Corrigendum: Safe and effective delivery of supplemental iron to healthy adults: a two-phase, randomized, double-blind trial - the safe iron study.

[This corrects the article DOI: 10.3389/fnut.2023.1230061.].

Safe and effective delivery of supplemental iron to healthy adults: a two-phase, randomized, double-blind trial - the safe iron study.

Introduction: The safety of novel forms of iron in healthy, iron-replete adults as might occur if used in population-based iron supplementation programs was examined. We tested the hypotheses that supplementation with nanoparticulate iron hydroxide adipate tartrate (IHAT), an iron-enriched Aspergillus oryzae product (ASP), or ferrous sulphate heptahydrate (FS) are safe as indicated by erythrocyte susceptibility to malarial infection, bacterial proliferation, and gut inflammation. Responses to FS administered daily or weekly, and with or without other micronutrients were compared. Methods: Two phases of randomized, double-blinded trials were conducted in Boston, MA. Phase I randomized 160 volunteers to six treatments: placebo, IHAT, ASP, FS, and FS plus a micronutrient powder (MNP) administrated daily at 60 mg Fe/day; and FS administered as a single weekly dose of 420 mg Fe. Phase II randomized 86 volunteers to IHAT, ASP, or FS administered at 120 mg Fe/day. Completing these phases were 151 and 77 participants, respectively. The study was powered to detect effects on primary endpoints: susceptibility of participant erythrocytes to infection by Plasmodium falciparum, the proliferation potential of selected pathogenic bacteria in sera, and markers of gut inflammation. Secondary endpoints for which the study was not powered included indicators of iron status and gastrointestinal symptoms. Results: Supplementation with any form of iron did not affect any primary endpoint. In Phase I, the frequency of gastrointestinal symptoms associated with FS was unaffected by dosing with MNP or weekly administration; but participants taking IHAT more frequently reported abdominal pain (27%, p < 0.008) and nausea (4%, p = 0.009) than those taking FS, while those taking ASP more frequently reported nausea (8%, p = 0.009). Surprisingly, only 9% of participants taking IHAT at 120 mg Fe/day (Phase II) reported abdominal pain and no other group reported that symptom. Discussion: With respect to the primary endpoints, few differences were found when comparing these forms of iron, indicating that 28 days of 60 or 120 mg/day of IHAT, ASP, or FS may be safe for healthy, iron-replete adults. With respect to other endpoints, subjects receiving IHAT more frequently reported abdominal pain and nausea, suggesting the need for further study. Clinical Trial Registration: ClinicalTrials.gov, NCT03212677; registered: 11 July 2017.

Dematin Regulates Calcium Mobilization, Thrombosis, and Early Akt Activation in Platelets.

The complex intrinsic and extrinsic pathways contributing to platelet activation profoundly impact hemostasis and thrombosis. Detailed cellular mechanisms that regulate calcium mobilization, Akt activation, and integrin signaling in platelets remain incompletely understood. Dematin is a broadly expressed actin binding and bundling cytoskeletal adaptor protein regulated by phosphorylation via cAMP-dependent protein kinase. Here, we report the development of a conditional mouse model specifically lacking dematin in platelets. Using the new mouse model termed PDKO, we provide direct evidence that dematin is a major regulator of calcium mobilization, and its genetic deletion inhibits the early phase of Akt activation in response to collagen and thrombin agonists in platelets. The aberrant platelet shape change, clot retraction, and in vivo thrombosis observed in PDKO mice will enable future characterization of dematin-mediated integrin activation mechanisms in thrombogenic as well as nonvascular pathologies.

Calpain-1 ablation partially rescues disease-associated hallmarks in models of Machado-Joseph disease.

