A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, al... more A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, also called porcino, cep or penny bun). The lectin was structurally characterized i.e its amino acid sequence and three-dimensional structure were determined. The new protein is a homodimer and each protomer folds as β-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) β-trefoil to distinguish it from the other lectin that has been described in these mushrooms. The lectin has potent anti-proliferative effects on human cancer cells, which confers to it an interesting therapeutic potential as an antineoplastic agent. Several crystal forms of the apoprotein and of complexes with different carbohydrates were studied by X-ray diffraction. The structure of the apoprotein was solved at 1.12 Å resolution. The interaction of the lectin with lactose, galactose, N-acetylgalactosamine and T-antigen disaccharide, Galβ1-3GalNAc, was examined in detail. All the three potential binding sites present in the β-trefoil fold are occupied in at least one crystal form and are described in detail in this paper. No important conformational changes are observed in the lectin when comparing its co-crystals with carbohydrates with those of the ligand-free protein.
The combinatorial assembly of protein complexes is at the heart of chromatin biology. Lysine deme... more The combinatorial assembly of protein complexes is at the heart of chromatin biology. Lysine demethylase LSD1(KDM1A)/CoREST beautifully exemplifies this concept. The active site of the enzyme tightly associates to the N-terminal domain of transcription factors of the SNAIL1 family, which therefore can competitively inhibit the binding of the N-terminal tail of the histone substrate. Our enzymatic, crystallographic, spectroscopic, and computational studies reveal that LSD1/CoREST can bind to a hexapeptide derived from the SNAIL sequence through recognition of a positively charged α-helical turn that forms upon binding to the enzyme. Variations in sequence and length of this six amino acid ligand modulate affinities enabling the same binding site to differentially interact with proteins that exert distinct biological functions. The discovered short peptide inhibitors exhibit antiproliferative activities and lay the foundation for the development of peptidomimetic small molecule inhibitors of LSD1.
A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus e... more A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galβ1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 Å resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer.
AIMS
Long-term mycophenolate mofetil (MMF) therapy may induce inosine
5′-monophosphate dehydrogen... more AIMS Long-term mycophenolate mofetil (MMF) therapy may induce inosine 5′-monophosphate dehydrogenase (IMPDH) activity in peripheral blood mononuclear cells (PBMCs), thus decreasing MMF immunosuppressive properties. Pharmacodynamic monitoring was used to investigate whether biological activity is altered after long-term therapy. METHODS IMPDH activity was measured in PBMC samples from 54 stable kidney transplant patients, already on MMF (for at least 3 months), before (t0) and 2 h after (t2) MMF morning dose administration; levels were monitored for up to 15 months, together with total mycophenolic acid (MPA) and free MPA concentrations. RESULTS During the 15 months’ monitoring, t0 IMPDH activity in transplant recipients increased from 5.9 3.7 nmol h-1 mg-1 [95% confidence interval (CI) 4.9, 6.9] to 9.0 3.9 nmol h-1 mg-1 (95% CI 7.2, 10.8), with an intra- and interpatient variability of 28% and 42%. Five patients experienced acute rejection during the follow-up: t0 IMPDH activity was increased during rejection vs. nonrejection, and the trend was significantly higher in rejecting than in nonrejecting subjects for the whole monitoring period. CONCLUSIONS Even though a correlation has been found between IMPDH activity and rejection, its efficacy as a predictive tool in long-term transplant outcomes may be affected by high interpatient variability; on the other hand, continuous monitoring of the IMPDH trend could make an effective prognostic parameter of rejection. Other trials also including pre-transplant data on both IMPDH expression and activity are warranted to better assess their role as biomarkers for MPA effect in clinical practice.
The re-emergence of tuberculosis in recent years led the World Health Organization (WHO) to launc... more The re-emergence of tuberculosis in recent years led the World Health Organization (WHO) to launch the Stop TB Strategy program. Beside repurposing the existing drugs and exploring novel molecular combinations, an essential step to face the burden of tuberculosis will be to develop new drugs by identifying vulnerable bacterial targets. Recent studies have focused on decaprenylphosphoryl-D-ribose oxidase (DprE1) of Mycobacterium tuberculosis, an essential enzyme involved in cell wall metabolism, for which new promising molecules have proved efficacy as antitubercular agents. This review summarizes the state of the art concerning DprE1 in terms of structure, enzymatic activity and inhibitors. This enzyme is emerging as one of the most vulnerable target in M. tuberculosis.
