Heterodimeric Protein Research Articles

Structural and evolutionary insights into the functioning of glycoprotein hormones and their receptors.

The neuroendocrine system that comprises the glycoprotein hormones (GpHs) and their receptors is essential for reproduction and metabolism. Each GpH hormone is an αβ heterodimer of cystine-knot proteins and its cognate receptor is a G-protein coupled receptor (GPCR) distinguished by a large leucine-rich-repeat (LRR) extracellular domain that binds the hormone and a class A GPCR transmembrane domain that signals through an associating heterotrimeric G protein. Hence, the receptors are called LRR-containing GPCRs-LGRs. The vertebrate GpHs and LGRs have co-evolved from homologs in the earliest metazoan animals, including sponges and comb jellies, but these are absent from unicellular organisms and plants. The two GpH subunits and accompanying LGR receptor of the nematode Caenorhabditis elegans are representative of the invertebrate evolutionary predecessors of human GpH proteins and their receptors, for example follicle-stimulating hormone (FSH) and the FSH receptor (FSHR). Atomic structures of the human GpHs and their receptors, which have been determined by X-ray crystallography and cryogenic electron microscopy (cryo-EM), inform the evolutionary process and provide a mechanistic understanding of the transmission of biochemical signals of hormone binding at the cell surface to the elicitation of second messengers such as cyclic AMP in the cytoplasm. There is compelling biochemical and cellular evidence for the importance of receptor dimers in GpH signaling in cells; yet, all of the human receptors are monomeric as defined beautifully by cryo-EM. Fortunately, the LGR of C. elegans is a stable dimer and its structure, when analyzed in the context of structural information from the human counterparts, predicts a hypothetical model for functionally relevant dimeric associations of the human GpH receptors.

Structure of an LGR dimer - an evolutionary predecessor of glycoprotein hormone receptors.

The glycoprotein hormones of humans, produced in the pituitary and acting through receptors in the gonads to support reproduction and in the thyroid gland for metabolism, have co-evolved from invertebrate counterparts 1,2 . These hormones are heterodimeric cystine-knot proteins; and their receptors bind the cognate hormone at an extracellular domain and transmit the signal of this binding through a transmembrane domain that interacts with a heterotrimeric G protein. Structures determined for the human receptors as isolated for cryogenic electron microscopy (cryo-EM) are all monomeric 3-6 despite compelling evidence for their functioning as dimers 7-10 . Here we describe the cryo-EM structure of the homologous receptor from a neuroendocrine pathway that promotes growth in a nematode 11 . This structure is an asymmetric dimer that can be activated by the hormone from that worm 12 , and it shares features especially like those of the thyroid stimulating hormone receptor (TSHR). When studied in the context of the human homologs, this dimer provides a structural explanation for the transactivation evident from functional complementation of binding-deficient and signaling-deficient receptors 7 , for the negative cooperativity in hormone action that is manifest in the 1:2 asymmetry of primary TSH:TSHR complexes 8,9 , and for switches in G-protein usage that occur as 2:2 complexes form 9,10 .

Intestinal oxygen utilisation and cellular adaptation during intestinal ischaemia-reperfusion injury.

The gastrointestinal tract can be deranged by ailments including sepsis, trauma and haemorrhage. Ischaemic injury provokes a common constellation of microscopic and macroscopic changes that, together with the paradoxical exacerbation of cellular dysfunction and death following restoration of blood flow, are collectively known as ischaemia-reperfusion injury (IRI). Although much of the gastrointestinal tract is normally hypoxemic, intestinal IRI results when there is inadequate oxygen availability due to poor supply (pathological hypoxia) or abnormal tissue oxygen use and metabolism (dysoxia). Intestinal oxygen uptake usually remains constant over a wide range of blood flows and pressures, with cellular function being substantively compromised when ischaemia leads to a>50% decline in intestinal oxygen consumption. Restoration of perfusion and oxygenation provokes additional injury, resulting in mucosal damage and disruption of intestinal barrier function. The primary cellular mechanism for sensing hypoxia and for activating a cascade of cellular responses to mitigate the injury is a family of heterodimer proteins called hypoxia-inducible factors (HIFs). The HIF system is connected to numerous biochemical and immunologic pathways induced by IRI and the concentration of those proteins increases during hypoxia and dysoxia. Activation of the HIF system leads to augmented transcription of specific genes in various types of affected cells, but may also augment apoptotic and inflammatory processes, thus aggravating gut injury. KEY POINTS: During intestinal ischaemia, mitochondrial oxygen uptake is reduced when cellular oxygen partial pressure decreases to below the threshold required to maintain normal oxidative metabolism. Upon reperfusion, intestinal hypoxia may persist because microcirculatory flow remains impaired and/or because available oxygen is consumed by enzymes, intestinal cells and neutrophils.

