Protein Family Research Articles

Cis inhibition of co-expressed DIPs and Dprs shapes neural development.

In Drosophila , two interacting adhesion protein families, Dprs and DIPs, coordinate the assembly of neural networks. While intercellular DIP/Dpr interactions have been well characterized, DIPs and Dprs are often co-expressed within the same cells, raising the question as to whether they also interact in cis . We show, in cultured cells and in vivo, that DIP-α and DIP-δ can interact in cis with their ligands, Dpr6/10 and Dpr12, respectively. When co-expressed in cis with their cognate partners, these Dprs regulate the extent of trans binding, presumably through competitive cis interactions. We demonstrate the neurodevelopmental effects of cis inhibition in fly motor neurons and in the mushroom body. We further show that a long disordered region of DIP-α at the C-terminus is required for cis but not trans interactions, likely because it alleviates geometric constraints on cis binding. Thus, the balance between cis and trans interactions plays a role in controlling neural development.

Deficiency of CAMSAP2 impairs olfaction and the morphogenesis of mitral cells

In developing olfactory bulb (OB), mitral cells (MCs) remodel their dendrites to establish the precise olfactory circuit, and these circuits are critical for individuals to sense odors and elicit behaviors for survival. However, how microtubules (MTs) participate in the process of dendritic remodeling remains elusive. Here, we reveal that calmodulin-regulated spectrin-associated proteins (CAMSAPs), a family of proteins that bind to the minus-end of the noncentrosomal MTs, play a crucial part in the development of MC dendrites. We observed that Camsap2 knockout (KO) males are infertile while the reproductive tract is normal. Further study showed that the infertility was due to the severe defects of mating behavior in male mice. Besides, mice with loss-of-function displayed defects in the sense of smell. Furthermore, we found that the deficiency of CAMSAP2 impairs the classical morphology of MCs, and the CAMSAP2-dependent dendritic remodeling process is responsible for this defect. Thus, our findings demonstrate that CAMSAP2 plays a vital role in regulating the development of MCs.

Sequence-developability mapping of affibody and fibronectin paratopes via library-scale variant characterization.

Protein developability is requisite for use in therapeutic, diagnostic, or industrial applications. Many developability assays are low throughput, which limits their utility to the later stages of protein discovery and evolution. Recent approaches enable experimental or computational assessment of many more variants, yet the breadth of applicability across protein families and developability metrics is uncertain. Here, three library-scale assays-on-yeast protease, split green fluorescent protein (GFP), and non-specific binding-were evaluated for their ability to predict two key developability outcomes (thermal stability and recombinant expression) for the small protein scaffolds affibody and fibronectin. The assays' predictive capabilities were assessed via both linear correlation and machine learning models trained on the library-scale assay data. The on-yeast protease assay is highly predictive of thermal stability for both scaffolds, and the split-GFP assay is informative of affibody thermal stability and expression. The library-scale data was used to map sequence-developability landscapes for affibody and fibronectin binding paratopes, which guides future design of variants and libraries.

The monodomain Kunitz protein EgKU-7 from the dog tapeworm Echinococcus granulosus is a high-affinity trypsin inhibitor with two interaction sites.

Typical Kunitz proteins (I2 family of the MEROPS database, Kunitz-A family) are metazoan competitive inhibitors of serine peptidases that form tight complexes of 1:1 stoichiometry, mimicking substrates. The cestode Echinococcus granulosus, the dog tapeworm causing cystic echinococcosis in humans and livestock, encodes an expanded family of monodomain Kunitz proteins, some of which are secreted to the dog host interface. The Kunitz protein EgKU-7 contains, in addition to the Kunitz domain with the anti-peptidase loop comprising a critical arginine, a C-terminal extension of ∼20 amino acids. Kinetic, electrophoretic, and mass spectrometry studies using EgKU-7, a C-terminally truncated variant, and a mutant in which the critical arginine was substituted by alanine, show that EgKU-7 is a tight inhibitor of bovine and canine trypsins with the unusual property of possessing two instead of one site of interaction with the peptidases. One site resides in the anti-peptidase loop and is partially hydrolyzed by bovine but not canine trypsins, suggesting specificity for the target enzymes. The other site is located in the C-terminal extension. This extension can be hydrolyzed in a particular arginine by cationic bovine and canine trypsins but not by anionic canine trypsin. This is the first time to our knowledge that a monodomain Kunitz-A protein is reported to have two interaction sites with its target. Considering that putative orthologs of EgKU-7 are present in other cestodes, our finding unveils a novel piece in the repertoire of peptidase-inhibitor interactions and adds new notes to the evolutionary host-parasite concerto.