Proteolytic fragmentation of polyglutamine-expanded ataxin-3 is a concomitant and modifier of the molecular pathogenesis of Machado-Joseph disease (MJD), the most common autosomal dominant cerebellar ataxia. Calpains, a group of calcium-dependent cysteine proteases, are important mediators of ataxin-3 cleavage and implicated in multiple neurodegenerative conditions. Pharmacologic and genetic approaches lowering calpain activity showed beneficial effects on molecular and behavioural disease characteristics in MJD model organisms. However, specifically targeting one of the calpain isoforms by genetic means has not yet been evaluated as a potential therapeutic strategy. In our study, we tested whether calpains are overactivated in the MJD context and if reduction or ablation of calpain-1 expression ameliorates the disease-associated phenotype in MJD cells and mice. In all analysed MJD models, we detected an elevated calpain activity at baseline. Lowering or removal of calpain-1 in cells or mice counteracted calpain system overactivation and led to reduced cleavage of ataxin-3 without affecting its aggregation. Moreover, calpain-1 knockout in YAC84Q mice alleviated excessive fragmentation of important synaptic proteins. Despite worsening some motor characteristics, YAC84Q mice showed a rescue of body weight loss and extended survival upon calpain-1 knockout. Together, our findings emphasize the general potential of calpains as a therapeutic target in MJD and other neurodegenerative diseases.

LDL receptor related protein 1 requires the I3 domain of discs-large homolog 1/DLG1 for interaction with the kinesin motor protein KIF13B.

KIF13B, a kinesin-3 family motor, was originally identified as GAKIN due to its biochemical interaction with human homolog of Drosophila discs-large tumor suppressor (hDLG1). Unlike its homolog KIF13A, KIF13B contains a carboxyl-terminal CAP-Gly domain. To investigate the function of the CAP-Gly domain, we developed a mouse model that expresses a truncated form of KIF13B protein lacking its CAP-Gly domain (KIF13BΔCG), whereas a second mouse model lacks the full-length KIF13A. Here we show that the KIF13BΔCG mice exhibit relatively higher serum cholesterol consistent with the reduced uptake of [3H]CO-LDL in KIF13BΔCG mouse embryo fibroblasts. The plasma level of factor VIII was not significantly elevated in the KIF13BΔCG mice, suggesting that the CAP-Gly domain region of KIF13B selectively regulates LRP1-mediated lipoprotein endocytosis. No elevation of either serum cholesterol or plasma factor VIII was observed in the full length KIF13A null mouse model. The deletion of the CAP-Gly domain region caused subcellular mislocalization of truncated KIF13B concomitant with the mislocalization of LRP1. Mechanistically, the cytoplasmic domain of LRP1 interacts specifically with the alternatively spliced I3 domain of DLG1, which complexes with KIF13B via their GUK-MBS domains, respectively. Importantly, double mutant mice generated by crossing KIF13A null and KIF13BΔCG mice suffer from perinatal lethality showing potential craniofacial defects. Together, this study provides first evidence that the carboxyl-terminal region of KIF13B containing the CAP-Gly domain is important for the LRP1-DLG1-KIF13B complex formation with implications in the regulation of metabolism, cell polarity, and development.

Safe and effective delivery of supplemental iron to healthy older adults: The double-blind, randomized, placebo-controlled trial protocol of the Safe Iron Study.

The forms of iron currently available to correct iron deficiency have adverse effects, including infectious diarrhea, increased susceptibility to malaria, inflammation and detrimental changes to the gut microbiome. These adverse effects limit their use such that the growing burden of iron deficiency has not abated in recent decades. Here, we summarize the protocol of the "Safe Iron Study", the first clinical study examining the safety and efficacy of novel forms of iron in healthy, iron-replete adults. The Safe Iron Study is a double-blind, randomized, placebo-controlled trial conducted in Boston, MA, USA. This study compares ferrous sulfate heptahydrate (FeSO 4·H 2O) with two novel forms of iron supplements (iron hydroxide adipate tartrate (IHAT) and organic fungal iron metabolite (Aspiron™ Natural Koji Iron)). In Phase I, we will compare each source of iron administrated at a low dose (60 mg Fe/day). We will also determine the effect of FeSO 4 co-administrated with a multiple micronutrient powder and weekly administration of FeSO 4. The forms of iron found to produce no adverse effects, or adverse effects no greater than FeSO 4 in Phase I, Phase II will evaluate a higher, i.e., a therapeutic dose (120 mg Fe/day). The primary outcomes of this study include ex vivo malaria ( Plasmodium falciparum) infectivity of host erythrocytes, ex vivo bacterial proliferation (of selected species) in presence of host plasma and intestinal inflammation assessed by fecal calprotectin. This study will test the hypotheses that the novel forms of iron, administered at equivalent doses to FeSO 4, will produce similar increases in iron status in iron-replete subjects, yet lower increases in ex vivo malaria infectivity, ex vivo bacterial proliferation, gut inflammation. Ultimately, this study seeks to contribute to development of safe and effective forms of supplemental iron to address the global burden of iron deficiency and anemia. Registration: ClinicalTrials.gov identifier: NCT03212677; registered: 11 July 2017.

