The L279P Mutation of Nuclear Distribution Gene C (NudC) Influences Its Chaperone Activity and Lissencephaly Protein 1 (LIS1) Stability

LIS1, a gene mutated in classical lissencephaly, plays essential roles in cytoplasmic dynein regulation, mitosis and cell migration. However, the regulation of LIS1 (lissencephaly protein 1) protein remains largely unknown. Genetic studies in Aspergillus nidulans have uncovered that the Nud (nuclear distribution) pathway is involved in the regulation of cytoplasmic dynein complex and a temperature-sensitive mutation in the nudC gene (L146P) greatly reduces the protein levels of NudF, an Aspergillus ortholog of LIS1. Here, we showed that L146 in Aspergillus NudC and its flanking region were highly conservative during evolution. The similar mutation in human NudC (L279P) obviously led to reduced LIS1 and cellular phenotypes similar to those of LIS1 down-regulation. To explore the underlying mechanism, we found that the p23 domain-containing protein NudC bound to the molecular chaperone Hsp90, which is also associated with LIS1. Inhibition of Hsp90 chaperone function by either geldanamycin or radicicol resulted in a decrease in LIS1 levels. Ectopic expression of Hsp90 partially reversed the degradation of LIS1 caused by overexpression of NudC-L279P. Furthermore, NudC was found to regulate the ATPase activity of Hsp90, which was repressed by the mutation of L279P. Interestingly, NudC itself was shown to possess a chaperone function, which also was suppressed by the L279P mutation. Together, these data suggest that NudC may be involved in the regulation of LIS1 stability by its chaperone function.     

Structural features and chaperone activity of the NudC protein family

The NudC family consists of four conserved proteins with representatives in all eukaryotes. The archetypal nudC gene from Aspergillus nidulans is a member of the nud gene family that is involved in the maintenance of nuclear migration. This family also includes nudF, whose human orthologue, Lis1, codes for a protein essential for brain cortex development. Three paralogues of NudC are known in vertebrates: NudC, NudC-like (NudCL), and NudC-like 2 (NudCL2). The fourth distantly related member of the family, CML66, contains a NudC-like domain. The three principal NudC proteins have no catalytic activity but appear to play as yet poorly defined roles in proliferating and dividing cells. We present crystallographic and NMR studies of the human NudC protein and discuss the results in the context of structures recently deposited by structural genomics centers (i.e., NudCL and mouse NudCL2). All proteins share the same core CS domain characteristic of proteins acting either as cochaperones of Hsp90 or as independent small heat shock proteins. However, while NudC and NudCL dimerize via an N-terminally located coiled coil, the smaller NudCL2 lacks this motif and instead dimerizes as a result of unique domain swapping. We show that NudC and NudCL, but not NudCL2, inhibit the aggregation of several target proteins, consistent with an Hsp90-independent heat shock protein function. Importantly, and in contrast to several previous reports, none of the three proteins is able to form binary complexes with Lis1. The availability of structural information will be of help in further studies on the cellular functions of the NudC family.     

NudCL2 is an autophagy receptor that mediates selective autophagic degradation of CP110 at mother centrioles to promote ciliogenesis

Primary cilia extending from mother centrioles are essential for vertebrate development and homeostasis maintenance. Centriolar coiled-coil protein 110 (CP110) has been reported to suppress ciliogenesis initiation by capping the distal ends of mother centrioles. However, the mechanism underlying the specific degradation of mother centriole-capping CP110 to promote cilia initiation remains unknown. Here, we find that autophagy is crucial for CP110 degradation at mother centrioles after serum starvation in MEF cells. We further identify NudC-like protein 2 (NudCL2) as a novel selective autophagy receptor at mother centrioles, which contains an LC3-interacting region (LIR) motif mediating the association of CP110 and the autophagosome marker LC3. Knockout of NudCL2 induces defects in the removal of CP110 from mother centrioles and ciliogenesis, which are rescued by wild-type NudCL2 but not its LIR motif mutant. Knockdown of CP110 significantly attenuates ciliogenesis defects in NudCL2-deficient cells. In addition, NudCL2 morphants exhibit ciliation-related phenotypes in zebrafish, which are reversed by wild-type NudCL2, but not its LIR motif mutant. Importantly, CP110 depletion significantly reverses these ciliary phenotypes in NudCL2 morphants. Taken together, our data suggest that NudCL2 functions as an autophagy receptor mediating the selective degradation of mother centriole-capping CP110 to promote ciliogenesis, which is indispensable for embryo development in vertebrates.Subject terms: Cilia, Centrosome     