A synthetic protein-level neural network in mammalian cells.

Artificial neural networks provide a powerful paradigm for nonbiological information processing. To understand whether similar principles could enable computation within living cells, we combined de novo-designed protein heterodimers and engineered viral proteases to implement a synthetic protein circuit that performs winner-take-all neural network classification. This "perceptein" circuit combines weighted input summation through reversible binding interactions with self-activation and mutual inhibition through irreversible proteolytic cleavage. These interactions collectively generate a large repertoire of distinct protein species stemming from up to eight coexpressed starting protein species. The complete system achieves multi-output signal classification with tunable decision boundaries in mammalian cells and can be used to conditionally control cell death. These results demonstrate how engineered protein-based networks can enable programmable signal classification in living cells.

A novel mutation, Ile344Asn, in microsomal triglyceride transfer protein abolishes binding to protein disulfide isomerase.

Microsomal triglyceride transfer protein (MTP) plays crucial roles in the assembly and secretion of apolipoprotein B-containing lipoproteins and loss of function MTP variants are associated with abetalipoproteinemia, a disease characterized by the absence of these lipoproteins. MTP is a heterodimeric protein of two subunits, MTP and protein disulfide isomerase (PDI). In this study, we report a proband with abetalipoproteinemia who was monitored annually over a period of ten years in her third decade and had very low plasma lipids and undetectable apoB-containing lipoproteins. Genetic testing revealed biallelic variants in the MTTP gene. She has a well-documented nonsense mutation Gly865* that does not interact with the PDI subunit. She also has a novel missense MTP mutation, Ile344Asn. We show that this mutation abrogates lipid transfer activity in MTP and does not support apolipoprotein B secretion. This residue is present in the central α-helical domain of MTP and the substitution of Ile with Asn at this position disrupts interactions between MTP and PDI subunits. Ile344 is away from the known MTP:PDI interacting sites identified in the crystal structure of MTP suggesting that MTP:PDI interactions are more dynamic than previously envisioned. Identification of more missense mutations will enhance our understanding about the structure-function of MTP and the role of critical residues in these interactions between the two subunits. This knowledge may guide us in developing novel treatment modalities to reduce plasma lipids and atherosclerosis.

The Impact of a Catalytic Site Mutation on the Shape and Mechanics of Tubulin Protofilaments

Alpha-beta heterodimers of tubulin proteins serve as the building blocks of microtubules, which are key biopolymers forming one of the principal systems of the cellular cytoskeleton. A detailed study of these building blocks, as well as their alterations caused by point mutations, contributes to a deeper understanding of physiological and pathological processes related to the cytoskeleton. This study presents an analysis of the impact of the E254N point mutation in the catalytic site of α-tubulin on the bending conformations of human recombinant tubulin tetramers using molecular dynamics methods. The models were constructed based on high-resolution cryo-electron microscopy data, allowing the reconstruction of three-dimensional structures of both wild-type and mutant tetramers. The simulations revealed that the primary difference between wild-type and mutant tubulin lies in the equilibrium bending direction of the protofilaments, while the bending amplitude, twisting, and associated stiffness remain largely unchanged. We propose that the observed differences in bending directions may be related to variations in protofilament tilts within microtubules, which aligns with previously published cryo-electron microscopy data. These findings provide valuable insights into the principles underlying the formation of the polymeric structure of microtubules based on the properties of their individual building blocks.