Structure and topography of the synaptic V-ATPase-synaptophysin complex.

Synaptic vesicles are organelles with a precisely defined protein and lipid composition1,2, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here, we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains3. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the protein gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters4,5. Synaptophysin6 and its paralogs synaptoporin7 and synaptogyrin8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin.

A General Mechanism for the General Stress Response in Bacteria.

The General Stress Response promotes survival of bacteria in adverse conditions, but how sensor proteins transduce species-specific signals to initiate the response is not known. The serine/threonine phosphatase RsbU initiates the General Stress Response in B. subtilis upon binding a partner protein (RsbT) that is released from sequestration by environmental stresses. We report that RsbT activates RsbU by inducing otherwise flexible linkers of RsbU to form a short coiled-coil that dimerizes and activates the phosphatase domains. Importantly, we present evidence that related coiled-coil linkers and phosphatase dimers transduce signals from diverse sensor domains to control the General Stress Response and other signaling across bacterial phyla. These results additionally resolve the mystery of how shared sensory domains control serine/threonine phosphatases, diguanylate cyclases and histidine kinases, revealing a common coiled-coil linker transduction mechanism. We propose that this provides bacteria with a modularly exchangeable toolkit for the evolution of diverse signaling pathways.

Navigating the Maze of Kinases: CaMK-like Family Protein Kinases and Their Role in Atherosclerosis.

Circulating low-density lipoprotein (LDL) levels are a major risk factor for cardiovascular diseases (CVD), and even though current treatment strategies focusing on lowering lipid levels are effective, CVD remains the primary cause of death worldwide. Atherosclerosis is the major cause of CVD and is a chronic inflammatory condition in which various cell types and protein kinases play a crucial role. However, the underlying mechanisms of atherosclerosis are not entirely understood yet. Notably, protein kinases are highly druggable targets and represent, therefore, a novel way to target atherosclerosis. In this review, the potential role of the calcium/calmodulin-dependent protein kinase-like (CaMKL) family and its role in atherosclerosis will be discussed. This family consists of 12 subfamilies, among which are the well-described and conserved liver kinase B1 (LKB1) and 5' adenosine monophosphate-activated protein kinase (AMPK) subfamilies. Interestingly, LKB1 plays a key role and is considered a master kinase within the CaMKL family. It has been shown that LKB1 signaling leads to atheroprotective effects, while, for example, members of the microtubule affinity-regulating kinase (MARK) subfamily have been described to aggravate atherosclerosis development. These observations highlight the importance of studying kinases and their signaling pathways in atherosclerosis, bringing us a step closer to unraveling the underlying mechanisms of atherosclerosis.

Comparative analysis of Bcl-2 family protein overexpression in CAR T cells alone and in combination with BH3 mimetics.

Approximately 50% of patients with hematologic malignancies relapse after chimeric antigen receptor (CAR) T cell treatment; mechanisms of failure include loss of CAR T persistence and tumor resistance to apoptosis. We hypothesized that both of these challenges could potentially be overcome by overexpressing one or more of the Bcl-2 family proteins in CAR T cells to reduce their susceptibility to apoptosis, both alone and in the presence of BH3 mimetics, which can be used to activate apoptotic machinery in malignant cells. We comprehensively investigated overexpression of different Bcl-2 family proteins in CAR T cells with different signaling domains as well as in different tumor types. We found that Bcl-xL and Bcl-2 overexpression in CAR T cells bearing a 4-1BB costimulatory domain resulted in increased expansion and antitumor activity, reduced exhaustion, and decreased apoptotic priming. In addition, CAR T cells expressing either Bcl-xL or a venetoclax-resistant Bcl-2 variant led to enhanced antitumor efficacy and survival in murine xenograft models of lymphoma and leukemia in the presence or absence of the BH3 mimetic venetoclax, a clinically approved BH3 mimetic. In this setting, Bcl-xL overexpression had stronger effects than overexpression of Bcl-2 or the Bcl-2(G101V) variant. These findings suggest that CAR T cells could be optimally engineered by overexpressing Bcl-xL to enhance their persistence while opening a therapeutic window for combination with BH3 mimetics to prime tumors for apoptosis.