Distinct Functions of Nuclear Distribution Proteins LIS1, Ndel1 and NudCL in Regulating Axonal Mitochondrial Transport

Neurons critically depend on the long‐distance transport of mitochondria. Motor proteins kinesin and dynein control anterograde and retrograde mitochondrial transport, respectively in axons. The regulatory molecules that link them to mitochondria need to be better characterized. Nuclear distribution (Nud) family proteins LIS1, Ndel1 and NudCL are critical components of cytoplasmic dynein complex. Roles of these Nud proteins in neuronal mitochondrial transport are unknown. Here we report distinct functions of LIS1, Ndel1 and NudCL on axonal mitochondrial transport in cultured hippocampal neurons. We found that LIS1 interacted with kinsein family protein KIF5b. Depletion of LIS1 enormously suppressed mitochondrial motility in both anterograde and retrograde directions. Inhibition of either Ndel1 or NudCL only partially reduced retrograde mitochondrial motility. However, knocking down both Ndel1 and NudCL almost blocked retrograde mitochondrial transport, suggesting these proteins may work together to regulate retrograde mitochondrial transport through linking dynein‐LIS1 complex. Taken together, our results uncover novel roles of LIS1, Ndel1 and NudCL in the transport of mitochondria in axons.      

Characterization and function analysis of Hsp60 and Hsp10 under different acute stresses in black tiger shrimp, <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Heat shock proteins (Hsps) are a class of highly conserved proteins produced in virtually all living organisms from bacteria to humans. Hsp60 and Hsp10, the most important mitochondrial chaperones, participate in environmental stress responses. In this study, the full-length complementary DNAs (cDNAs) of Hsp60 (PmHsp60) and Hsp10 (PmHsp10) were cloned from Penaeus monodon. Sequence analysis showed that PmHsp60 and PmHsp10 encoded polypeptides of 578 and 102 amino acids, respectively. The expression profiles of PmHsp60 and PmHsp10 were detected in the gills and hepatopancreas of the shrimps under pH challenge, osmotic stress, and heavy metal exposure, and results suggested that PmHsp60 and PmHsp10 were involved in the responses to these stimuli. ATPase and chaperone activity assay indicated that PmHsp60 could slow down protein denaturation and that Hsp60/Hsp10 may be combined to produce a chaperone complex with effective chaperone and ATPase activities. Overall, this study provides useful information to help further understand the functional mechanisms of the environmental stress responses of Hsp60 and Hsp10 in shrimp.     

Molecular characterization and expression analysis of <Emphasis Type="Italic">Hsp90</Emphasis> in <Emphasis Type="Italic">Schizothorax prenanti</Emphasis>

Aquatic animals suffer from various environmental stresses because the aquatic environment is a very complex system. To monitor the health status of fish, Hsp90 a potential early warning marker was determined in Schizothorax prenanti after infection with a bacterium. In this study, we cloned Hsp90 from S. prenanti for the first time. The full-length cDNA sequence of SpHsp90 was 2663 bp, contains an open reading frame of 2181 bp, and has a gene encoding 726 amino acids, an estimated molecular mass of 83.38 kDa, and a theoretical isoelectric point of 4.91. The SpHsp90 amino acid sequence has five conserved HSP90 family signatures and shares 87.0–95.5 % identity with other vertebrates. Phylogenetic analysis and structure comparison indicated that SpHsp90 should be a β isoform of the HSP90 family. SpHsp90 was ubiquitously expressed in all examined tissues, and the highest level of expression was in the kidney. After Streptococcus agalactiae infection, the level of SpHsp90 expression had significant changes (P < 0.05) in the hepatopancreas, spleen, kidney, and blood. The expression increased to the highest level at 6 h in the blood and at 24 h in the hepatopancreas, spleen, and kidney. The results suggested that the SpHsp90 gene could be induced by S. agalactiae in S. prenanti and that SpHsp90 may be involved in resistance to bacterial infection and provide an early warning information. The kidney is the most suitable for detecting SpHsp90 after bacterial infection.     