Calcineurin-fusion facilitates cryo-EM structure determination of a Family A GPCR

Advances in singe-particle cryo-electron microscopy (cryo-EM) have made it possible to solve the structures of numerous Family A and Family B G protein-coupled receptors (GPCRs) in complex with G proteins and arrestins, as well as several Family C GPCRs. Determination of these structures has been facilitated by the presence of large extramembrane components (such as G protein, arrestin, or Venus flytrap domains) in these complexes that aid in particle alignment during the processing of the cryo-EM data. In contrast, determination of the inactive state structure of Family A GPCRs is more challenging due to the relatively small size of the seven transmembrane domain (7TM) and to the surrounding detergent micelle that, in the absence of other features, make particle alignment impossible. Here, we describe an alternative protein engineering strategy where the heterodimeric protein calcineurin is fused to a GPCR by three points of attachment, the cytoplasmic ends of TM5, TM6, and TM7. This three-point attachment provides a more rigid link with the GPCR transmembrane domain that facilitates particle alignment during data processing, allowing us to determine the structures of the β2 adrenergic receptor (β2AR) in the apo, antagonist-bound, and agonist-bound states. We expect that this fusion strategy may have broad application in cryo-EM structural determination of other Family A GPCRs.

Design, synthesis, molecular dynamics and gene silencing studies of novel therapeutic HIF-1α siRNAs in hypoxic cancer cells

Hypoxia inducible factors (HIFs) are heterodimeric proteins that belong to a small group of transcription factors, which mainly regulates transcription of genes under hypoxic conditions. Particularly, oxygen sensing subunit of HIF-1α is a predominant subtype that heterodimerizes with oxygen-independent HIF-1β subunit, to trigger the transcription of hypoxia responsive genes. Due to poor supply of blood and rapid division of cancerous cells, tumor microenvironment exhibits low oxygen condition and therefore increased levels of HIF-1α. One of the promising therapeutic strategies to cancer is modulation of HIF-1α signaling pathway. Small interfering RNA (siRNA) mediated downregulation of HIF-1α has been reported to prevent growth and progression of various types of cancer and holds great promise in the cancer treatment. In this study, computational approaches were used to design potential siRNAs targeting HIF-1α and investigate their interaction with the human argonaute-2 (hAgo2). Molecular dynamic simulation of HIF-1α siRNAs-hAgo2 complexes revealed key interactions required for the efficient binding of guide strand to hAgo2 protein. Two siRNAs (S2 and S5) exhibiting strong binding with hAgo2 were further considered. Subsequently, we transfected the MCF-7 cell line with both standard HIF-1α and our designed siRNAs (S2 and S5). Following transfection, translation changes in the MCF-7 cells were assessed through western blotting. S2 and S5 efficiently reduced the expression of HIF-1α in hypoxic conditions. The aim of the present study is to understand the siRNA-hAgo2 interaction. It is also focused on the desiging of effective siRNA against HIF-1α.

Scalable protein design using optimization in a relaxed sequence space.

Machine learning (ML)-based design approaches have advanced the field of de novo protein design, with diffusion-based generative methods increasingly dominating protein design pipelines. Here, we report a "hallucination"-based protein design approach that functions in relaxed sequence space, enabling the efficient design of high-quality protein backbones over multiple scales and with broad scope of application without the need for any form of retraining. We experimentally produced and characterized more than 100 proteins. Three high-resolution crystal structures and two cryo-electron microscopy density maps of designed single-chain proteins comprising up to 1000 amino acids validate the accuracy of the method. Our pipeline can also be used to design synthetic protein-protein interactions, as validated experimentally by a set of protein heterodimers. Relaxed sequence optimization offers attractive performance with respect to designability, scope of applicability for different design problems, and scalability across protein sizes.

DNA fragmentation factor 40-based therapeutic approaches for cancer: a review article.

