Cells synthesize various distinct kinds of proteins for developing and maintaining cell morphology and functions. To distribute these proteins to their proper destinations, many motor proteins are recruited to the microtubule cytoskeleton, whose plus ends generally point to the cell periphery or axon tips of the neurons. One of the most important and well-studied motor families is kinesin superfamily proteins (KIFs). Kinesin was discovered decades ago. So far, 15 mammalian kinesin families (kinesin 1-13, 14A and 14B) have been identified, which consist of totally 45 mammalian KIF genes. All the members of KIFs have so-called "motor domain" that generally moves along the microtubules, driving through the energy from ATP hydrolysis. Although these motor domains are conserved, regions outside the motor domains can be quite divergent and unique to individual motors, enabling various cargoes to be bound. Up to now, members of KIFs have been shown to transport synaptic vesicle precursors, mitochondria, membrane organelles, cilia components, and mRNAs. In addition to function as motor proteins, a member of KIFs (KIF26A) has been also found to be a signaling regulator.

KIF12 belongs to the kinesin-12 family and contains 642 amino acid residues. It consists of an N-terminal head domain, a stalk domain and a C-terminal tail domain. Kinesin motor (22-288 aa) is located to the head domain. Coiled-coil region (376-465 aa) forms the stalk domain. And in the C-terminal, a tail domain has been identified, including a proline rich domain (469-554 aa). KIF12 was firstly identified by degenerate PCR. According to a quantitative trait loci (QTL) gene discovery system established by the Complex Trait Consortium, it has been proposed as a cpk (congenital polycystic kidney) modifier gene. Kif12 transcription is regulated by HNF1α/β, which expresses in kidney and pancreatic islets. However, the molecular function and the physiological relevance of KIF12 have been still very elusive.

The peroxisome is a metabolic organelle found in all eukaryotes except the Archaezoa. Peroxisomes contain catalase, essential for turnover of H2O2 to reduce the oxidative stress. It also contains several β-oxidation enzymes essentially metabolizing very long chain fatty acids (VLCFA). Deficiency in peroxisome assembly or functions was reported to result in inherited disorders such as Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD) and acatalasemia, and to increase the risk to develop type 2 diabetes. Because peroxisomes do not contain genome or protein synthesis machinery, most peroxisomal enzymes are synthesized on ribosomes free in the cytosol, and targeted to peroxisomes according to the peroxisome targeting signals (PTSs) on the proteins. So far two kinds of PTSs have been identified, called PTS1 and PTS2. The PTS1 signal is generally a SKL (Ser-Lys-Leu) tripeptide or its variants and located to the extreme carboxyl terminal of the protein. Catalase targets to the peroxisomes by its PTS1 signal. On the other hand, the PTS2 signal is a nonapeptide sequence located near the N terminus or at internal locations of proteins, sharing the consensus sequence (R/K) (L/V/I) (X)5 (H/Q) (L/A) (X = any amino acid). Peroxisomal enzyme acetyl-CoA acyltransferase 1 (ACAA1) targets to the peroxisomes by its PTS2 signal.

Peroxisome matrix proteins are targeted to peroxisomes properly and efficiently to maintain peroxisome morphology and functions. How proteins import into the peroxisomes and how this targeting is regulated are two key problems about the peroxisome matrix protein targeting. Now, most studies have been focusing on the mechanism by which the peroxisome matrix protein import was accomplished. Only a few works were about the regulation the peroxisome targeting.

The purpose of the present study is to establish a good system for revealing the molecular function and the physiological relevance of KIF12, by which I found a new function of KIF12 in peroxisome matrix protein targeting though its cytoplasmic binding partner.

In this study, I firstly generated Kif12 knockout (KO) mice. Using homologous recombination, the genomic DNA fragment covers from 2nd to 11th exons is replaced by a selection cassette, which consists of a promoter trapping β Geo and a PGK promoter-driven puro, called "βGeo/puro". This results in a translational frame shift at the downstream of the first exon of Kif12 gene, and thus KIF12 protein is eliminated from the KO mouse. Results from Southern blotting, genotyping PCR and immunoblotting strongly suggest that Kif12-deficient mouse were successfully generated in this study.

Two synthesized peptides were designed for generating rabbit anti-mouse KIF12 antibodies. Peptide P12PR is located to the PRD domain of KIF12, which generates antibody 12PR. And peptide P12T is located to the C-terminal of KIF12 protein, which generates antibody 12T.

KIF12 tissue distribution assay was performed by using 12T antibody. Results showed that strong expression of KIF12 in mouse pancreatic islet and kidney. However, the lysates of mouse heart, brain, lung, spleen, fat, muscle and liver did not show any specific signals. Then I have further characterized an insulinoma-derived pancreatic beta cell line MIN6, and detected that MIN6 cells had the highest expression level of KIF12 among the analyzed samples. These results suggest that KIF12 plays some roles in maintaining appropriate function of pancreatic beta cells and kidneys. As pancreatic beta cells have the highest expression of KIF12, I used pancreatic beta cells as the experimental system for this study.