Calpain-1 regulates platelet function in a humanized mouse model of sickle cell disease.

One of the major contributors to sickle cell disease (SCD) pathobiology is the hemolysis of sickle red blood cells (RBCs), which release free hemoglobin and platelet agonists including adenosine 5'-diphosphate (ADP) into the plasma. While platelet activation/aggregation may promote tissue ischemia and pulmonary hypertension in SCD, modulation of sickle platelet dysfunction remains poorly understood. Calpain-1, a ubiquitous calcium-activated cysteine protease expressed in hematopoietic cells, mediates aggregation of platelets in healthy mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice to investigate the role of calpain-1 in steady state and hypoxia/reoxygenation (H/R)-induced sickle platelet activation and aggregation, clot retraction, and pulmonary arterial hypertension. Using multi-electrode aggregometry, which measures platelet adhesion and aggregation in whole blood, we determined that steady state SSCKO mice exhibit significantly impaired PAR4-TRAP-stimulated platelet aggregation as compared to Townes sickle (SS) and humanized control (AA) mice. Interestingly, the H/R injury induced platelet hyperactivity in SS and SSCKO, but not AA mice, and partially rescued the aggregation defect in SSCKO mice. The PAR4-TRAP-stimulated GPIIb-IIIa (αIIbß3) integrin activation was normal in SSCKO platelets suggesting that an alternate mechanism mediates the impaired platelet aggregation in steady state SSCKO mice. Taken together, we provide the first evidence that calpain-1 regulates platelet hyperactivity in sickle mice, and may offer a viable pharmacological target to reduce platelet hyperactivity in SCD.

Gene disruption of dematin causes precipitous loss of erythrocyte membrane stability and severe hemolytic anemia.

Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin-actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin's actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate-dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex.

Leukocyte Calpain Deficiency Reduces Angiotensin II-Induced Inflammation and Atherosclerosis But Not Abdominal Aortic Aneurysms in Mice.

OBJECTIVE: Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. APPROACH AND RESULTS: To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. CONCLUSIONS: The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Calpain-1 contributes to IgE-mediated mast cell activation.

Mast cells play a central role in allergy through secretion of both preformed and newly synthesized mediators. Mast cell mediator secretion is controlled by a complex network of signaling events. Despite intensive studies, signaling pathways in the regulation of mast cell mediator secretion remain incompletely defined. In this study, we examined the role of calpain in IgE-dependent mast cell activation. IgE-mediated activation of mouse bone marrow-derived mast cells enhanced calpain activity. Inhibition of calpain activity by a number of calpain inhibitors reduced IgE-mediated mast cell degranulation both in vitro and in vivo. Calpain inhibitors blocked IgE-mediated TNF and IL-6 production in vitro and reduced late-phase allergic response in vivo. Importantly, mouse calpain-1 null bone marrow-derived mast cells showed reduced IgE-mediated mast cell degranulation in vitro and in vivo, diminished cytokine and chemokine production in vitro, and impaired late-phase allergic response in vivo. Further studies revealed that calpain-1 deficiency led to specific attenuation of IκB-NF-κB pathway and IKK-SNAP23 pathway, whereas calcium flux, MAPK, Akt, and NFAT pathway proceed normally in IgE-activated calpain-1 null mast cells. Thus, calpain-1 is identified as a novel regulator in IgE-mediated mast cell activation and could serve as a potential therapeutic target for the management of allergic inflammation.