DNA Fragmentation Factor (DFF) is a heterodimer protein involved in DNA fragmentation during apoptosis, which acts as a trigger downstream of caspase-3 activation. DFF40 catalytically active homo-oligomers break down chromosomal DNA. Previous scientific investigations have revealed a link between DFF40 expression changes and various cancers. DFF40 deletion or down-regulation has been observed in some cancers. Consequently, therapeutic strategies involving the DFF40 molecule compensating led to an increased rate of cancer cell apoptosis. In this review article, we aimed to introduce cancers with low expression of this protein first. The second part of this paper focuses on studies that utilized exogenous DFF40 protein produced by recombinant DNA technology and surveyed during in vitro and in vivo tests. Finally, compensation for diminished expression of the mentioned protein via gene therapy-based techniques to make up for this apoptotic molecule's low expression is the topic of thelast part of this review article.

The relationship between interleukin 1-B and calprotecin in periodontal Disease

Backgrounds: One of the most common diseases affecting the oral cavity in humans is periodontitis. Researchers looked at the effects of calprotectin and IL-1B in human gingival Among the cytokines involved in the pathogenesis of periodontal disease, IL-1β, an inflammatory cytokine, can be highlighted for its contribution to stimulating the recruitment and differentiation of osteoblasts in tissues. Calprotectin is a heterodimer protein consisting of two calcium-bindinglinkages, belonging to the S-100 family.Materials and method: Ninety samples (patient and control) were obtained for the study between July 2023 and December 2024. Of them, sixty samples came from patients who saw periodontists in outpatient clinics and at Balad General Hospital. A sterile cotton swab (transport cotton swab) was used to obtain the sample. Using a syringe and a 5-milliliter Slim syringe, blood was drawn for 79 medical devices and then put into gelatinous gel tubes. The blood was then allowed to clot for fifteen minutes, sediment on the central computer at 3500 for one minute, and the serum was separated and kept in Eppendorf tubes with a 2-milliliter top at room temperature. The bacterial isolates were identified using physiological and biochemical assays, and the sample was held for 20 AD before being employed in the decimal filter. immunological examination The levels of immunological proteins calprotectin and human interleukin (IL-1β) were measured using ELISA technique. Results: IL-1B level in patients with Periodontitis recorded that (50.3±26.7) in comparison with control group that were (49.8±21.8,9.85±4.28) .While calprotectin level in patients with Periodontitis recorded that (10.79±2.69) in comparison with control group that were (9.85±4.28). Conclusion: Most common causes of Periodontitis was Prophyromonas gingivalis ,Staph. Aureus, Strep. Pyogenes. functional polymorphisms in the IL-1 B were not related to the calprotectin in development in Periodontitis prevalent diseases.

The prodomain of bone morphogenetic protein 2 promotes dimerization and cleavage of BMP6 homodimers and BMP2/6 heterodimers

Bone morphogenetic protein 2 (BMP2) and BMP6 are key regulators of systemic iron homeostasis. All BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments, but nothing is known about how BMP2 or BMP6 homodimeric or heterodimeric precursor proteins are proteolytically activated. Here, we conducted in vitro cleavage assays, which revealed that BMP2 is sequentially cleaved by furin at two sites, initially at a site upstream of the mature ligand, and then at a site adjacent to the ligand domain, while BMP6 is cleaved at a single furin motif. Cleavage of both sites of BMP2 is required to generate fully active BMP2 homodimers when expressed in Xenopus embryos or liver endothelial cells, and fully active BMP2/6 heterodimers in Xenopus. We analyzed BMP activity in Xenopus embryos expressing chimeric proteins consisting of the BMP2 prodomain and BMP6 ligand domain, or vice versa. We show that the prodomain of BMP2 is necessary and sufficient to generate active BMP6 homodimers and BMP2/6 heterodimers, whereas the BMP6 prodomain cannot generate active BMP2 homodimers or BMP2/6 heterodimers. We examined BMP2 and BMP6 homodimeric and heterodimeric ligands generated from native and chimeric precursor proteins expressed in Xenopus embryos. Whereas native BMP6 is not cleaved when expressed alone, it is cleaved to generate BMP2/6 heterodimers when co-expressed with BMP2. Furthermore, BMP2-6 chimeras are cleaved to generate BMP6 homodimers. Our findings reveal an important role for the BMP2 prodomain in dimerization and proteolytic activation of BMP6.

