Identification of nanobodies against hepatocellular carcinoma marker glypican-3.

Glypican-3 (GPC3) is a highly specific diagnostic marker for hepatocellular carcinoma (HCC) diagnosis and a potential target in HCC therapy. Nanobodies (Nbs) are promising targeting molecules due to their high specificity and strong affinities to antigens, high stability, deep tissue penetration, and low immunogenicity. In this study, we isolated Nbs against GPC3 marker protein from a synthetic Nb library by phage display. To characterize these Nbs, we performed enzyme-linked immunosorbent assay, immunoprecipitation assay, and immunofluorescent assay to demonstrate that four (G8, G10, G11, and G64) of them bound specifically to recombinant as well as endogenous GPC3, and epitope mapping showed they all bound to N-terminal subunit of GPC3. Furthermore, we found that G64 exhibited high protein stability and GPC3 binding activity in serum at 37℃ for at least 96 h, and G64 did not affect the proliferation of HEK293T cells and HCC cell line HepG2. Our study provides four anti-GPC3 Nbs as promising targeting molecules for HCC diagnostic and therapeutic drugs.

Applications of nanobodies in plant science and biotechnology.

KEY MESSAGE: Present review summarizes the current applications of nanobodies in plant science and biotechnology, including plant expression of nanobodies, plant biotechnological applications, nanobody-based immunodetection, and nanobody-mediated resistance against plant pathogens. Nanobodies (Nbs) are variable domains of heavy chain-only antibodies (HCAbs) isolated from camelids. In spite of their single domain structure, nanobodies display many unique features, such as small size, high stability, and cryptic epitopes accessibility, which make them ideal for sophisticated applications in plants and animals. In this review, we summarize the current applications of nanobodies in plant science and biotechnology, focusing on nanobody expression in plants, plant biotechnological applications, determination of plant toxins and pathogens, and nanobody-mediated resistance against plant pathogens. Prospects and challenges of nanobody applications in plants are also discussed.

Transcriptional factor Nrf2 is essential for aggresome formation during proteasome inhibition.

Aggrephagy, the aggresome-related protein degradation system, represents a protective cellular response to shuttle misfolded proteins into the microtubule-organizing center for degradation through the autophagic pathway during stress conditions, including heat shock, oxidative stress and proteasome inhibition. In response to proteasome failure, many genes are transcriptionally activated to facilitate ubiquitinated proteins to be cleared via the aggrephagy pathway. Although many regulators involved in aggresome formation have been identified, the mechanism how transcriptional activation promotes aggresome formation remains unknown. Here, we have demonstrated that nuclear factor erythroid 2-related factor 2 (Nrf2) accumulated in the nucleus and activated the transcription of sequestosome-1 (p62) during proteasome inhibition in 293 cells. Loss of Nrf2 resulted in failure of aggresome formation and cell death; whereas overexpression of p62 alleviated Nrf2 knockdown-induced aggresome formation defects and promoted cell survival. Notably, blocking Nrf2 activation using a p38/MAPK inhibitor prevented proteasome inhibitor-induced aggresome formation. These findings suggested that Nrf2 may be a critical regulator of aggresome formation, which protects cells from proteasome dysfunction-induced stress.

p38δ MAPK regulates aggresome biogenesis by phosphorylating SQSTM1 in response to proteasomal stress.

Aggresome formation is a major strategy to enable cells to cope with proteasomal stress. Misfolded proteins are assembled into micro-aggregates and transported to the microtubule organizing center (MTOC) to form perinuclear aggresomes before their degradation through autophagy. So far, multiple factors have been identified as the activators of micro-aggregate formation, but much less is known about the regulatory mechanisms of their transport. Here, we report that proteasomal stress leads to the activation of p38 MAPK family members. Two of them, p38γ (MAPK12) and p38δ (MAPK13), are dispensable for micro-aggregate formation but are required for their targeting to the MTOC. Interestingly, p38δ promotes micro-aggregate transport by phosphorylating SQSTM1, a major scaffold protein that assembles soluble ubiquitylated proteins into micro-aggregates. Expression of the phospho-mimetic mutant of SQSTM1 in p38δ-knockout cells completely rescued their aggresome formation defects and enhanced their resistance to proteasomal stress to wild-type levels. This study reveals p38δ-mediated SQSTM1 phosphorylation as a critical signal for the targeting of micro-aggregates to the MTOC and provides direct evidence for the survival advantages associated with aggresome formation in cells under proteasomal stress.

Numb positively regulates autophagic flux via regulating lysosomal function.