A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, al... more A novel lectin was purified from the fruiting bodies of king bolete mushrooms (Boletus edulis, also called porcino, cep or penny bun). The lectin was structurally characterized i.e its amino acid sequence and three-dimensional structure were determined. The new protein is a homodimer and each protomer folds as β-trefoil domain and therefore we propose the name Boletus edulis lectin (BEL) β-trefoil to distinguish it from the other lectin that has been described in these mushrooms. The lectin has potent anti-proliferative effects on human cancer cells, which confers to it an interesting therapeutic potential as an antineoplastic agent. Several crystal forms of the apoprotein and of complexes with different carbohydrates were studied by X-ray diffraction. The structure of the apoprotein was solved at 1.12 Å resolution. The interaction of the lectin with lactose, galactose, N-acetylgalactosamine and T-antigen disaccharide, Galβ1-3GalNAc, was examined in detail. All the three potential binding sites present in the β-trefoil fold are occupied in at least one crystal form and are described in detail in this paper. No important conformational changes are observed in the lectin when comparing its co-crystals with carbohydrates with those of the ligand-free protein.
The combinatorial assembly of protein complexes is at the heart of chromatin biology. Lysine deme... more The combinatorial assembly of protein complexes is at the heart of chromatin biology. Lysine demethylase LSD1(KDM1A)/CoREST beautifully exemplifies this concept. The active site of the enzyme tightly associates to the N-terminal domain of transcription factors of the SNAIL1 family, which therefore can competitively inhibit the binding of the N-terminal tail of the histone substrate. Our enzymatic, crystallographic, spectroscopic, and computational studies reveal that LSD1/CoREST can bind to a hexapeptide derived from the SNAIL sequence through recognition of a positively charged α-helical turn that forms upon binding to the enzyme. Variations in sequence and length of this six amino acid ligand modulate affinities enabling the same binding site to differentially interact with proteins that exert distinct biological functions. The discovered short peptide inhibitors exhibit antiproliferative activities and lay the foundation for the development of peptidomimetic small molecule inhibitors of LSD1.
A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus e... more A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galβ1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 Å resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer.
AIMS
Long-term mycophenolate mofetil (MMF) therapy may induce inosine
5′-monophosphate dehydrogen... more AIMS Long-term mycophenolate mofetil (MMF) therapy may induce inosine 5′-monophosphate dehydrogenase (IMPDH) activity in peripheral blood mononuclear cells (PBMCs), thus decreasing MMF immunosuppressive properties. Pharmacodynamic monitoring was used to investigate whether biological activity is altered after long-term therapy. METHODS IMPDH activity was measured in PBMC samples from 54 stable kidney transplant patients, already on MMF (for at least 3 months), before (t0) and 2 h after (t2) MMF morning dose administration; levels were monitored for up to 15 months, together with total mycophenolic acid (MPA) and free MPA concentrations. RESULTS During the 15 months’ monitoring, t0 IMPDH activity in transplant recipients increased from 5.9 3.7 nmol h-1 mg-1 [95% confidence interval (CI) 4.9, 6.9] to 9.0 3.9 nmol h-1 mg-1 (95% CI 7.2, 10.8), with an intra- and interpatient variability of 28% and 42%. Five patients experienced acute rejection during the follow-up: t0 IMPDH activity was increased during rejection vs. nonrejection, and the trend was significantly higher in rejecting than in nonrejecting subjects for the whole monitoring period. CONCLUSIONS Even though a correlation has been found between IMPDH activity and rejection, its efficacy as a predictive tool in long-term transplant outcomes may be affected by high interpatient variability; on the other hand, continuous monitoring of the IMPDH trend could make an effective prognostic parameter of rejection. Other trials also including pre-transplant data on both IMPDH expression and activity are warranted to better assess their role as biomarkers for MPA effect in clinical practice.