Distinct roles for µ-calpain and m-calpain in synaptic NMDAR-mediated neuroprotection and extrasynaptic NMDAR-mediated neurodegeneration.

Prolonged calpain activation is widely recognized as a key component of neurodegeneration in a variety of pathological conditions. Numerous reports have also indicated that synaptic activation of NMDA receptors (NMDARs) provides neuroprotection against a variety of insults. Here, we report the paradoxical finding that such neuroprotection involves calpain activation. NMDAR activation in cultured rat cortical neurons was neuroprotective against starvation and oxidative stress-induced damage. It also resulted in the degradation of two splice variants of PH domain and Leucine-rich repeat Protein Phosphatase 1 (PHLPP1), PHLPP1α and PHLPP1ß, which inhibit the Akt and ERK1/2 pathways. Synaptic NMDAR-induced neuroprotection and PHLPP1 degradation were blocked by calpain inhibition. Lentiviral knockdown of PHLPP1 mimicked the neuroprotective effects of synaptic NMDAR activation and occluded the effects of calpain inhibition on neuroprotection. In contrast to synaptic NMDAR activation, extrasynaptic NMDAR activation had no effect on PHLPP1 and the Akt and ERK1/2 pathways, but resulted in calpain-mediated degradation of striatal-enriched protein tyrosine phosphatase (STEP) and neuronal death. Using µ-calpain- and m-calpain-selective inhibitors and µ-calpain and m-calpain siRNAs, we found that µ-calpain-dependent PHLPP1 cleavage was involved in synaptic NMDAR-mediated neuroprotection, while m-calpain-mediated STEP degradation was associated with extrasynaptic NMDAR-induced neurotoxicity. Furthermore, m-calpain inhibition reduced while µ-calpain knockout exacerbated NMDA-induced neurotoxicity in acute mouse hippocampal slices. Thus, synaptic NMDAR-coupled µ-calpain activation is neuroprotective, while extrasynaptic NMDAR-coupled m-calpain activation is neurodegenerative. These results help to reconcile a number of contradictory results in the literature and have critical implications for the understanding and potential treatment of neurodegenerative diseases.

Calpain-2 compensation promotes angiotensin II-induced ascending and abdominal aortic aneurysms in calpain-1 deficient mice.

BACKGROUND AND OBJECTIVE: Recently, we demonstrated that angiotensin II (AngII)-infusion profoundly increased both aortic protein and activity of calpains, calcium-activated cysteine proteases, in mice. In addition, pharmacological inhibition of calpain attenuated AngII-induced abdominal aortic aneurysm (AA) in mice. Recent studies have shown that AngII infusion into mice leads to aneurysmal formation localized to the ascending aorta. However, the precise functional contribution of calpain isoforms (-1 or -2) in AngII-induced abdominal AA formation is not known. Similarly, a functional role of calpain in AngII-induced ascending AA remains to be defined. Using BDA-410, an inhibitor of calpains, and calpain-1 genetic deficient mice, we examined the relative contribution of calpain isoforms in AngII-induced ascending and abdominal AA development. METHODOLOGY/RESULTS: To investigate the relative contribution of calpain-1 and -2 in development of AngII-induced AAs, male LDLr -/- mice that were either calpain-1 +/+ or -/- were fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min) for 4 weeks. Calpain-1 deficiency had no significant effect on body weight or blood pressure during AngII infusion. Moreover, calpain-1 deficiency showed no discernible effects on AngII-induced ascending and abdominal AAs. Interestingly, AngII infusion induced increased expression of calpain-2 protein, thus compensating for total calpain activity in aortas of calpain-1 deficient mice. Oral administration of BDA-410, a calpain inhibitor, along with AngII-infusion significantly attenuated AngII-induced ascending and abdominal AA formation in both calpain-1 +/+ and -/- mice as compared to vehicle administered mice. Furthermore, BDA-410 administration attenuated AngII-induced aortic medial hypertrophy and macrophage accumulation. Western blot and immunostaining analyses revealed BDA-410 administration attenuated AngII-induced C-terminal fragmentation of filamin A, an actin binding cytoskeletal protein in aorta. CONCLUSION: Calpain-2 compensates for loss of calpain-1, and both calpain isoforms are involved in AngII-induced aortic aneurysm formation in mice.