Serum Calprotectin Level in Type 2 Diabetic Patients with and without Diabetic Peripheral Neuropathy: A Comparison Study

Abstract Background: Peripheral neuropathy is one of the microvascular complications that affects patients with diabetes mellitus and involves both sensory and motor nerves. The development and the progress of diabetic peripheral neuropathy (DPN) were ascribed to the inflammatory activity of the immune cells in the nerves. Calprotectin (CLP) is a heterodimer protein found in the membranes of monocytes and other inflammatory cells and the cytosol of neutrophils and released from them up to activation. Aim of Study: Evaluation of serum CLP level as a potential inflammatory biomarker for the occurrence of DPN in type 2 diabetic patients. Patients and Methods: one hundred and twenty-six patients diagnosed with type 2 diabetes mellitus were randomly selected from those who attended the National Diabetic Center between December 2022 and July 2023. Michigan Neuropathy Screening Instrument (MNSI) and nerve conduction study (NCS) were used for grouping the patients. Enzyme-linked immunosorbent assay technique has been used to measure serum CLP levels. Results: Serum levels of CLP showed no significant differences among patients with and those without diabetic peripheral neuropathy according to their NCS findings and the total scores for each and for both in combination according to the subgroups (P > 0.05). Conclusions and Recommendations: The serum level of CLP in type 2 diabetic patients was not affected by the occurrence of diabetic peripheral neuropathy. Further studies are required on newly diagnosed patients and a larger sample size.

Differential interference with actin-binding protein function by acute Cytochalasin B.

Dynamic actin filament remodeling is crucial for a plethora of fundamental cell biological processes, ranging from cell division and migration to cell communication, intracellular trafficking or tissue development. Cytochalasin B and -D are fungal secondary metabolites frequently used for interference with such processes. Although generally assumed to block actin filament polymerization at their rapidly growing barbed ends and compete with regulators at these sites, our molecular understanding of their precise effects in dynamic actin structures is scarce. Here we combine live cell imaging and analysis of fluorescent actin-binding protein dynamics with acute treatment of lamellipodia in migrating cells with cytochalasin B. Our results show that in spite of an abrupt halt of lamellipodium protrusion, cytochalasin B affects various actin filament barbed end-binding proteins in a differential fashion. Cytochalasin B enhances instead of diminishes the accumulation of prominent barbed end-binding factors such as Ena/VASP family proteins and heterodimeric capping protein (CP) in the lamellipodium. Similar results were obtained with cytochalasin D. All these effects are highly specific, as cytochalasin-induced VASP accumulation requires the presence of CP, but not vice versa , and coincides with abrogation of both actin and VASP turnover. Cytochalasin B can also increase apparent barbed end interactions with the actin-binding β-tentacle of CP and partially mimic its Arp2/3 complex-promoting activity in the lamellipodium. In conclusion, our results reveal a new spectrum of cytochalasin activities on barbed end-binding factors, with important implications for the interpretation of their effects on dynamic actin structures.

Broad-spectrum coronavirus neutralization induced by hetero RBD-Fc protein vaccine.

In the landscape of infectious diseases, human coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2 pose significant threats, characterized by severe respiratory illnesses and notable resistance to conventional treatments due to their rapid evolution and the emergence of diverse variants, particularly within SARS-CoV-2. This study investigated the development of broad-spectrum coronavirus vaccines using heterodimeric RBD-Fc proteins engineered through the "Knob-into-Hole" technique. We constructed various recombinant proteins incorporating the receptor-binding domains (RBDs) of different coronaviruses. Heterodimers combining RBDs from SARS-CoV-2 with those of SARS-CoV or MERS-CoV elicited superior neutralizing responses compared to homodimeric proteins in murine models. Additionally, heterotetrameric proteins, specifically D614G_Delta/BA.1_XBB.1.5-RBD and MERS_D614G/BA.1_XBB.1.5-RBD, elicited remarkable breadth and potency in neutralizing all known SARS-CoV-2 variants, SARS-CoV, related sarbecoviruses like GD-Pangolin and WIV1, and even MERS-CoV pseudoviruses. Furthermore, these heterotetrameric proteins also demonstrated enhanced cellular immune responses. These findings underscore the potential of recombinant hetero proteins as a universal vaccine strategy against current and future coronavirus threats.