Autophagy is a lysosome-dependent catabolic process involving in the degradation and recycling of unnecessary or damaged proteins and organelles. Emerging evidence indicates that autophagy dysfunction is closely related to various human diseases including cancer, aging, myopathies and neurodegenerative disorders. Here, using genetic knockdown, we uncover the role of Numb, an endocytic adaptor protein, in regulating the late steps of autophagy. We found that Numb depletion led to the accumulation of autophagic vacuole, as verified by RFP-LC3 staining combined with transmission electron microscopy. Further investigation indicated that Numb depletion impaired autophagic degradation through inhibiting the activities of lysosomal enzymes (Cathepsin D, ß-glucuronidase and ß-glucosidase). Moreover, Numb depletion induced elevation of lysosomal pH values and decrease of glycosylated lysosome-associated membrane proteins. We further observed that Rab7 activity was inhibited in Numb-depleted cells. Together, our findings revealed a novel function of Numb and its likely mechanism in regulation of autophagy events.

Loss of PINK1 inhibits apoptosis by upregulating α-synuclein in inflammation-sensitized hypoxic-ischemic injury in the immature brains.

The incidence of preterm birth is rising worldwide. Among preterm infants, many face a lifetime of neurologic impairments. Recent studies have revealed that systemic inflammation can sensitize the immature brain to hypoxic-ischemic (HI) injury. Therefore, it is important to identify the mechanisms involved in inflammation-sensitized HI injury in immature brains. PTEN-induced putative kinase 1 (PINK1) is a regulatory protein that is highly expressed in the brain. We have previously found that PINK1 gene knockout can protect matured brains from HI injury in postnatal day 10 mice. However, the mechanisms are unknown. In this study, we employed an inflammation-sensitized HI injury model using postnatal day 3 mice to study the roles and mechanisms that PINK1 plays in the immature brains. Lipopolysaccharide (LPS) was injected intraperitoneally into the mice before HI treatment to set up the model. We found that PINK1-knockout mice had fewer brain infarcts and less cell apoptosis than did the wild-type mice. Furthermore, we found that α-synuclein was markedly higher in the PINK1-knockout mice than in the wild-type mice, and inhibition of α-synuclein through small interfering RNA (siRNA) reversed the protective effect in the PINK1-knockout mice. Collectively, these findings indicate that loss of PINK1 plays a novel role in the protection of inflammation-sensitized HI brain damage.

Cytosolic PINK1 promotes the targeting of ubiquitinated proteins to the aggresome-autophagy pathway during proteasomal stress.

During proteasomal stress, cells can alleviate the accumulation of polyubiquitinated proteins by targeting them to perinuclear aggresomes for autophagic degradation, but the mechanism underlying the activation of this compensatory pathway remains unclear. Here we report that PINK1-s, a short form of Parkinson disease (PD)-related protein kinase PINK1 (PTEN induced putative kinase 1), is a major regulator of aggresome formation. PINK1-s is extremely unstable due to its recognition by the N-end rule pathway, and tends to accumulate in the cytosol during proteasomal stress. Overexpression of PINK1-s induces aggresome formation in cells with normal proteasomal activities, while loss of PINK1-s function leads to a significant decrease in the efficiency of aggresome formation induced by proteasomal inhibition. PINK1-s exerts its effect through phosphorylation of the ubiquitin-binding protein SQSTM1 (sequestosome 1) and increasing its ability to sequester polyubiquitinated proteins into aggresomes. These findings pinpoint PINK1-s as a sensor of proteasomal activities that transduces the proteasomal impairment signal to the aggresome formation machinery.

Numb regulates vesicular docking for homotypic fusion of early endosomes via membrane recruitment of Mon1b.

Numb is an endocytic protein that plays crucial roles in diverse cellular processes such as asymmetric cell division, cell migration and differentiation. However, the molecular mechanism by which Numb regulates endocytic trafficking is poorly understood. Here, we demonstrate that Numb is a docking regulator for homotypic fusion of early endosomes (EEs). Numb depletion causes clustered but unfused EEs, which can be rescued by overexpressing cytosolic Numb 65 and Numb 71 but not plasma membrane-attached Numb 66 or Numb 72. Time-lapse analysis reveals that paired vesicles tend to tether but not fuse with each other in the absence of Numb. We further show that Numb binds to another docking regulator, Mon1b, and is required for the recruitment of cytosolic Mon1b to the EE membrane. Consistent with this, deletion of Mon1b causes similar defects in EE fusion. Our study thus identifies a novel mechanism by which Numb regulates endocytic sorting by mediating EE fusion.

Parkin-induced ubiquitination of Mff promotes its association with p62/SQSTM1 during mitochondrial depolarization.