Pharmacological inhibition of calpain-1 prevents red cell dehydration and reduces Gardos channel activity in a mouse model of sickle cell disease.

Sickle cell disease (SCD) is a globally distributed hereditary red blood cell (RBC) disorder. One of the hallmarks of SCD is the presence of circulating dense RBCs, which are important in SCD-related clinical manifestations. In human dense sickle cells, we found reduced calpastatin activity and protein expression compared to either healthy RBCs or unfractionated sickle cells, suggesting an imbalance between activator and inhibitor of calpain-1 in favor of activator in dense sickle cells. Calpain-1 is a nonlysosomal cysteine proteinase that modulates multiple cell functions through the selective cleavage of proteins. To investigate the relevance of this observation in vivo, we evaluated the effects of the orally active inhibitor of calpain-1, BDA-410 (30 mg/kg/d), on RBCs from SAD mice, a mouse model for SCD. In SAD mice, BDA-410 improved RBC morphology, reduced RBC density (D(20); from 1106 ± 0.001 to 1100 ± 0.001 g/ml; P<0.05) and increased RBC-K(+) content (from 364 ± 10 to 429 ± 12.3 mmol/kg Hb; P<0.05), markedly reduced the activity of the Ca(2+)-activated K(+)channel (Gardos channel), and decreased membrane association of peroxiredoxin-2. The inhibitory effect of calphostin C, a specific inhibitor of protein kinase C (PKC), on the Gardos channel was eliminated after BDA-410 treatment, which suggests that calpain-1 inhibition affects the PKC-dependent fraction of the Gardos channel. BDA-410 prevented hypoxia-induced RBC dehydration and K(+) loss in SAD mice. These data suggest a potential role of BDA-410 as a novel therapeutic agent for treatment of SCD.

Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology.

Pharmacological inhibitors of cysteine proteases have provided useful insights into the regulation of calpain activity in erythrocytes. However, the precise biological function of calpain activity in erythrocytes remains poorly understood. Erythrocytes express calpain-1, an isoform regulated by calpastatin, the endogenous inhibitor of calpains. In the present study, we investigated the function of calpain-1 in mature erythrocytes using our calpain-1-null [KO (knockout)] mouse model. The calpain-1 gene deletion results in improved erythrocyte deformability without any measurable effect on erythrocyte lifespan in vivo. The calcium-induced sphero-echinocyte shape transition is compromised in the KO erythrocytes. Erythrocyte membrane proteins ankyrin, band 3, protein 4.1R, adducin and dematin are degraded in the calcium-loaded normal erythrocytes but not in the KO erythrocytes. In contrast, the integrity of spectrin and its state of phosphorylation are not affected in the calcium-loaded erythrocytes of either genotype. To assess the functional consequences of attenuated cytoskeletal remodelling in the KO erythrocytes, the activity of major membrane transporters was measured. The activity of the K+-Cl- co-transporter and the Gardos channel was significantly reduced in the KO erythrocytes. Similarly, the basal activity of the calcium pump was reduced in the absence of calmodulin in the KO erythrocyte membrane. Interestingly, the calmodulin-stimulated calcium pump activity was significantly elevated in the KO erythrocytes, implying a wider range of pump regulation by calcium and calmodulin. Taken together, and with the atomic force microscopy of the skeletal network, the results of the present study provide the first evidence for the physiological function of calpain-1 in erythrocytes with therapeutic implications for calcium imbalance pathologies such as sickle cell disease.

Targeted gene inactivation of calpain-1 suppresses cortical degeneration due to traumatic brain injury and neuronal apoptosis induced by oxidative stress.