The Id protein Extramacrochaetae restrains the E protein Daughterless to regulate Notch, Rap1, and Sevenless within the R7 equivalence group of the Drosophila eye.

The Drosophila Id gene extramacrochaetae (emc) is required during Drosophila eye development for proper cell fate specification within the R7 equivalence group. Without emc, R7 cells develop like R1/6 cells, and there are delays and deficits in differentiation of non-neuronal cone cells. Although emc encodes an Inhibitor of DNA-binding (Id) protein that is known to antagonize proneural bHLH protein function, no proneural gene is known for R7 or cone cell fates. These fates are also independent of daughterless (da), which encodes the ubiquitous E protein heterodimer partner of proneural bHLH proteins. We report here that the effects of emc mutations disappear in the absence of da, and are partially mimicked by forced expression of Da dimers, indicating that emc normally restrains da from interfering with R7 and cone cell specification, as occurs in emc mutants. emc, and da, regulate three known contributors to R7 fate, which are Notch signaling, Rap1, and Sevenless. R7 specification is partially restored to emc mutant cells by mutation of RapGap1, confirming that Rap1 activity, in addition to Notch activity, is a critical target of emc. These findings exemplify how mutations of an Id protein gene can affect processes that do not require any bHLH protein, by restraining Da activity within physiological bounds.

Computational structural prediction and chemical inhibition of the human mitochondrial pyruvate carrier protein heterodimer complex.

The mitochondrial pyruvate carrier (MPC) plays a role in numerous diseases including neurodegeneration, metabolically dependent cancers, and the development of insulin resistance. Several previous studies in genetic mouse models or with existing inhibitors suggest that inhibition of the MPC could be used as a viable therapeutic strategy in these diseases. However, the MPC structure is unknown, making it difficult to screen for and develop therapeutically viable inhibitors. Currently known MPC inhibitors would make for poor drugs due to their poor pharmacokinetic properties, or in the case of the thiazolidinediones (TZDs), off-target specificity for peroxisome-proliferator activated receptor gamma (PPARgamma) leads to unwanted side effects. In this study, we develop several structural models for the MPC heterodimer complex and investigate the chemical interactions required for the binding of these known inhibitors to MPC and PPARgamma. Based on these models, the MPC most likely takes on outward-facing (OF) and inward-facing (IF) conformations during pyruvate transport, and inhibitors likely plug the carrier to inhibit pyruvate transport. Although some chemical interactions are similar between MPC and PPARgamma binding, there is likely enough difference to reduce PPARgamma specificity for future development of novel, more specific MPC inhibitors.

New Benzylidene‐Triazole Tethered Coumarin Molecular Hybrids: Synthesis, SAR, Molecular Modelling Simulations Targeting Tubulin α/β Dimer and its Antimicrobial Evaluation