Association between circulating levels of heat-shock protein 27 and aggressive periodontitis

Heat-shock protein (Hsp) 27 is a major intracellular molecular chaperone and controller of intracellular responses to inflammatory signals. In the extracellular space, recombinant Hsp27 has been described to exert anti-inflammatory activities. The aim of this study was to assess the association between circulating levels of Hsp27 and different types of periodontitis. Pro- and anti-inflammatory cytokines and the stress proteins Hsp27 and Hsp60 with proposed anti- and pro-inflammatory properties, respectively, were measured by two-site ELISA in the serum of patients with aggressive periodontitis (AgP, n?=?30), chronic periodontitis (CP, n?=?29) and periodontally healthy controls (H, n?=?28). Furthermore, Hsp27 and Hsp60 levels were also measured longitudinally in 12 AgP patients at 6 time points up to 3 months after treatment. AgP patients had lower levels of Hsp27 compared to CP patients and healthy subjects (adjusted one-way ANOVA, p?<?0.001, followed by post hoc Tukey HSD comparisons), while no differences in levels of Hsp60 or cytokines between the three groups were detected. In CP patients and H subjects, the systemic Hsp27 levels correlated with Hsp60 (r?=?0.43, p?<?0.001; r?=?0.59, p?<?0.001, respectively) and with pro-inflammatory cytokines TNF-α (r?=?0.48, p?<?0.001; r?=?0.55, p?<?0.001, respectively) and IL-6 (r?=?0.44, p?<?0.01). However, no such correlations were detected in AgP cases. No consistent temporal patterns of changes of Hsp27 concentration were detected across AgP patients following periodontal treatment. This study provides the first evidence that Hsp27 may be differentially expressed and regulated in AgP patients as compared with CP patients and healthy individuals.     

Genomic,evolutionary and expression profile analysis of <Emphasis Type="Italic">Hsp70</Emphasis> superfamily in A and D genome of cotton (<Emphasis Type="Italic">Gossypium</Emphasis> spp.) under the challenge of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

In this study, we comparatively analyzed the 115 Hsp70 genes identified in Gossypium raimondii, Gossypium hirsutum and Gossypium arboreum genomes. Those Hsp70 genes unequally distributed among chromosomes in A and D genome of cotton (Gossypium spp.), and were classified into 29 groups according to the homology of them. Based on the localization information of the orthologs in Arabidopsis, the Hsp70 proteins were predicted to locate in cytosol, endoplasmic reticulum, mitochondrion or chloroplast. Homologous analysis indicated the evolutionary conservation of Hsp70 in cotton. In addition, those Hsp70 genes were differently expressed in Suyuan-045, Hai-7124 and TM-1, which were highly resistant, resistant, and sensitive to Verticillium dahliae respectively. The expressions of 26 Hsp70 genes were induced by Verticillium dahliae except for Hsp70-07/16/25/26, and the result suggested the potential involvement of them in responding to Verticillium wilt. Hsp70-08/30/31 was highly expressed in both Suyuan-045 and Hai-7124, and it was hypothesized that they might be involved in the resistance to the invasion of Verticillium dahliae. 144h after inoculation with Verticillium dahliae, the expression of Hsp70-13/14/15 was only up-regulated in Suyuan-045, and it was assumed that they might be involved in resistance to the extension of Verticillium dahliae. Further study on those Hsp70 genes would be valuable to reveal the role of them in Verticillium wilt resistance.     

Small phosphatidate phosphatase (<Emphasis Type="Italic">TtPAH2</Emphasis>) of <Emphasis Type="Italic">Tetrahymena</Emphasis> complements respiratory function and not membrane biogenesis function of yeast <Emphasis Type="Italic">PAH1</Emphasis>

Phosphatidate phosphatases (PAH) play a central role in lipid metabolism and intracellular signaling. Herein, we report the presence of a low-molecular-weight PAH homolog in the single-celled ciliate Tetrahymena thermophila. In vitro phosphatase assay showed that TtPAH2 belongs to the magnesium-dependent phosphatidate phosphatase (PAP1) family. Loss of function of TtPAH2 did not affect the growth of Tetrahymena. Unlike other known PAH homologs, TtPAH2 did not regulate lipid droplet number and ER morphology. TtPAH2 did not rescue growth and ER/nuclear membrane defects of the pah1? yeast cells, suggesting that the phosphatidate phosphatase activity of the protein is not sufficient to perform these cellular functions. Surprisingly, TtPAH2 complemented the respiratory defect in the pah1? yeast cells indicating a specific role of TtPAH2 in respiration. Overall, our results indicate that TtPAH2 possesses the minimal function of PAH protein family in respiration. We suggest that the amino acid sequences absent from TtPAH2 but present in all other known PAH homologs are critical for lipid homeostasis and membrane biogenesis.     

