Dysfunction of endocytic kinase AAK1 in ALS.

Mechanisms of human mutant superoxide dismutase 1 (SOD1)-induced toxicity in causing the familial form of amyotrophic lateral sclerosis (ALS) remain elusive. Identification of new proteins that can selectively interact with mutant SOD1s and investigation of their potential roles in ALS are important to discover new pathways that are involved in disease pathology. Using the yeast two-hybrid system, we identified the adaptor-associated kinase 1 (AAK1), a regulatory protein in clathrin-coated vesicle endocytic pathway that selectively interacted with the mutant but not the wild-type SOD1. Using both transgenic mouse and rat SOD1-linked familial ALS (FALS) models, we found that AAK1 was partially colocalized with the endosomal and presynaptic protein markers under the normal physiological condition, but was mislocated into aggregates that contained mutant SOD1s and the neurofilament proteins in rodent models of ALS in disease. AAK1 protein levels were also decreased in ALS patients. These results suggest that dysfunction of a component in the endosomal and synaptic vesicle recycling pathway is involved in ALS pathology.

The Joubert syndrome-associated missense mutation (V443D) in the Abelson-helper integration site 1 (AHI1) protein alters its localization and protein-protein interactions.

BACKGROUND: Missense mutations in AHI1 result in the neurodevelopmental ciliopathy called Joubert syndrome. RESULTS: Mutations in AHI1 decrease cilia formation, alter its localization and stability, and change its binding to HAP1 and NPHP1. CONCLUSION: Mutations in AHI1 affect ciliogenesis, AHI1 protein localization, and AHI1-protein interactions. SIGNIFICANCE: This study begins to describe how missense mutations in AHI1 can cause Joubert syndrome. Mutations in AHI1 cause Joubert syndrome (JBTS), a neurodevelopmental ciliopathy, characterized by midbrain-hindbrain malformations and motor/cognitive deficits. Here, we show that primary cilia (PC) formation is decreased in fibroblasts from individuals with JBTS and AHI1 mutations. Most missense mutations in AHI1, causing JBTS, occur in known protein domains, however, a common V443D mutation in AHI1 is found in a region with no known protein motifs. We show that cells transfected with AHI1-V443D, or a new JBTS-causing mutation, AHI1-R351L, have aberrant localization of AHI1 at the basal bodies of PC and at cell-cell junctions, likely through decreased binding of mutant AHI1 to NPHP1 (another JBTS-causing protein). The AHI1-V443D mutation causes decreased AHI1 stability because there is a 50% reduction in AHI1-V443D protein levels compared with wild type AHI1. Huntingtin-associated protein-1 (Hap1) is a regulatory protein that binds Ahi1, and Hap1 knock-out mice have been reported to have JBTS-like phenotypes, suggesting a role for Hap1 in ciliogenesis. Fibroblasts and neurons with Hap1 deficiency form PC with normal growth factor-induced ciliary signaling, indicating that the Hap1 JBTS phenotype is likely not through effects at PC. These results also suggest that the binding of Ahi1 and Hap1 may not be critical for ciliary function. However, we show that HAP1 has decreased binding to AHI1-V443D indicating that this altered binding could be responsible for the JBTS-like phenotype through an unknown pathway. Thus, these JBTS-associated missense mutations alter their subcellular distribution and protein interactions, compromising functions of AHI1 in cell polarity and cilium-mediated signaling, thereby contributing to JBTS.

Brefeldin A-inhibited guanine exchange factor 2 regulates filamin A phosphorylation and neuronal migration.

Periventricular heterotopia (PH) is a human malformation of cortical development associated with gene mutations in ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2 encodes for Big2 protein) and Filamin A (FLNA). PH is thought to derive from neuroependymal disruption, but the extent to which neuronal migration contributes to this phenotype is unknown. Here, we show that Arfgef2 null mice develop PH and exhibit impaired neural migration with increased protein expression for both FlnA and phosphoFlnA at Ser2152. Big2 physically interacts with FlnA and overexpression of phosphomimetic Ser2512 FLNA impairs neuronal migration. FlnA phosphorylation directs FlnA localization toward the cell cytoplasm, diminishes its binding affinity to actin skeleton, and alters the number and size of paxillin focal adhesions. Collectively, our results demonstrate a molecular mechanism whereby Big2 inhibition promotes phosphoFlnA (Ser2152) expression, and increased phosphoFlnA impairs its actin binding affinity and the distribution of focal adhesions, thereby disrupting cell intrinsic neuronal migration.

Species differences in the expression of Ahi1, a protein implicated in the neurodevelopmental disorder Joubert syndrome, with preferential accumulation to stigmoid bodies.

