Kinesin is a motor protein that uses the energy from ATP hydrolysis to move along the microtubule system. To investigate how the chemical energy stored in ATP is converted to mechanical movement, the corresponding N-terminal region of rat brain kinesin was expressed in BL21-Codon Plus (DE3)-RP competent cells. After SP-cation exchange chromatography and size exclusion chromatography, the protein yield reached 10 mg/L culture with the purity above 95%. The purified protein had ATPase activity and specifically reacted with the kinesin antibody in the Western blotting analysis. The purified kinesin was crystallized under the following condition: 1.7 mol/L (NH4)2SO4, 500 mmol/L NaCl, 20% glycerol. The kinesin crystal can diffract up to 2.0 angstroms resolution.
Animals ; Brain ; enzymology ; Crystallization ; Hydrolysis ; Kinesin ; chemistry ; isolation & purification ; Rats

The kinesin superfamily is a class of motor proteins moving along microtubule filaments and playing essential roles in mitosis of eukaryotic cells. In the cancer biology, mitotic activity is an essential factor for development and metastasis of various cancers. Therefore, the inhibition of kinesin activity is suggested as an alternative cancer therapy. Accumulated clinical evidences have proved the potency of kinesin inhibitors in cancer treatments. In this review, we provided an overview of kinesins that play a critical role in the pathophysiology of various cancers and described the beneficial vs. side effects of their inhibitors that have been tested in both basic science and clinical studies.
Biology ; Eukaryotic Cells ; Kinesin* ; Microtubules ; Mitosis ; Neoplasm Metastasis ; Translational Medical Research*

The Kinesin superfamily proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF1Balpha is a monomeric motor that conveys mitochondria and plays an important role in cellular function. Here, we used the yeast two-hybrid system to identify the proteins that interacts with KIF1Balpha and found a specific interaction with the mammalian LIN-7 (MALS)-3/vertebrate homology of LIN-7 (Veri) and synaptic scaffolding molecule (S-SCAM). MALS-3 protein bound to the tail region of KIF1Balpha but not to other kinesin family members in the yeast two-hybrid assay. The "T-X-V" motif at the C-terminal end of KIF1Balpha is essential for interaction with MALS-3. In addition, this protein showed specific interactions in the Glutathione S-transferase (GST) pull-down assay. An antibody to MALS-3 specifically coimmunoprecipitated KIF1Balpha associated with MALS-3 from mouse brain extracts. These results suggest that MALS-3, as KIF1Balpha receptor, is involved in the KIF1Balpha-mediated transport.
Animals ; Brain ; Glutathione Transferase ; Humans ; Kinesin* ; Mice ; Microtubules ; Mitochondria ; Organelles ; PDZ Domains* ; Two-Hybrid System Techniques

The kinesin proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF1A is a monomeric motor that conveys synaptic vesicle precursors and plays an important role in neuronal function. Here, we used the yeast two-hybrid system to identify the neuronal protein (s) that interacts with the tail region of KIF1A and found a specific interaction with synaptotagmin XI. The amino acid residues between 830 and 1300 of KIF1A are required for the interaction with synaptotagmin XI. KIF1A also bound to the tail region of synaptotagmin IV but not to other synaptotagmin in the yeast two-hybrid assay. KIF1A interacted with GST-synaptotagim XI fusion proteins, but not with GST alone. An antibody to synaptotagmin XI specifically co-mmunoprecipitated KIF1A associated with synaptotagimin from mouse brain extracts. These results suggest that KIF1A motor protein transports of synaptotagmin XI-containing synaptic vesicle precursors along microtubule.
Animals ; Brain ; Kinesin* ; Mice ; Microtubules ; Neurons ; Organelles ; Protein Transport ; Synaptic Vesicles ; Synaptotagmins* ; Two-Hybrid System Techniques