In order to assess the possible changes in organelle biogenesis and distribution, I observed the organelles and microtubules in primary cultured pancreatic islet cells. I labeled the Golgi complexes by anti-GM-130 antibody, the insulin granules by anti-insulin antibody, the lysosomes by LysoTracker, the mitochondria by MitoTracker, microtubules by anti-α-tubulin antibody. Results showed that all above mentioned organelles or microtubules are not different in WT and KO islets cells. These results suggested that KIF12 is dispensable for biogenesis of major organelles and microtubules.

However, the down-regulation of the signal strength of a peroxisomal enzyme was specifically distinguished in the KO cells. Peroxisomal enzyme acetyl-CoA acyltransferase 1 (ACAA1) targets peroxisomes by its PTS2 signal, and catalyzes the first step of peroxisomal beta-oxidation. For peroxisome visualization, I labeled ACAA1 by its antibody to reveal its dotty signals in the cytoplasm. Although the distribution of the peroxisomes was not apparently changed, the signal strength of ACAA1 was significantly decreased in the KO cells. In order to test whether this decrease was truly due to the loss of KIF12 protein, I performed a rescue experiment using a full-length Kif12 cDNA adenoviral vector, pKIF12FL-mCitrine. As a result, the overexpression of pKIF12FL-mCitrine in KO islet cells rescued the mean ACAA1 level to normality. These results implied that peroxisome targeting of ACAA1 was impaired in Kif12 KO cells.

KIF12 binding partner was reported to be a regulator of peroxisome matrix protein targeting. I further performed immunofluorescence microscopy of primary cultured pancreatic islet cells with its antibody, which revealed its significant decrease in Kif12 KO cells. And this decrease could be rescued by KIF12 overexpression.

To further confirm the impaired peroxisome targeting and its connection with KIF12 binding protein, I performed biochemical studies. As pancreatic islets are rather small compared with the brain, the intrinsic tissue is difficult to be handled as a starting material. Therefore I used mouse insulinoma-derived MIN6 cells for the biochemical studies. Results showed that Kif12 knockdown decreased the expression levels of KIF12 binding partner and ACAA1, and this decreasing could be rescued by overexpression of KIF12 protein.

Above data suggested that KIF12 controls peroxisome matrix protein targeting through regulating the intracellular level of its binding protein. Many explanations could account for its down-regulation in KIF12 KO or knockdown cells. One is that KIF12 functions as a transcription regulator of these proteins. Another one is that KIF12 binds to these proteins, and keep it away from protein degradation machinery. To verify the first possibility, I performed quantitative real-time RT-PCR, to compare the transcription of this protein in WT and KIF12 KD MIN6 cells, using β-actin as control. Results suggested that its down-regulation is independent of KIF12. Therefore, I assessed if KIF12 could bind to it and stabilize it. To confirm this, intrinsic IP experiments and proximity ligation assay were performed. Results strongly suggested their binding in vivo. This finding suggests that KIF12 mediated-protein downregulation may be due to its selective stabilization on this protein in the cytoplasm. Future studies will reveal the precise mechanistic links between KIF12 and stabilization of KIF12 binding protein.

In the present study, I established Kif12-deficient models. To investigate the physiological relevance of KIF12 in development and metabolism, the newly established Kif12 KO mice will be quite feasible, as it apparently gives no significant abnormality in development, viability and reproduction. In addition, I have generated a specific knockdown system in MIN6 beta cells with miRNA vector and its rescue system using an RNAi-immune expression vector. This miRNA vector specifically knockdown the KIF12 protein in MIN6 beta cells to 90% of the control, which could be rescued to the intrinsic level of the protein by RNAi-immune KIF12 vector infection. This knockdown system would be useful for biochemical studies, as pancreatic islets are too small to be handled as a starting material. These model systems will allow us to study the physiological relevance and molecular pathways of KIF12 throughout different levels, from molecules to individuals.

To search for the possible cellular abnormality in Kif12-deficient islet cells, I performed immunofluorescence staining to visualize major subcellular organelles and cytoskeleton, such as Golgi complex, insulin granules, lysosomes, mitochondria, microtubules and peroxisomes. Majority of the subcellular structures were not apparently changed in Kif12-deficient pancreatic islet cells, except for the peroxisome. The most significant finding in this study was a specific decrease in the amount of proteins targeted to peroxisomes, but the distribution was apparently unaltered.

This study will be the first one which shows the functional relevance of KIF12 in peroxisome matrix protein targeting. The specificity and feasibility of these finding has been supported by the following evidences: [1] abnormality was specifically found in peroxisomes; [2] impaired peroxisome targeting was morphologically and biochemically described; [3] it was found in two different systems of cell culture, islet primary culture and the MIN6 cell line; [4] it had been confirmed by two different methods, knockout and knockdown; and [5] it could be rescued in above cases.

Specific mutations in Pex5 could impair the PTS1 protein targeting in S. cerevisiae. Similar phenotype was also found in plants, Chinese hamster, and human. Thus, Pex5 is essential for importing peroxisome matrix proteins carrying PTS1 or PTS2 signals. In the present study, I found that knockdown of KIF12 in MIN6 cells reduced the Pex5 protein level and the impaired peroxisomal targeting of ACAA1 protein. It is likely that this down-regulation of Pex5 protein in Kif12-deficient cells could be a possible cause of impaired peroxisome matrix protein targeting.