Combination of polymeric micelle formulation of TGFβ receptor inhibitors and paclitaxel produces consistent response across different mouse models of Triple‐negative breast cancer

AbstractTriple‐negative breast cancer (TNBC) is notoriously difficult to treat due to the lack of targetable receptors and sometimes poor response to chemotherapy. The transforming growth factor beta (TGFβ) family of proteins and their receptors (TGFRs) are highly expressed in TNBC and implicated in chemotherapy‐induced cancer stemness. Here, we evaluated combination treatments using experimental TGFR inhibitors (TGFβi), SB525334 (SB), and LY2109761 (LY) with paclitaxel (PTX) chemotherapy. These TGFβi target TGFR‐I (SB) or both TGFR‐I and TGFR‐II (LY). Due to the poor water solubility of these drugs, we incorporated each of them in poly(2‐oxazoline) (POx) high‐capacity polymeric micelles (SB‐POx and LY‐POx). We assessed their anticancer effect as single agents and in combination with micellar PTX (PTX‐POx) using multiple immunocompetent TNBC mouse models that mimic human subtypes (4T1, T11‐Apobec and T11‐UV). While either TGFβi or PTX showed a differential effect in each model as single agents, the combinations were consistently effective against all three models. Genetic profiling of the tumors revealed differences in the expression levels of genes associated with TGFβ, epithelial to mesenchymal transition (EMT), TLR‐4, and Bcl2 signaling, alluding to the susceptibility to specific gene signatures to the treatment. Taken together, our study suggests that TGFβi and PTX combination therapy using high‐capacity POx micelle delivery provides a robust antitumor response in multiple TNBC subtype mouse models.

PGK1 Is Involved in the HIF-1 Signaling Pathway as a Hub Gene for Ferroptosis After Traumatic Brain Injury.

The pathogenesis of ferroptosis in traumatic brain injury (TBI) is unclear; therefore, we aimed to identify key molecules associated with ferroptosis in TBI using bioinformatics analysis to determine its underlying mechanisms. GSE128543 dataset was downloaded from the Gene Expression Omnibus (GEO) database, and TBI-associated modules were obtained by weighted gene co-expression network analysis (WGCNA). We identified 60 differentially expressed genes (DEGs) by intersecting the modules with ferroptosis and glycolysis/gluconeogenesis gene libraries. The hypoxia-inducible factor-1 (HIF-1) signaling pathway was identified to be critical for ferroptosis post-TBI, and protein-protein interaction (PPI) network identified 20 hub genes, including phosphoglycerate kinase 1 (PGK1), ribosomal protein (RP) family, pyruvate kinase M1/2 (PKM), hypoxia-inducible factor 1α subunit (HIF-1α), and MYC genes. In this study, we further explored the role of PGK1, a gene involved in HIF-1 signaling pathway; however, its role and mechanism in TBI are still unclear. Moreover, we constructed a TBI mouse model and examined PGK1 and HIF-1α expression levels, and the results revealed their expressions increased after cortical injury in mice and they co-localized in the same cells. Furthermore, we examined the expressions of PGK1 in the cerebrospinal fluid of 20 clinical patients with different degrees of brain injuries within 48h of surgery and examined the cognitive function of patients according to the Glasgow Coma Scale (GCS). The results revealed that PGK1 expression level was negatively correlated with the severity of the brain injury. These findings suggest that PGK1 may become a potential hub gene for ferroptosis via the HIF-1 signaling pathway, second to neurological injury after TBI, thereby affecting patient prognosis.

Perilipin1 inhibits Nosema bombycis proliferation by promoting Domeless- and Hop-mediated JAK-STAT pathway activation in Bombyx mori.

