation in receptor activation, intracellular Ca2+rise, and its subsequent downstream signaling. The mutant 183MDYS186-AAAA in ECL2 showed Gi-biased signaling while 268KTL270-AAA in ECL3 showed Gq biasing. C281A mutant in ECL3 was deficient in all assays. CONCLUSION: Conserved residues in the ECL2 of APJR are key for ligand binding, activation mechanism, and selective downstream signaling. Additionally, we demonstrate that Cys281 (in ECL3) mediated disulfide linkage is important for ligand recognition and receptor activation. GENERAL SIGNIFICANCE: This work explains the importance of extracellular loop domains in ligand binding, receptor activation and downstream signaling of human APJR.