Genome-wide identification and expression analysis of the molecular chaperone binding protein <Emphasis Type="Italic">BiP</Emphasis> genes in <Emphasis Type="Italic">Citrus</Emphasis>

Here, we report for the first time the genome-wide identification and expression analysis of the molecular chaperone BiP genes in Citrus. Six genes encoding the conserved protein domain family GPR78/BiP/KAR2 were identified in the genome of Citrus sinensis and C. clementina. Two of them, named here as CsBiP1 and CsBiP2, were classified as true BiPs based on their deduced amino acid sequences. Alignment of the deduced amino acid sequences of CsBiP1 and CsBiP2 with BiP homologs from soybean and Arabidopsis showed that they contain all the conserved functional motifs of BiPs. Analysis of the promoter region of CsBiPs revealed the existence of cis-acting regulatory sequences involved in abiotic, heat-shock, and endoplasmic reticulum (ER) stress responses. Publicly available RNA-seq data indicated that CsBiP1 is abundantly expressed in leaf, flower, fruit, and callus, whereas CsBiP2 expression is rarely detected in any tissues under normal conditions. Comparative quantitative real-time PCR (qPCR) analysis of expression of these genes between C. sinensis grafted on the drought-tolerant “Rangpur” lime (C. limonia) and -sensitive “Flying Dragon” trifoliate orange (Poncirus trifoliata) rootstocks showed that CsBiP1 was upregulated by drought stress on the former but downregulated on the latter, whereas the CsBiP2 mRNA levels were downregulated on drought-stressed “Flying Dragon,” but remained constant on “Rangpur.” CsBiP2 upregulation was only observed in C. sinensis seedlings subjected to osmotic and cold treatments. Taken together, these results indicate the existence of two highly conserved BiP genes in Citrus that are differentially regulated in the different tissues and in response to abiotic stresses.     

<Emphasis Type="Italic">t</Emphasis>-Specific <Emphasis Type="Italic">D17Leh66</Emphasis> DNA Elements in the Family Muridae

Blot-hybridization analysis with the use of the t-specific probe D17Leh66 has been used to study DNA of various representatives of family Muridae. Hamsters from genus Phodopus have no homologs of this probe, whereas African rats from genus Lophuromys have some homologous elements. This indicates that sequence D17Leh66 is ancient and was probably present in the common ancestor of family Muridae.     

<Emphasis Type="Italic">TaELF3-1DL</Emphasis>, a homolog of <Emphasis Type="Italic">ELF3</Emphasis>, is associated with heading date in bread wheat

Early flowering 3 (ELF3) is a regulator to modulate photoperiod flowering in Arabidopsis. The homologs of ELF3 in rice and barley also have been identified essential for regulation of flowering time. In the current study, TaELF3 genes, homologs of ELF3 in bread wheat (Triticum aestivum L.), were cloned by a comparative genomics approach and located on homologous group 1 chromosomes, designated as TaELF3-1AL, TaELF3-1BL, and TaELF3-1DL, respectively. A sequence-tagged site (STS) marker was developed based on sequence polymorphism at the TaELF3-1DL locus. A quantitative trait locus (QTL) for heading date (HD) co-segregating with TaELF3-1DL explained 7.7–20.6% of the phenotypic variance in a RIL mapping population derived from the Gaocheng 8901/Zhoumai 16 cross genotyped using the wheat 90K iSelect assay. The late HD allele of TaELF3-1DL was prevalently selected in China’s specific wheat-growing regions and other countries. This study produces novel information in better understanding HD and provides a reliable functional marker for molecular marker-assisted selection in wheat breeding.     

Hsp90 depletion goes wild

Hsp90 reveals phenotypic variation in the laboratory, but is Hsp90 depletion important in the wild? Recent work from Chen and Wagner in BMC Evolutionary Biology has discovered a naturally occurring Drosophila allele that downregulates Hsp90, creating sensitivity to cryptic genetic variation. Laboratory studies suggest that the exact magnitude of Hsp90 downregulation is important. Extreme Hsp90 depletion might reactivate transposable elements and/or induce aneuploidy, in addition to revealing cryptic genetic variation.See research article http://wwww.biomedcentral.com/1471-2148/12/25