Lipid droplets (LDs) are dynamic organelles that participate in the regulation of lipid metabolism and cellular homeostasis inside of cells. LD-associated proteins, also known as perilipins (PLINs), are a family of proteins found on the surface of LDs that regulate lipid metabolism, immunity, and other functions. In silkworms, pébrine disease caused by infection by the microsporidian Nosema bombycis (Nb) is a severe threat to the sericultural industry. Although we found that Nb relies on lipids from silkworms to facilitate its proliferation, the relationship between PLINs and Nb proliferation remains unknown. Here, we found Nb infection caused the accumulation of LDs in the fat bodies of silkworm larvae. The characterized perilipin1 gene (plin1) promotes the accumulation of intracellular LDs and is involved in Nb proliferation. plin1 is similar to perilipin1 in humans and is conserved in all insects. The expression of plin1 was mostly enriched in the fat body rather than in other tissues. Knockdown of plin1 enhanced Nb proliferation, whereas overexpression of plin1 inhibited its proliferation. Furthermore, we confirmed that plin1 increased the expression of the Domeless and Hop in the JAK-STAT immune pathway and inhibited Nb proliferation. Taken together, our current findings demonstrate that plin1 inhibits Nb proliferation by promoting the JAK-STAT pathway through increased expression of Domeless and Hop. This study provides new insights into the complicated connections among microsporidia pathogens, LD surface proteins, and insect immunity.IMPORTANCELipid droplets (LDs) are lipid storage sites in cells and are present in almost all animals. Many studies have found that LDs may play a role in host resistance to pathogens and are closely related to innate immunity. The present study found that a surface protein of insect lipid droplets could not only regulate the morphological changes of lipid droplets but also inhibit the proliferation of a microsporidian pathogen Nosema bombycis (Nb) by activating the JAK-STAT signaling pathway. This is the first discovery of the relationship between microsporidian pathogen and insect lipid surface protein perilipin and insect immunity.

ATG16L1 induces the formation of phagophore-like membrane cups.

The hallmark of non-selective autophagy is the formation of cup-shaped phagophores that capture bulk cytoplasm. The process is accompanied by the conjugation of LC3B to phagophores by an E3 ligase complex comprising ATG12-ATG5 and ATG16L1. Here we combined two complementary reconstitution approaches to reveal the function of LC3B and its ligase complex during phagophore expansion. We found that LC3B forms together with ATG12-ATG5-ATG16L1 a membrane coat that remodels flat membranes into cups that closely resemble phagophores. Mechanistically, we revealed that cup formation strictly depends on a close collaboration between LC3B and ATG16L1. Moreover, only LC3B, but no other member of the ATG8 protein family, promotes cup formation. ATG16L1 truncates that lacked the C-terminal membrane binding domain catalyzed LC3B lipidation but failed to assemble coats, did not promote cup formation and inhibited the biogenesis of non-selective autophagosomes. Our results thus demonstrate that ATG16L1 and LC3B induce and stabilize the characteristic cup-like shape of phagophores.

The OsZIP2 transporter is involved in root-to-shoot translocation and intervascular transfer of cadmium in rice.

Cadmium (Cd) is a toxic metal that poses serious threats to human health. Rice is a major source of dietary Cd but how rice plants transport Cd to the grain is not fully understood. Here, we characterize the function of the ZIP (ZRT, IRT-like protein) family protein, OsZIP2, in the root-to-shoot translocation of Cd and intervascular transfer of Cd in nodes. OsZIP2 is localized at the plasma membrane and exhibited Cd2+ transport activity when heterologously expressed in yeast. OsZIP2 is strongly expressed in xylem parenchyma cells in roots and in enlarged vascular bundles in nodes. Knockout of OsZIP2 significantly enhanced root-to-shoot translocation of Cd and alleviated the inhibition of root elongation by excess Cd stress; whereas overexpression of OsZIP2 decreased Cd translocation to shoots and resulted in Cd sensitivity. Knockout of OsZIP2 increased Cd allocation to the flag leaf but decreased Cd allocation to the panicle and grain. We further reveal that the variation of OsZIP2 expression level contributes to grain Cd concentration among rice germplasms. Our results demonstrate that OsZIP2 functions in root-to-shoot translocation of Cd in roots and intervascular transfer of Cd in nodes, which can be used for breeding low Cd rice varieties.

RhoMax: Computational Prediction of Rhodopsin Absorption Maxima Using Geometric Deep Learning.