Calpains are calcium-regulated cysteine proteases that have been implicated in the regulation of cell death pathways. Here, we used our calpain-1 null mouse model to evaluate the function of calpain-1 in neural degeneration following a rodent model of traumatic brain injury. In vivo, calpain-1 null mice show significantly less neural degeneration and apoptosis and a smaller contusion 3 days post-injury than wild type littermates. Protection from traumatic brain injury corroborated with the resistance of calpain-1 neurons to apoptosis induced by oxidative stress. Biochemical analysis revealed that caspase-3 activation, extracellular calcium entry, mitochondrial membrane permeability, and release of apoptosis-inducing factor from mitochondria are partially blocked in the calpain-1 null neurons. These findings suggest that the calpain-1 knock-out mice may serve as a useful model system for neuronal protection and apoptosis in traumatic brain injury and other neurodegenerative disorders in which oxidative stress plays a role.

Identification of a specific region of Plasmodium falciparum EBL-1 that binds to host receptor glycophorin B and inhibits merozoite invasion in human red blood cells.

The malaria parasite Plasmodium falciparum invades human erythrocytes through multiple pathways utilizing several ligand-receptor interactions. These interactions are broadly classified in two groups according to their dependency on sialic acid residues. Here, we focus on the sialic acid-dependent pathway by using purified glycophorins and red blood cells (RBCs) to screen a cDNA phage display library derived from P. falciparum FCR3 strain, a sialic acid-dependent strain. This screen identified several parasite proteins including the erythrocyte-binding ligand-1, EBL-1. The phage cDNA insert encoded the 69-amino acid peptide, termed F2i, which is located within the F2 region of the DBL domain, designated here as D2, of EBL-1. Recombinant D2 and F2i polypeptides bound to purified glycophorins and RBCs, and the F2i peptide was found to interfere with binding of D2 domain to its receptor. Both D2 and F2i polypeptides bound to trypsin-treated but not neuraminidase or chymotrypsin-treated erythrocytes, consistent with known glycophorin B resistance to trypsin, and neither the D2 nor F2i polypeptide bound to glycophorin B-deficient erythrocytes. Importantly, purified D2 and F2i polypeptides partially inhibited merozoite reinvasion in human erythrocytes. Our results show that the host erythrocyte receptor glycophorin B directly interacts with the DBL domain of parasite EBL-1, and the core binding site is contained within the 69 amino acid F2i region (residues 601-669) of the DBL domain. Together, these findings suggest that a recombinant F2i peptide with stabilized structure could provide a protective function at blood stage infection and represents a valuable addition to a multi-subunit vaccine against malaria.

Phosphorylation-independent dual-site binding of the FHA domain of KIF13 mediates phosphoinositide transport via centaurin alpha1.

Phosphatidylinositol 3,4,5-triphosphate (PIP3) plays a key role in neuronal polarization and axon formation. PIP3-containing vesicles are transported to axon tips by the kinesin KIF13B via an adaptor protein, centaurin α1 (CENTA1). KIF13B interacts with CENTA1 through its forkhead-associated (FHA) domain. We solved the crystal structures of CENTA1 in ligand-free, KIF13B-FHA domain-bound, and PIP3 head group (IP4)-bound conformations, and the CENTA1/KIF13B-FHA/IP4 ternary complex. The first pleckstrin homology (PH) domain of CENTA1 specifically binds to PIP3, while the second binds to both PIP3 and phosphatidylinositol 3,4-biphosphate (PI(3,4)P(2)). The FHA domain of KIF13B interacts with the PH1 domain of one CENTA1 molecule and the ArfGAP domain of a second CENTA1 molecule in a threonine phosphorylation-independent fashion. We propose that full-length KIF13B and CENTA1 form heterotetramers that can bind four phosphoinositide molecules in the vesicle and transport it along the microtubule.

Receptor-independent cardiac protein kinase Calpha activation by calpain-mediated truncation of regulatory domains.