The re-emergence of tuberculosis in recent years led the World Health Organization (WHO) to launc... more The re-emergence of tuberculosis in recent years led the World Health Organization (WHO) to launch the Stop TB Strategy program. Beside repurposing the existing drugs and exploring novel molecular combinations, an essential step to face the burden of tuberculosis will be to develop new drugs by identifying vulnerable bacterial targets. Recent studies have focused on decaprenylphosphoryl-D-ribose oxidase (DprE1) of Mycobacterium tuberculosis, an essential enzyme involved in cell wall metabolism, for which new promising molecules have proved efficacy as antitubercular agents. This review summarizes the state of the art concerning DprE1 in terms of structure, enzymatic activity and inhibitors. This enzyme is emerging as one of the most vulnerable target in M. tuberculosis.
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Papers by Laurent Chiarelli
Long-term mycophenolate mofetil (MMF) therapy may induce inosine
5′-monophosphate dehydrogenase (IMPDH) activity in peripheral blood
mononuclear cells (PBMCs), thus decreasing MMF immunosuppressive
properties. Pharmacodynamic monitoring was used to investigate
whether biological activity is altered after long-term therapy.
METHODS
IMPDH activity was measured in PBMC samples from 54 stable kidney
transplant patients, already on MMF (for at least 3 months), before
(t0) and 2 h after (t2) MMF morning dose administration; levels were
monitored for up to 15 months, together with total mycophenolic acid
(MPA) and free MPA concentrations.
RESULTS
During the 15 months’ monitoring, t0 IMPDH activity in transplant
recipients increased from 5.9 3.7 nmol h-1 mg-1 [95% confidence
interval (CI) 4.9, 6.9] to 9.0 3.9 nmol h-1 mg-1 (95% CI 7.2, 10.8), with
an intra- and interpatient variability of 28% and 42%. Five patients
experienced acute rejection during the follow-up: t0 IMPDH activity
was increased during rejection vs. nonrejection, and the trend was
significantly higher in rejecting than in nonrejecting subjects for the
whole monitoring period.
CONCLUSIONS
Even though a correlation has been found between IMPDH activity and
rejection, its efficacy as a predictive tool in long-term transplant
outcomes may be affected by high interpatient variability; on the other
hand, continuous monitoring of the IMPDH trend could make an
effective prognostic parameter of rejection. Other trials also including
pre-transplant data on both IMPDH expression and activity are warranted
to better assess their role as biomarkers for MPA effect in clinical practice.
Long-term mycophenolate mofetil (MMF) therapy may induce inosine
5′-monophosphate dehydrogenase (IMPDH) activity in peripheral blood
mononuclear cells (PBMCs), thus decreasing MMF immunosuppressive
properties. Pharmacodynamic monitoring was used to investigate
whether biological activity is altered after long-term therapy.
METHODS
IMPDH activity was measured in PBMC samples from 54 stable kidney
transplant patients, already on MMF (for at least 3 months), before
(t0) and 2 h after (t2) MMF morning dose administration; levels were
monitored for up to 15 months, together with total mycophenolic acid
(MPA) and free MPA concentrations.
RESULTS
During the 15 months’ monitoring, t0 IMPDH activity in transplant
recipients increased from 5.9 3.7 nmol h-1 mg-1 [95% confidence
interval (CI) 4.9, 6.9] to 9.0 3.9 nmol h-1 mg-1 (95% CI 7.2, 10.8), with
an intra- and interpatient variability of 28% and 42%. Five patients
experienced acute rejection during the follow-up: t0 IMPDH activity
was increased during rejection vs. nonrejection, and the trend was
significantly higher in rejecting than in nonrejecting subjects for the
whole monitoring period.
CONCLUSIONS
Even though a correlation has been found between IMPDH activity and
rejection, its efficacy as a predictive tool in long-term transplant
outcomes may be affected by high interpatient variability; on the other
hand, continuous monitoring of the IMPDH trend could make an
effective prognostic parameter of rejection. Other trials also including
pre-transplant data on both IMPDH expression and activity are warranted
to better assess their role as biomarkers for MPA effect in clinical practice.