Microbial rhodopsins (MRs) are a diverse and abundant family of photoactive membrane proteins that serve as model systems for biophysical techniques. Optogenetics utilizes genetic engineering to insert specialized proteins into specific neurons or brain regions, allowing for manipulation of their activity through light and enabling the mapping and control of specific brain areas in living organisms. The obstacle of optogenetics lies in the fact that light has a limited ability to penetrate biological tissues, particularly blue light in the visible spectrum. Despite this challenge, most optogenetic systems rely on blue light due to the scarcity of red-shifted opsins. Finding additional red-shifted rhodopsins would represent a major breakthrough in overcoming the challenge of limited light penetration in optogenetics. However, determining the wavelength absorption maxima for rhodopsins based on their protein sequence is a significant hurdle. Current experimental methods are time-consuming, while computational methods lack accuracy. The paper introduces a new computational approach called RhoMax that utilizes structure-based geometric deep learning to predict the absorption wavelength of rhodopsins solely based on their sequences. The method takes advantage of AlphaFold2 for accurate modeling of rhodopsin structures. Once trained on a balanced train set, RhoMax rapidly and precisely predicted the maximum absorption wavelength of more than half of the sequences in our test set with an accuracy of 0.03 eV. By leveraging computational methods for absorption maxima determination, we can drastically reduce the time needed for designing new red-shifted microbial rhodopsins, thereby facilitating advances in the field of optogenetics.

Crosstalk between Regnase-1 and -3 shapes mast cell survival and cytokine expression.

Post-transcriptional regulation of immune-related transcripts by RNA-binding proteins (RBPs) impacts immune cell responses, including mast cell functionality. Despite their importance in immune regulation, the functional role of most RBPs remains to be understood. By manipulating the expression of specific RBPs in murine mast cells, coupled with mass spectrometry and transcriptomic analyses, we found that the Regnase family of proteins acts as a potent regulator of mast cell physiology. Specifically, Regnase-1 is required to maintain basic cell proliferation and survival, whereas both Regnase-1 and -3 cooperatively regulate the expression of inflammatory transcripts upon activation, with Tnf being a primary target in both human and mouse cells. Furthermore, Regnase-3 directly interacts with Regnase-1 in mast cells and is necessary to restrain Regnase-1 expression through the destabilization of its transcript. Overall, our study identifies protein interactors of endogenously expressed Regnase factors, characterizes the regulatory interplay between Regnase family members in mast cells, and establishes their role in the control of mast cell homeostasis and inflammatory responses.

MARCH1 and MARCH2 inhibit pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in trans-Golgi network

The membrane-associated RING-CH (MARCH) family of proteins are members of the E3 ubiquitin ligase family and are essential for a variety of biological functions. Currently, MARCH proteins are discovered to execute antiviral functions by directly triggering viral protein degradation or blocking the furin cleavage of viral class I fusion proteins. Here, we report a novel antiviral mechanism of MARCH1 and MARCH2 (MARCH1/2) in the replication of Pseudorabies virus (PRV), a member of the Herpesviridae family. We discovered MARCH1/2 restrict PRV replication at the cell-to-cell fusion step. Furthermore, MARCH1/2 block gB cleavage, and this is dependent on their E3 ligase activity. Interestingly, the blocking of gB cleavage by MARCH1/2 does not contribute to their antiviral activity in vitro. We discovered that MARCH1/2 are associated with the cell-to-cell fusion complex of gB, gD, gH, and gL and trap these viral proteins in the trans-Golgi network (TGN) rather than degrading them. Overall, we conclude that MARCH1/2 inhibit PRV by trapping the viral cell-to-cell fusion complex in TGN.

Protein family annotation for the Unified Human Gastrointestinal Proteome by DPCfam clustering

Technological advances in massively parallel sequencing have led to an exponential growth in the number of known protein sequences. Much of this growth originates from metagenomic projects producing new sequences from environmental and clinical samples. The Unified Human Gastrointestinal Proteome (UHGP) catalogue is one of the most relevant metagenomic datasets with applications ranging from medicine to biology. However, the low levels of sequence annotation may impair its usability. This work aims to produce a family classification of UHGP sequences to facilitate downstream structural and functional annotation. This is achieved through the release of the DPCfam-UHGP50 dataset containing 10,778 putative protein families generated using DPCfam clustering, an unsupervised pipeline grouping sequences into single or multi-domain architectures. DPCfam-UHGP50 considerably improves family coverage at protein and residue levels compared to the manually curated repository Pfam. In the hope that DPCfam-UHGP50 will foster future discoveries in the field of metagenomics of the human gut, we release a FAIR-compliant database of our results that is easily accessible via a searchable web server and Zenodo repository.