AbstractA series of new 7‐{3‐[3‐(Benzylidene‐amino)‐[1,2,4]triazole‐1‐yl]‐propoxy}‐4‐methyl‐chromen‐2‐one molecular hybrids 7(a–k) were synthesized using molecular hybridization approach. The synthesized new benzylidene‐triazole tethered coumarin molecular hybrids were characterized by spectroscopic studies and elemental analysis. The coumarin molecular hybrids have been explored for their antifungal efficacy in terms of tubulin inhibitors using molecular modelling studies against tubulin alphabeta hetero dimer protein. The results clearly demonstrated strong binding affinity of coumarin molecular hybrids with indole substituted and 2,4‐dihydroxy substituted as antagonists with respective binding scores of −10.5 and −10.7 kcal/mol towards tubulin alphabeta hetero dimer protein which was higher in comparison to standards griseofulvin (−8.2 kcal/mol) and fluconazole (−8.1 kcal/mol). In addition, broth dilution method was used to conduct in‐vitro antifungal studies against three fungal strains namely Candida albicans, Aspergillus niger and Aspergillus fumigatus. The results obtained clearly illustrate the antifungal activity of indole and 2,4‐dihydroxy substituted compounds which are consistent with docking simulations. Out of all the synthesized molecular hybrids, indole and 2,4‐dihydroxy substituted compounds depict outstanding antifungal activity with Minimum Inhibitory Concentration (MIC) value of 31.25 μg/ml and 62.5 μg/ml towards C.albicans and with MIC value of 31.25 for both these molecules towards A.niger in each case which is quite higher than griseofulvin and fluconazole respectively. Present work clearly demonstrates the potential efficacy of synthesized indole and 2,4‐dihydroxy substituted coumarin molecular hybrids to be considered as lead compounds for the development of new agents against resistant strains for the treatment of fungal infection.

Zebrafish do not have calprotectin.

The protein heterodimer calprotectin and its component proteins, S100A8 and S100A9, play important antibacterial and proinflammatory roles in the mammalian innate immune response. Gaining mechanistic insights into the regulation and biological function of calprotectin will help facilitate patient diagnostics and therapy and further our understanding of the host-microbe interface. Recent literature has identified zebrafish s100a10b as zebrafish calprotectin based on sequence similarity, genomic context, and transcriptional upregulation during the immune response to bacterial infections. The field would benefit from expanding the breadth of calprotectin studies into a zebrafish innate immunity model. Here, we carefully evaluated the possibility that zebrafish possess a calprotectin. We found that zebrafish do not possess an ortholog of mammalian S100A8 or S100A9. We then identified four zebrafish s100 proteins- including s100a10b-that are expressed in immune cells and upregulated during the immune response. We recombinantly expressed and purified these proteins and measured the antimicrobial and proinflammatory characteristics. We found that none of the zebrafish proteins exhibited activity comparable to mammalian calprotectin. Our work demonstrates conclusively that zebrafish have no ortholog of calprotectin, and the most plausible candidate proteins have not convergently evolved similar functions.

The BMP2 prodomain promotes dimerization and cleavage of BMP6 homodimers and BMP2/6 heterodimers in vivo.

Bone morphogenetic protein 2 (BMP2) and BMP6 are key regulators of systemic iron homeostasis. All BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments, but nothing is known about how BMP2 or BMP6 homodimeric or heterodimeric precursor proteins are proteolytically activated. Here, we conducted in vitro cleavage assays, which revealed that BMP2 is sequentially cleaved by furin at two sites, initially at a site upstream of the mature ligand, and then at a site adjacent to the ligand domain, while BMP6 is cleaved at a single furin motif. Cleavage of both sites of BMP2 is required to generate fully active BMP2 homodimers when expressed in Xenopus embryos or liver endothelial cells, and fully active BMP2/6 heterodimers in Xenopus . We analyzed BMP activity in Xenopus embryos expressing chimeric proteins consisting of the BMP2 prodomain and BMP6 ligand domain, or vice versa. We show that the prodomain of BMP2 is necessary and sufficient to generate active BMP6 homodimers and BMP2/6 heterodimers, whereas the BMP6 prodomain cannot generate active BMP2 homodimers or BMP2/6 heterodimers. We examined BMP2 and BMP6 homodimeric and heterodimeric ligands generated from native and chimeric precursor proteins expressed in Xenopus embryos. Whereas native BMP6 is not cleaved when expressed alone, it is cleaved to generate BMP2/6 heterodimers when co-expressed with BMP2. Furthermore, BMP2-6 chimeras are cleaved to generate BMP6 homodimers. Our findings reveal an important role for the BMP2 prodomain in dimerization and proteolytic activation of BMP6.