The ubiquitin ligase Parkin and autophagic adapter protein p62 are known to function in a common pathway controlling mitochondrial autophagy (mitophagy). However, the evidence supporting that p62 is directly recruited by ubiquitinated proteins remains undetermined. Here, we demonstrate that mitochondrial fission factor (Mff) associates with Parkin and carbonyl cyanide m-chlorophenyl hydrazone treatment significantly increases the affinity of Parkin with Mff. After recruitment to depolarized mitochondria, Parkin mediates poly-ubiquitination of Mff at lysine 251. Replacement of lysine 251 by arginine (K251R) totally abrogates Parkin-stimulated ubiquitination of Mff. Subsequently, the ubiquitinated Mff promotes its association with p62. Mff knockout interferes with p62 translocation to damaged mitochondria. Only re-transfection of Mff WT, but not K251R mutant, rescues this phenotype. Furthermore, loss of Mff results in failure of Parkin translocation and final clearance of damaged mitochondria. Thus, our data reveal functional links among Mff, p62, and the selective autophagy of mitochondria, which are implicated in the pathogenesis of neurodegeneration diseases.

Antioxidant properties of high-density lipoproteins are impaired in women with polycystic ovary syndrome.

OBJECTIVE: To determine the relationships among the inflammatory index, intrinsic oxidation levels, lipid and apolipoprotein (apo)A-I concentrations of high-density lipoprotein (HDL), and polycystic ovary syndrome (PCOS). DESIGN: Cross-sectional study. SETTING: University hospital. PATIENT(S): A total of 425 patients with PCOS and 441 control women were included. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The HDL inflammatory index (HII) was determined using a cell-free fluorometric assay. Intrinsic HDL oxidation levels, HDL-free cholesterol, HDL-cholesterol ester, HDL-triglyceride, serum apoA-I, and malondialdehyde levels were also measured. RESULT(S): The mean HII value and the frequency of HII ≥1 were significantly higher in the PCOS group (0.77 ± 0.54, 27.1%) than in the control group (0.53 ± 0.37, 8.4%). These values were also higher in each of the 4 PCOS phenotypes based on the Rotterdam criteria than in the controls, and higher in patients with hyperandrogenism (HA) + oligo- and/or anovulation (OA) phenotype than in those with OA + polycystic ovary (PCO) phenotype. Furthermore, patients with PCOS with OA + PCO had lower malondialdehyde and intrinsic HDL oxidation levels compared with those with HA. Multivariate regression analysis demonstrated that PCOS, HDL-cholesterol ester, and E2 levels were the main predictors of HII value. CONCLUSION(S): The impairment of HDL antioxidant/anti-inflammatory function in PCOS is related to HA status, increased oxidative stress, and abnormalities in HDL components and thus may contribute to PCOS pathogenesis and increase the risks of future cardiovascular diseases.

[Promotion of Pink1S Auto-phosphorylation with CK2ß].

The aim of this study is to determine the regulatory mechanism of PTEN-induced putative kinase protein 1 short isoform (PINK1S) in cytoplasm. By co-immunoprecipitation (Co-IP) assay, we identified that PINK1S interacted with the beta regulatory subunit of Casein Kinase 2 (CK2ß), but not with the catalytic subunits CK2α1 and CK2α2. Furthermore, cells were transfected with PINK1S and CK2ß, and then PINK1S was purified by immunoprecipitation. After detecting the phosphorylated proteins by Phos-tag Biotin, we found that CK2ß overexpression increased auto-phosphorylation of PINK1S. Finally, we generated CK2ß knockdown cell lines by RNA interference. Purified PINK1S from CK2ß knockdown cells significantly reduced its auto-phosphorylation compared with control cells. These results suggested that CK2ß functions as a regulatory subunit of PINK1S kinase complex promoted its activation by self-phosphorylation.

[CK2beta promotes Pink1/Parkin-mediated MIRO1 degradation].

PTEN-induced putative kinase 1 (PINK1), a Parkinson's disease (PD)-related protein, has two isoforms, the mitochondria-localized full-length isoform PINK1FL and the cytoplasm-localized short isoform PINK1-cyto. Studies have suggested that PINK1FL can selectively accumulate at the surface of damaged mitochondria and cooperate with another Parkinson's Disease-related protein PARKIN to trigger the degradation of MIRO1, a mitochondria trafficking regulator. The functions of PINK1-cyto are, however, not yet clear. To investigate the functions of PINK1-cyto, we expressed different proteins in cultured HEK293 cells by transfecting it with different plasmids, and detected the protein levels by Western blot after expressing for 24 h. We found that in cultured HEK293 cells, PINK1-cyto could also cooperate with PARKIN degrade MIRO1 in the presence of CK23, and the regulatory subunit of Casein Kinase II. Interestingly, this function of CK2P was not dependent on CK2alpha, the catalytic subunit of Casein Kinase II. We also found that CK2P could promote the direct interaction between PINK1-cyto and MIRO1 by immunocoprecipitation analysis. This result suggested that in addition to CK2alpha, CK2beta could also form a kinase complex.