Proteomic profiling of TBC1 domain family member 21-null sperms reveals the critical roles of TEKT 1 in their tail defects.

Approximately 7% of the males exhibit reduced fertility; however, the regulatory genes and pathways involved remain largely unknown. TBC1 domain family member 21 (TBC1D21) contains a conserved RabGAP catalytic domain that induces GDP/GTP exchange to inactivate Rabs by interacting with microtubules. We previously reported that Tbc1d21-null mice exhibit severe sperm tail defects with a disrupted axoneme, and that TBC1D21 interacts with RAB10. However, the pathological mechanisms underlying the Tbc1d21 loss-induced sperm tail defects remain unknown. Murine sperm from wild-type and Tbc1d21-null mice were comparatively analyzed using proteomic assays. Over 1600 proteins were identified, of which 15 were significantly up-regulated in Tbc1d21-null sperm. Notably, several tektin (TEKT) family proteins, belonging to a type of intermediate filament critical for stabilizing the microtubular structure of cilia and flagella, were significantly up-regulated in Tbc1d21-/- sperm. We also found that TBC1D21 interacts with TEKT1. In addition, TEKT1 co-localized with RAB10 during sperm tail formation. Finally, we found Tbc1d21-null sperm exhibited abnormal accumulation of TEKT1 in the midpiece region, accompanied by disrupted axonemal structures. These results reveal that TBC1D21 modulates TEKTs protein localization in the axonemal transport system during sperm tail formation.

Pore-Forming VDAC Proteins of the Outer Mitochondrial Membrane: Regulation and Pathophysiological Role.

Voltage-dependent anion channels (VDAC1-3) of the outer mitochondrial membrane are a family of pore-forming β-barrel proteins that carry out controlled "filtration" of small molecules and ions between the cytoplasm and mitochondria. Due to the conformational transitions between the closed and open states and interaction with cytoplasmic and mitochondrial proteins, VDACs not only regulate the mitochondrial membrane permeability for major metabolites and ions, but also participate in the control of essential intracellular processes and pathological conditions. This review discusses novel data on the molecular structure, regulatory mechanisms, and pathophysiological role of VDAC proteins, as well as future directions in this area of research.

Natural genetic diversity of the DBL domain of a novel member of the plasmodium vivax erythrocyte binding-like proteins (EBP2) in the Amazon rainforest

In malaria parasites, the erythrocyte binding-like proteins (EBL) are a family of invasion proteins that are attractive vaccine targets. In the case of Plasmodium vivax, the widespread malaria parasite, blood-stage vaccines have been largely focused on a single EBL candidate, the Duffy binding-like domain (DBL) of the Duffy binding protein (DBPII), due to its well-characterized role in the reticulocyte invasion. A novel P. vivax EBL family member, the Erythrocyte binding protein (EBP2, also named EBP or DBP2), binds preferentially to reticulocytes and may mediate an alternative P. vivax invasion pathway. To gain insight into the natural genetic diversity of the DBL domain of EBP2 (region II; EBP2-II), we analyzed ebp2-II gene sequences of 71 P. vivax isolates collected in different endemic settings of the Brazilian Amazon rainforest, where P. vivax is the predominant malaria-associated species. Although most of the substitutions in the ebp2-II gene were non-synonymous and suggested positive selection, the results showed that the DBL domain of the EBP2 was much less polymorphic than that of DBPII. The predominant EBP2 haplotype in the Amazon region corresponded to the C127 reference sequence first described in Cambodia (25% C127-like haplotype). An overview of ebp2-II gene sequences available at GenBank (n = 352) from seven countries (Cambodia, Madagascar, Myanmar, PNG, South Korea, Thailand, Vietnam) confirmed the C127-like haplotype as highly prevalent worldwide. Two out of 43 haplotypes (5 to 20 inferred per country) showed a global frequency of 60%. The results presented here open new avenues of research pursuit while suggesting that a vaccine based on the DBL domain of EBP2 should target a few haplotypes for broad coverage.