Takamasa Uekita

Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that degrades the pericellular extracellular matrix (ECM) and is expressed in many migratory cells, including invasive cancer cells. MT1-MMP has been shown to localize at the migration edge and to promote cell migration; however, it is not clear how the enzyme is regulated during the migration process. Here, we report that MT1-MMP is internalized from the surface and that this event depends on the sequence of its cytoplasmic tail. Di-leucine (Leu571–572 and Leu578–579) and tyrosine573 residues are important for the internalization, and the μ2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence. MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles. The mutations that disturb internalization caused accumulation of the enzyme at the adherent edge, though the net proteolytic activity was not affected much. Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity. These data indicate that dynamic turnover of MT1-MMP at the migration edge by internalization is important for proper enzyme function during cell migration and invasion.

Tetraspanin CD63 promotes targeting and lysosomal proteolysis of membrane-type 1 matrix metalloproteinase.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is known to be internalized from cell surface, however, the fate of internalized MT1-MMP is still unknown. Here we demonstrate that at least a part of internalized MT1-MMP is targeted for lysosomal proteolysis. Treatment with an inhibitor of lysosomal proteinases chloroquine suppressed degradation of internalized MT1-MMP and induced accumulation of MT1-MMP in CD63-positive lysosomes. Ectopic expression of CD63 accelerated degradation of MT1-MMP, which was blocked by chloroquine. MT1-MMP, and CD63 were shown to form a complex through hemopexin-like domain of MT1-MMP and N-terminal region of CD63, and thus accelerated degradation of MT1-MMP was not observed with mutants lacking these domains. CD63 mutant lacking lysosomal targeting motif was unable to promote MT1-MMP degradation. These results suggest that CD63 regulates MT1-MMP by targeting to lysosomes.

Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1 (MTCBP-1) acts as an eukaryotic aci-reductone dioxygenase (ARD) in the methionine salvage pathway

MTCBP‐1 was identified as a protein that binds the cytoplasmic tail of membrane‐type 1 matrix metalloproteinase (MT1‐MMP/MMP‐14). Since MTCBP‐1 has a putative β‐barrel structure, it is presumably a member of the recently proposed cupin superfamily that contains tremendously diverged functions of proteins in spite of their well‐conserved β‐barrel structure. MTCBP‐1 shows significant homology to the bacterial aci‐reductone dioxygenase (ARD) in the cupin family, which is an enzyme in the methionine salvage pathway (MTA cycle). Since it is difficult to speculate the functions of cupin proteins simply based on their sequence homology, we examined whether the eukaryotic ARD homologs surely function in the methionine metabolism. Under sulfur‐depleted conditions, yeast could grow when substrate of MTA cycle was provided. Disruption of the yeast ARD homolog, YMR009w gene, abolished ability of the cells to grow in this culture condition. Re‐expression of either the YMR009w or MTCBP‐1 gene restored the cell growth. Mutation analysis revealed that the glutamic acid residue in the β‐barrel fold and the N‐terminal extension from the β‐barrel fold were found to be important for the activity to restore the growth. Thus, MTCBP‐1 isolated as a binding protein for MT1‐MMP was demonstrated to function as an ARD‐like enzyme in the MTA cycle in yeast.

Regulated nucleo-cytoplasmic shuttling of human aci-reductone dioxygenase (hADI1) and its potential role in mRNA processing

Bacterial aci‐reductone dioxygenase (ARD), a member of the cupin superfamily, has evolutionarily primitive protein folding and functions in the methionine recycling pathway. Recently, a human ARD orthologue (human ADI1, hADI1) has been identified and exhibits functions other than ARD activity. The hADI1 localizes mainly to the cytoplasm, but a substantial fraction is nuclear, suggesting functions in both cellular compartments. In this study, we report that nucleo‐cytoplasmic transport of hADI1 is regulated by a non‐canonical nuclear export signal (NES) located in the N‐terminal region of hADI1. The NES is composed of multiple basic amino‐acid residues instead of the canonical leucine‐rich sequence. Nuclear export of hADI1 was not mediated by CRM1, a major transporter that binds to leucine‐rich NES. Substitution of the basic residues with alanines abolished NES activity. Mutant hADI1 accumulated in the nucleus and formed speckles frequently observed with splicing factors and some transcription factors. Indeed, hADI1 specifically co‐localized with the splicing factor U1‐70K to the nucleus but not with another splicing factor, SC35. U1‐70K over‐expression induced nuclear accumulation of hADI1. Nuclear hADI1 expression significantly altered the splicing pattern of the adenovirus E1A mini‐gene, which generates multiple alternatively spliced transcripts. Thus, hADI1 may have acquired a novel role in nuclear mRNA processing possibly by modulating U1‐70K‐related functions, an activity negatively regulated by a non‐classical NES sequence.

Studies on the Recovery of Genomic DNA and Functional Genes from Mammalian Pelt Specimens

絶滅種や稀少種の機能遺伝子を解析することは,遺伝子資源の保全や進化学的研究の見地から重要な課題である.本研究は,毛皮あるいは剥製標本の表皮から効率的にゲノムDNAを抽出する方法を開発し,絶滅種や稀少種の機能遺伝子を解析することを最終的な目的として行ったものである.一般に,絶滅種や希少種の剥製•毛皮標本は数も少く,貴重であるので,DNA抽出のような,破壊的解析のための材料を入手することは,困難な場合が多い.従来報告されている古代DNAの抽出法では,いずれも,抽出のために比較的大きな標本片を用いており,少量の標品しか入手できない場合には適用できない.本研究では,特に,原材料となる剥製や毛皮標本の形をできるだけ損傷しないことに留意し,約1mm角の毛皮断片からゲノムDNAを効率よく抽出する方法の確立と,PCR解析を行うための条件の検討を行った.また,本研究で確立した方法を用いて,製作年次の異なる食肉目動物毛皮標本から回収したゲノムDNAをテンプレートとして,歯のエナメル質タンパク質をコードしているアメロゲニン遺伝子断片の回収と塩基配列の解析を試みた.結果の一部として,モンゴルオオカミの毛皮標本から抽出したゲノムDNAのアメロゲニン遺伝子の配列の一部を解読することができたので,イヌ(ゴールデンリトリーバー)の血液から抽出したゲノムDNAのアメロゲニン遺伝子の配列と比較したところ,モンゴルオオカミとイヌの配列は100%一致したが,イヌの品種間,あるいは個体差による配列の多型が存在する可能性もある。イヌ,オオカミのアメロゲニン遺伝子の塩基配列は従来報告が無く,部分的ではあるが配列が決定されたのは本論文が始めてである.イヌとオオカミの種間の違い,および,イヌの品種間の多型については,今後,さらに検討が必要である.