The kinesin proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF5 is a heterotetrameric motor that conveys vesicles and plays an important role in neuronal function. Here, we used the yeast two-hybrid system to identify the neuronal protein (s) that interacts with the tail region of KIF5 and found a specific interaction with betaIII spectrin. The amino acid residues between 1394 and 1774 of betaIII spectrin were required for the interaction with KIF5C. betaIII spectrin also bound to the tail region of neuronal KIF5A and ubiquitous KIF5B but not to other kinesin family members in the yeast two-hybrid assay. In addition, these proteins showed specific interactions, confirmed by GST pull-down assay and co-immunoprecipitation. betaIII spectrin interacted with GST-KIF5 fusion proteins, but not with GST alone. An antibody to betaIII spectrin specifically co-immunoprecipitated KIF5s associated with betaIII spectrin from mouse brain extracts. These results suggest that KIF5 motor proteins transport vesicles or organelles that are coated with betaIII spectrin.
Animals ; Brain ; Humans ; Immunoprecipitation ; Kinesin* ; Mice ; Microtubules ; Neurons ; Organelles ; Spectrin* ; Transport Vesicles ; Two-Hybrid System Techniques

KIF1B beta is a member of the Kinesin superfamily proteins (KIFs), which are microtubule-dependent molecular motors that are involved in various intracellular organellar transport processes. KIF1B beta is not restricted to neuronal systems, however, is widely expressed in other tissues, even though the function of KIF1B beta is still unclear. To elucidate the KIF1B beta-binding proteins in non-neuronal cells, we used the yeast two-hybrid system, and found a specific interaction of KIF1B beta and the sorting nexin (SNX) 17. The C-terminal region of SNX17 is required for the binding with KIF1B beta. SNX17 protein bound to the specific region of KIF1B beta (813-916. aa), but not to other kinesin family members. In addition, this specific interaction was also observed in the Glutathione S-transferase pull-down assay. An antibody to SNX17 specifically co-immunoprecipitated KIF1B beta associated with SNX17 from mouse brain extracts. These results suggest that SNX17 might be involved in the KIF1B beta-mediated transport as a KIF1B beta adaptor protein.
Animals ; Brain ; Glutathione Transferase ; Humans ; Kinesin ; Mice ; Microtubules ; Neurons ; Proteins ; Sorting Nexins ; Two-Hybrid System Techniques

OBJECTIVETo study the relationship between SNP rs17401966 at the KIF1B gene and the genetic susceptibility to Hepatocellular carcinoma (HCC).

METHODSAll study objects were recruited from two Grade A hospitals of Amoy from January 2011 to October 2014.They were surveyed in individual matching case-control study. Accepting criterias in the cases: HCC was first diagnosed based on diagnostic basis during the investigations, over 18 years old, present addresses were as same as surveyed areas in the district (county) level range, no past history of cancers; Exclusion criterias: patients with other liver diseases. The tumor patients without HCC, patients with autoimmune hepatitis or toxic hepatitis, patients who refused to be investigated or too ill to be investigated. Accepting criterias in the controls: the control who passed the physical examination matched the case in ages (no more than 3 years old), sex, health screening in the same hospital over the same period and district (county); Exclusion criterias: people with liver disease or any history of cancers. This study consisted of 376 HCC patients and 403 controls, 5 ml morning fasting venous blood of all subjects were obtained to isolate cells and distribute genotype. The differences in general information between cases and controls were tested by χ² test and t-test. The association between SNP rs17401966 and the risk of developing HCC were assessed by using the multiple factors logistic regression.

RESULTSThe mean age and standard deviation for case and control groups were (61.7 ± 12.8) years and (60.6 ± 12.7) years (t = 1.15, P = 0.251), respectively. The proportion of family history of cancer [28.7% (108/376)] and the HBsAg positive rate [26.9 % (101/376)] in case group were higher than these in control group [15.9% (64/403), 2.7% (11/403)] (χ² = 18.65, 92.02, P < 0.001). In HBsAg carriers, GG genotype genetic susceptibility to HCC is 0.12 (0.02-0.75) times for AA genotype, and G allele susceptibility to HCC is 0.38 (0.15-0.98) times for A allelc. In HBsAg negative group, it showed no statistical significance in the relationship between SNP rs17401966 and susceptibility to HCC, and compared with the A allele, the risk for HCC of G allele is 0.79 (0.62-1.01).

