The Structural Determinants Accounting for the Broad Substrate Specificity of the Quorum Quenching Lactonase GcL

The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

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TheArabidopsisAtSTE24 Is a CAAXProtease with Broad Substrate Specificity

Journal of Biological Chemistry ◽

10.1074/jbc.m202916200 ◽

2002 ◽

Vol 277 (33) ◽

pp. 29856-29864 ◽

Cited By ~ 43

Author(s):

Keren Bracha ◽

Meirav Lavy ◽

Shaul Yalovsky

Keyword(s):

Substrate Specificity ◽

Broad Substrate Specificity

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First characterization of an archaeal amino acid racemase with broad substrate specificity from the hyperthermophile Pyrococcus horikoshii OT-3

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2017.02.004 ◽

2017 ◽

Vol 124 (1) ◽

pp. 23-27 ◽

Cited By ~ 3

Author(s):

Ryushi Kawakami ◽

Haruhiko Sakuraba ◽

Taketo Ohmori ◽

Toshihisa Ohshima

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Pyrococcus Horikoshii ◽

Broad Substrate Specificity ◽

Amino Acid Racemase

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Human and mouse granzyme M display divergent and species-specific substrate specificities

Biochemical Journal ◽

10.1042/bj20110210 ◽

2011 ◽

Vol 437 (3) ◽

pp. 431-442 ◽

Cited By ~ 26

Author(s):

Stefanie A.H. de Poot ◽

Marijn Westgeest ◽

Daniel R. Hostetter ◽

Petra van Damme ◽

Kim Plasman ◽

...

Keyword(s):

Substrate Specificity ◽

Specific Substrate ◽

Structural Determinants ◽

Cytotoxic Lymphocyte ◽

Potent Inducer ◽

Methionine Residue ◽

Positional Scanning ◽

Methionine Residues ◽

Species Specific ◽

Positional Scanning Libraries

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1′–P2′ residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.

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