[Autophagy promoted by Pakinson's disease related protein Pink1].

OBJECTIVE: To investigate the function of Parkinson's disease (PD)-related protein Pink1 in autophagy. METHODS: Pink1, autophagy inhibitor Bcl-XL and autophagy induced-factor Beclin1 were amplified with RT-PCR and contructed into pcDNA3. 1 (+) vector. The stable HEK293 cell line of Pink1 expression was established through the lentivirus system. Pink1, Bcl-XL and Beclin1 were tansfected into the HEK293 Cell Line. Co-Immunoprecipitation and Western blot were performed to determine the interaction between Pink1 and Bcl-XL, the effect of Pink1 on the interaction between Bcl-XL and Beclin1, and the function of Pink1 in autophagy. RESULTS: There was a new interaction between Pink1 and antophagy inhibitor Bcl-XL. Overexpressed Pink1 promoted the dissociation of autophagy induced-factor Beclin1 from Bcl-XL. Overexpressed Pink1 increased the autophagic protein LC3 II/LC3 I. CONCLUSION: Pakinson's disease related protein Pink1 promotes the dissociation of autophagy induced-factor Beclin1 from antophagy inhibitor Bcl-XL and promotes autophagy.

[The mechanism of PINK1 localization on the outer membrane of mitochondria].

OBJECTIVE: To study the specific mechanism of PTEM-induced putative kinase 1(PINK1) located to the outer membrane of damaged mitochondria. METHODS: Cultured HEK293T cells were transfected with plasmids expressing different proteins, following with DMSO or CCCP treatment. Western blot and coimmuoprecipitation were used to detect the expression and interaction of proteins. RESULTS: Full length PINK1, but not its mitochondria targeting sequence (MTS) & trans-membrane (TM) deleted forms or other outer mitochondria outer membrane proteins, could interact with Tom40 upon CCCP treatment and the interaction ability was more than 20 times stronger than that of DMSO control. When the added CCCP is washed out, the interaction between full length PINK1 and Tom40 declined rapidly. PINK1 with removed or mutated TM can interact with Tom40 even in the absence of CCCP. CONCLUSION: The accumulated PINK1 on the outer membrane of damaged mitochondria is just stuck on the TOM complex instead of integrated into the lipid bilayer.

Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning.

LRRK2 (PARK8) is the most common genetic determinant of Parkinson's disease (PD), with dominant mutations in LRRK2 causing inherited PD and sequence variation at the LRRK2 locus associated with increased risk for sporadic PD. Although LRRK2 has been implicated in diverse cellular processes encompassing almost all cellular compartments, the precise functions of LRRK2 remain unclear. Here, we show that the Drosophila homolog of LRRK2 (Lrrk) localizes to the membranes of late endosomes and lysosomes, physically interacts with the crucial mediator of late endosomal transport Rab7 and negatively regulates rab7-dependent perinuclear localization of lysosomes. We also show that a mutant form of lrrk analogous to the pathogenic LRRK2(G2019S) allele behaves oppositely to wild-type lrrk in that it promotes rather than inhibits rab7-dependent perinuclear lysosome clustering, with these effects of mutant lrrk on lysosome position requiring both microtubules and dynein. These data suggest that LRRK2 normally functions in Rab7-dependent lysosomal positioning, and that this function is disrupted by the most common PD-causing LRRK2 mutation, linking endolysosomal dysfunction to the pathogenesis of LRRK2-mediated PD.

Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin.

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinson's disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinson's disease-like pathology. Recently, multiple genes mediating familial forms of Parkinson's disease have been identified, including PTEN-induced kinase 1 (PINK1; PARK6) and parkin (PARK2), which are also associated with sporadic forms of Parkinson's disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. Here we show that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1-parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinson's disease pathogenesis.

Regulation and function of local protein synthesis in neuronal dendrites.

It has long been shown that protein synthesis can occur in neuronal dendrites, but its significance remained unclear until relatively recently. Studies suggest that local protein synthesis has crucial roles in synaptic plasticity, the change in neuronal communication efficiency that is probably a cellular basis of learning and memory. Induced by neuronal activity, local protein synthesis provides key factors for the modification of activated synapses. In this review, we summarize the evidence for local protein synthesis and its functions in synaptic plasticity. We also discuss the molecular mechanisms by which neuronal activity induces the synthesis of proteins that allow for changes in synaptic function.