CONCLUSIONThe results demonstrated that the presence of the GG genotype, the GA genotype and the G allele at rs17401966 of the KIF1B gene might decrease the risk for HCC.

Aged ; Alleles ; Carcinoma, Hepatocellular ; Case-Control Studies ; Genetic Predisposition to Disease ; Genotype ; Humans ; Kinesin ; Liver Neoplasms ; Middle Aged ; Polymorphism, Single Nucleotide

Kinesin spindle protein (KSP/Eg5) is essential for the formation and maintenance of bipolar spindles during mitosis. Inhibition of this protein leads to cell cycle arrest and apoptosis without interfering other microtubule-dependent processes. Therefore, it is a potential target in cancer therapy. Here, a series of tetrahydro-beta-carboline derivatives 5a - k were synthesized as kinesin spindle protein inhibitor. Their structures were confirmed with 1H NMR, ESI-MS and elemental analysis. The synthesized compounds were evaluated for their inhibition of KSP.
Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Carbolines ; chemical synthesis ; chemistry ; pharmacology ; Kinesin ; antagonists & inhibitors ; metabolism ; Molecular Structure

In association with microtubules, a variety of kinesins play important roles in cellular functions such as intracellular transport of organelles or vesicles, signal transduction, and cell division. In a previous study we revealed that human kinesin superfamily protein member 4 (KIF4) is a chromokinesin that binds to chromosomes. Since localization of several kinds of kinesin at midzone called central spindle, or midbody that connects two daughter cells, or both, suggests their implication in cell division, we investigated KIF4 localization of during mitosis and cytokinesis in Hela cells. In addition to association with segregating chromosomes through entire mitosis, it also localized to the midzone and to midbody at ana/telophase through cytokinesis. Especially in cells at cytokinesis, KIF4 appeared as a doublet facing each other at the apical ends of two daughter cells. Three- dimensional analysis of architectural relationship between microtubule bundles and KIF4 indicated that KIF4 forms a ring structure wrapping around the microtubule bundles. These results suggest that KIF4 is involved in cytokinesis, although direct evidence was not provided in this study.
Animals ; Cell Division/*physiology ; Hela Cells ; Humans ; Immunohistochemistry ; Kinesin/*metabolism ; Mitotic Spindle Apparatus/*metabolism ; Research Support, Non-U.S. Gov't

OBJECTIVE: To analyze the pathogenic variants of the KIF1A gene and its corresponding protein structure in an autism spectrum disorder (ASD) family trio carrying harmful missense variants in the KIF1A gene. METHODS: The peripheral blood DNA of the patient and his parents was extracted and sequenced using whole exome sequencing (WES) technology and verified by Sanger sequencing. Bioinformatics software SIFT, PolyPhen-2, Mutation Taster, and CADD software were used to analyze the harmfulness and conservation of variants. The Human Brain Transcriptome (HBT) database was used to analyze the expression of the KIF1A gene in the brain. PredictProtein and SWISS-MODEL were further used to predict the secondary structure and tertiary structure of KIF1A wild-type protein and variant protein. PyMOL V2.4 was utilized to investigate the change of hydrogen bond connection after protein variant. RESULTS: The WES sequencing revealed a missense variant c.664A>C (p.Asn222His) in the child's KIF1A gene, and this variant was a de novo variant. The harmfulness prediction results suggest that this variant is harmful. By analyzing expression level of KIF1A gene in the brain. It is found that KIF1A gene widely expressed in various brain regions during embryonic development. By analyzing the variant protein structure, the missense variant of KIF1A will cause many changes in the secondary structure of protein, such as alpha-helix, beta-strand, and protein binding domain. The connection of hydrogen bond and spatial structure will also change, thereby changing the original biological function. CONCLUSION The KIF1A gene may be a risk gene for ASD.
Autism Spectrum Disorder/genetics* ; Child ; Female ; Humans ; Kinesin/genetics* ; Mutation ; Mutation, Missense ; Pregnancy ; Protein Domains ; Whole Exome Sequencing