RATIONALE: Protein kinase (PK)Cs and calpain cysteine proteases are highly expressed in myocardium. Ischemia produces calcium overload that activates calpains and conventional PKCs. However, calpains can proteolytically process PKCs, and the potential in vivo consequences of this interaction are unknown. OBJECTIVE: To determine the biochemical and pathophysiological consequences of calpain-mediated cardiac PKCα proteolysis. METHODS AND RESULTS: Isolated mouse hearts subjected to global ischemia/reperfusion demonstrated cleavage of PKCα. Calpain 1 overexpression was not sufficient to produce PKCα cleavage in normal hearts, but ischemia-induced myocardial PKCα cleavage and myocardial injury were greatly increased by cardiac-specific expression of calpain 1. In contrast, calpain 1 gene ablation or inhibition with calpastatin prevented ischemia/reperfusion induced PKCα cleavage; infarct size was decreased and ventricular function enhanced in infarcted calpain 1 knockout hearts. To determine consequences of PKCα fragmentation on myocardial protein phosphorylation, transgenic mice were created conditionally expressing full-length PKCα or its N-terminal and C-terminal calpain 1 cleavage fragments. Two-dimensional mapping of ventricular protein extracts showed a distinct PKCα phosphorylation profile that was exaggerated and distorted in hearts expressing the PKCα C-terminal fragment. MALDI mass spectroscopy revealed hyperphosphorylation of myosin-binding protein C and phosphorylation of atypical substrates by the PKCα C-terminal fragment. Expression of parent PKCα produced a mild cardiomyopathy, whereas myocardial expression of the C-terminal PKCα fragment induced a disproportionately severe, rapidly lethal cardiomyopathy. CONCLUSIONS: Proteolytic processing of PKCα by calcium-activated calpain activates pathological cardiac signaling through generation of an unregulated and/or mistargeted kinase. Production of the PKCα C-terminal fragment in ischemic hearts occurs via a receptor-independent mechanism.

Erythrocyte scaffolding protein p55/MPP1 functions as an essential regulator of neutrophil polarity.

As mediators of innate immunity, neutrophils respond to chemoattractants by adopting a highly polarized morphology. Efficient chemotaxis requires the formation of one prominent pseudopod at the cell front characterized by actin polymerization, while local inhibition suppresses the formation of rear and lateral protrusions. This asymmetric control of signaling pathways is required for directional migration along a chemotactic gradient. Here, we identify the MAGUK protein p55/MPP1 as a mediator of the frontness signal required for neutrophil polarization. We developed a p55 knockout (p55(-/-)) mouse model, and demonstrate that p55(-/-) neutrophils form multiple transient pseudopods upon chemotactic stimulation, and do not migrate efficiently in vitro. Upon agonist stimulation, p55 is rapidly recruited to the leading edge of neutrophils in mice and humans. Total F-actin polymerization, along with Rac1 and RhoA activation, appear to be normal in p55(-/-) neutrophils. Importantly, phosphorylation of Akt is significantly decreased in p55(-/-) neutrophils upon chemotactic stimulation. The activity of immunoprecipitated phosphatidylinositol 3-kinase gamma (PI3Kgamma), responsible for chemoattractant-induced synthesis of PIP(3) and Akt phosphorylation, is unperturbed in p55(-/-) neutrophils. Although the total amount of PIP(3) is normal in p55(-/-) neutrophils, PIP(3) is diffusely localized and forms punctate aggregates in activated p55(-/-) neutrophils, as compared to its accumulation at the leading edge membrane in the wild type neutrophils. Together, these results show that p55 is required for neutrophil polarization by regulating Akt phosphorylation through a mechanism that is independent of PI3Kgamma activity.

Identification of erythrocyte p55/MPP1 as a binding partner of NF2 tumor suppressor protein/Merlin.

Neurofibromatosis type 2 is an inherited disorder characterized by the development of benign and malignant tumors on the auditory nerves and central nervous system with symptoms including hearing loss, poor balance, skin lesions, and cataracts. Here, we report a novel protein-protein interaction between NF2 protein (merlin or schwannomin) and erythrocyte p55, also designated as MPP1. The p55 is a conserved scaffolding protein with postulated functions in cell shape, hair cell development, and neural patterning of the retina. The FERM domain of NF2 protein binds directly to p55, and surface plasmon resonance analysis indicates a specific interaction with a kD value of 3.7 nM. We developed a specific monoclonal antibody against human erythrocyte p55, and found that both p55 and NF2 proteins are colocalized in the non-myelin-forming Schwann cells. This finding suggests that the p55-NF2 protein interaction may play a functional role in the regulation of apico-basal polarity and tumor suppression pathways in non-erythroid cells.