Joubert syndrome (JBTS) is an autosomal recessive disorder characterized by cerebellum and brainstem malformations. Individuals with JBTS have abnormal breathing and eye movements, ataxia, hypotonia, and cognitive difficulty, and they display mirror movements. Mutations in the Abelson-helper integration site-1 gene (AHI1) cause JBTS in humans, suggesting that AHI1 is required for hindbrain development; however AHI1 may also be required for neuronal function. Support for this idea comes from studies demonstrating that the AHI1 locus is associated with schizophrenia. To gain further insight into the function of AHI1 in both the developing and mature central nervous system, we determined the spatial and temporal expression patterns of the gene products of AHI1 orthologs throughout development, in human, mouse, and zebrafish. Murine Ahi1 was distributed throughout the cytoplasm, dendrites, and axons of neurons, but was absent in glial cells. Ahi1 expression in the mouse brain was observed as early as embryonic day 10.5 and persisted into adulthood, with peak expression during the first postnatal week. Murine Ahi1 was observed in neurons of the hindbrain, midbrain, and ventral forebrain. Generally, the AHI1/Ahi1/ahi1 orthologs had a conserved distribution pattern in human, mouse, and zebrafish, but mouse Ahi1 was not present in the developing and mature cerebellum. Ahi1 was also observed consistently in the stigmoid body, a poorly characterized cytoplasmic organelle found in neurons. Overall, these results suggest roles for AHI1 in neurodevelopmental processes that underlie most of the neuroanatomical defects in JBTS, and perhaps in neuronal functions that contribute to schizophrenia.

Isolation and functional identification of a novel human hepatic growth factor: hepatopoietin Cn.

UNLABELLED: Hepatic stimulating substance (HSS) was first isolated from weanling rat liver in 1975 and found to stimulate hepatic DNA synthesis both in vitro and in vivo. Since then, mammalian and human HSS have been investigated for their potential to treat hepatic diseases. However, the essential nature in composition and structure of HSS remain puzzling because HSS has not been completely purified. Heating, ethanol precipitation, and ion-exchange chromatographies had been carried out to isolate the protein with specific stimulating activity from newborn calf liver, and [(3)H]thymidine deoxyribose (TdR)/bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE)-based proliferation assay to determine the bioactivity in vitro and in vivo. We report the purification of a novel 30-kDa protein from a crude extract of calf liver HSS. This protein is a member of the leucine-rich acidic nuclear protein family (LANP) and has been named hepatopoietin Cn (HPPCn). Studies of partially hepatectomized (PH) mice show that levels of HPPCn messenger RNA (mRNA) increase after liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. CONCLUSION: HPPCn is a novel hepatic growth factor that plays a role in liver regeneration.

Locally activity-released bifunctional fusion protein enhances antithrombosis and alleviates bleeding risk.

Despite the fact that lytic therapy of thromboembolic disorder has been achieved, reocclusion of the damaged vessels and bleeding complication frequently reduce the therapeutic effect. In order to prevent the vessel reocclusion and enhance the therapeutic effect, combining the anticoagulant with the thrombolytic was assumed. Herein, we propose that restraining but locally releasing anticoagulant activity in the vicinity of thrombus is a way to alleviate the bleeding risk. A bifunctional fusion protein, termed as SFH (Staphylokinase (SAK) linked by FXa recognition peptide at N-terminus of Hirudin (HV)), was designed. SFH retained thrombolytic activity but no anticoagulant activity in thrombus-free blood due to the extension of the N-terminus of HV. However, it could locally liberate intact HV and exhibit anticoagulant activity when FXa or fresh thrombus was present. At equimolar dose, both improved antithrombotic and thrombolytic effects of SFH were observed in kappa-carrageenin inducing mouse-tail thrombosis model and rat inferior vena cava thrombosis model, respectively. Moreover, we observed significantly lower bleeding risk in mice and rats treated with SFH than with the mixture of SAK and HV with monitoring TT (P < 0.01), aPTT (P < 0.05) and PT (P < 0.05), and bleeding time (P < 0.05). In conclusion, SFH is a promising bifunctional therapeutic candidate with lower bleeding risk.

[Construction and expression of a fusion protein made of tissue-type plasminogen activator and hirudin in Pichia pastoris].

To combine the fibrinolytic with anticoagulant activities for therapy of thrombotic deseases, a fusion protein made of tissue-type plasminogen activator (t-PA) and hirudin was constructed and expressed in chia pastoris. To improve thrombolytic properties of t-PA and reduce bleeding side effect of hirudin, FXa-recognition sequence was introduced between t-PA and hirudin molecules.The anticoagulant activity of hirudin can be target-released through cleavage of FXa at thrombus site. t-PA gene and hirudin gene with FXa-recognition sequence at its 5'-terminal were obtained by RT-PCR and PCR respectively. The fusion protein gene was cloned into plasmid pIC9K and electroporated into the genome of Pichia pastoris GS115. The expression of fusion protein was induced by methanol in shaking flask and secreted into the culture medium. Two forms of the fusion protein, single-chain and double-chain linked by a disulfide bond (due to the cleveage of t-PA at Arg275-Ile276), were obtained. The intact fusion protein retained the fibrinolytic activity but lacked any anticoagulant activity. After cleavage by FXa, the fusion protein liberated intact free hirudin to exert its anticoagulant activity. So, the fusion protein is a bifunctional molecule having good prospect to develop into a new targeted therapeutic agent with reduced bleeding side effect for thrombotic diseases.