In the present study, I clarified KIF12 binding protein, and found that it was down-regulated in Kif12-deficient beta cells, whose mRNA levels were unchanged. These data suggested that KIF12 controls down-regulation of this protein through a post-transcriptional mechanism, such as excluding the binding of E3 ligase to this protein. These findings will collectively suggest that KIF12 acts as a new cochaperone of this protein to prevent degradation of Pex5 and maintains the appropriate levels of their expression. Because the peroxisome is an essential organelle for metabolic pathways, this cochaperone kinesin will serve for a new prevention mechanism from metabolic diseases.

In summary, I discovered a new function of KIF12 in peroxisome matrix protein targeting in pancreatic islet cells and MIN6 beta cells through regulating its binding partner. This proposes a new regulatory pathway of peroxisome targeting involving KIF12 protein.

本研究は新規キネシンスーパーファミリータンパク質であるKIF12の性質を明らかにするため、標的遺伝子組換え法を用いてノックアウトマウスを作成し、ノックアウト膵島ベータ細胞ならびにノックダウンベータ細胞株においてKIF12欠失の影響を細胞生物学的に解析したものであり、下記の結果を得ている。

1.マウスES細胞における標的遺伝子組換え法によりKif12遺伝子座に欠失を導入した相同組換えES細胞株を作成した。次にこれをマウス胚盤胞に注入しキメラマウスを得、これを掛け合わせることによってKIF12欠失マウスを得た。

2.KIF12タンパク質C末端の配列を有するペプチドを合成し、これをウサギに免疫することによってKIF12タンパク質の特異抗体を得た。

3.KIF12特異抗体を用いてKIF12ノックアウトマウスと野生型マウスの各組織をイムノブロッティングによって解析することにより、KIF12が膵島ベータ細胞ならびに腎臓に特異的に多く発現していることが示された。

4.KIF12ノックアウトマウスと野生型マウスより膵島細胞の一次培養を行い、各種オルガネラマーカーでこれを間接蛍光抗体法によって染色したものをコンフォーカル蛍光レーザ顕微鏡にて観察したところ、KIF12ノックアウト細胞では特異的にペルオキシゾームにおけるACAA1酵素に対する染色シグナルの有意な減弱が示された。またこの減弱はKif12 cDNAを発現することによって補完されるので、この表現型は確かにKif12遺伝子の欠失によって生じたことが示された。

5.ペルオキシゾーム酵素量を調節する機能が知られているタンパク質に対してこれら膵島細胞を染色したところ、同様にKIF12ノックアウト細胞における染色シグナルの有意な減弱が示された。またこの減弱はKIF12 cDNAを発現することによって補完されるので、この表現型は確かにKif12遺伝子の欠失によって生じたことが示された。

6.KIF12とペルオキシゾームとの関係を生化学的に解析するため、Kif12遺伝子のpol II miRNA法によるノックダウンアデノウイルスベクターを構築した。このベクターをマウス膵島細胞株MIN6に強制発現させることによって、KIF12タンパク量の有意な減少を得た。さらにこのmiRNA配列に抵抗性を持つKIF12発現ベクターをコドン置換法によって構築した。これらのノックダウンベクターと発現ベクターの共発現によって、KIF12タンパク量が野生型と同等レベルにまで回復することが示された。

7.MIN6細胞におけるKif12ノックダウン系をイムノブロッティング法にて解析することにより、KIF12タンパク質の不足によって特異的にS1分画における調節タンパク質およびPex5タンパク質のタンパク量およびP2分画におけるACAA1タンパク量が有意に減少することが示された。またこれらの減少はKif12 cDNAを発現することによって補完されるので、この表現型は確かにKIF12タンパク質の不足によって生じたことが示された。

8.KIF12タンパク質の不足による調節タンパク質の減少が転写レベルの調節によるものかを調べるため、上記ノックダウン系においてリアルタイムRT-PCR法によって調節タンパク質遺伝子の転写量を測定した。その結果、野生型とノックダウン細胞では有意な差がないことが示された。

9.KIF12タンパク質と調節タンパク質との特異的な分子間相互作用を免疫沈降法および近接ライゲーションアッセイ法を用いて解析した。その結果、これらのタンパク質が細胞質において分子複合体を形成していることが示され、この分子複合体形成が細胞質において直接調節タンパク質のタンパク量を制御していることが示唆された。

以上、本論文は新規に樹立したKIF12ノックアウトマウスの膵島ベータ細胞ならびにベータ細胞株のKIF12ノックダウン系において、KIF12との分子複合体の形成による調節タンパク質量のモジュレーションを介したペルオキシゾーム酵素量の新規調節系を明らかにしたものである。本研究はこれまで未知に等しかったペルオキシゾーム酵素量の調節系について、キネシンスーパーファミリータンパク質の分子遺伝学の観点からまったく新しい経路の同定に至ったものであり、脂質糖代謝のシグナルネットワークの解析に重要な貢献をなすと考えられ、学位の授与に値するものと考えられる。