Inhibitor Report for: PMSF

Differential inhibition of brain versus peripheral acetylcholinesterase (AChE) by phenylmethylsulfonyl fluoride (PMSF) suggested that PMSF might preferentially inhibit different AChE molecular forms. AChE inhibition was examined after systemic and in vitro PMSF treatment. Systemic administration resulted in no overt behavioral changes but produced a 71% reduction in brain AChE; hemidiaphragm, extensor digitorum longus and soleus muscles showed 65, 50 and 41% reductions. Muscle asymmetric AChE was reduced to the greatest extent (50-80%). The tetrameric form was inhibited in brain and hemidiaphragm (60-76%) but spared in other muscles (18-22%). Monomeric AChE was spared in all tissues. When PMSF was added to a muscle homogenate all forms were inhibited equally. Purified monomer and tetramer forms were inhibited equally in vitro. These results suggest that PMSF inhibition of AChE is a consequence of a selective inhibition of membrane-associated forms and that the apparent brain selectivity is related to the greater fraction of membrane-associated AChE in brain.

The lipase gene from Psychrobacter celer PU3 was cloned into pET-28a(+) expression vector and overexpressed in E. coli BL21 (DE3) pLysS cells. The purified Psychrobacter celer lipase (PCL) was characterized as an alkaline active enzyme and has a molecular mass of around 30 kDa. The PCL was active even at a low temperature and the optimum range was observed between 10 and 40 degreesC temperatures. MALDI-TOF and phylogenetic analysis ensued that Psychrobacter celer PU3 lipase (PCL) was closely related to P. aureginosa lipase (PAL). MD simulation results suggests that temperature change did not affect overall structure of PCL, but it may alter temperature- dependent PCL structural changes. R(1) (129-135 AA) and R(2) (187-191 AA) regions could be important for temperature-dependent PCL function as they fluctuate much at 35 degreesC temperature. PMSF completely inhibited PCL lipase activity and it demonstrates the presence of serine residues in the active site of PCL. PCL is moderately halophilic and most of the tested organic solvents found to be inhibiting the lipase activity except the solvents ethanol and methanol. PCL activity was increased with surfactants (SDS and CTAB) and bleaching agents (hydrogen peroxide). The effect of different metal ions on PCL resulted that only mercuric chloride was found as the enhancer of the lipase activity. Antibiofilm property of PCL was evaluated against pathogenic Vibrio parahaemolyticus isolated from the diseased shrimp and MIC value was 500 U. PCL significantly altered the morphology and biofilm density of V. parahaemolyticus and the same was observed through scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) imaging. RT-PCR analysis revealed that the mRNA expression level of biofilm, colony morphology and major toxin-related (aphA, luxS, opaR, tolC, toxR) genes of V. parahaemolyticus were significantly downregulated with PCL treatment.

Acne is one of the most common dermatological conditions, but the details of its pathology are unclear, and current management regimens often have adverse effects. Cutibacterium acnes is known as a major acne-associated bacterium that derives energy from lipase-mediated sebum lipid degradation. C. acnes is commensal, but lipase activity has been observed to differ among C. acnes types. For example, higher populations of the type IA strains are present in acne lesions with higher lipase activity. In the present study, we examined a conserved lipase in type IB and II, but truncated in type IA C. acnes strains. Closed, blocked, and open structures of C. acnes ATCC11828 lipases were elucidated by X-ray crystallography at 1.6-2.4 A. The closed crystal structure, which is the most common form in aqueous solution, revealed that hydrophobic lid domain shields the active site. By comparing closed, blocked, and open structures, we found that the lid domain-opening mechanisms of C. acnes lipases involve the lid-opening residues, Phe- 179 and Phe-211. To the best of our knowledge, this is the first structure-function study of C. acnes lipases, which may help shed light on the mechanisms involved in acne development and may aid in future drug design.

The aggregation of alpha-synuclein (alpha-Syn) is a characteristic of Parkinson's disease (PD). alpha-Syn oligomerization/aggregation is accelerated by the serine peptidase, prolyl oligopeptidase (POP). Factors that affect POP conformation, including most of its inhibitors and an impairing mutation in its active site, influence the acceleration of alpha-Syn aggregation resulting from the interaction of these proteins. It is noteworthy, however, that alpha-Syn is not cleaved by POP. Prolyl endopeptidase-like (PREPL) protein is structurally related to the serine peptidases belonging to the POP family. Based on the alpha-Syn-POP studies and knowing that PREPL may contribute to the regulation of synaptic vesicle exocytosis, when this protein can encounter alpha-Syn, we investigated the alpha-Syn-PREPL interaction. The binding of these two human proteins was observed with an apparent affinity constant of about 5.7 muM and, as in the alpha-Syn assays with POP, the presence of PREPL accelerated the oligomerization/aggregation events, with no alpha-Syn cleavage. Furthermore, despite this lack of hydrolytic cleavage, the serine peptidase active site inhibitor phenylmethylsulfonyl fluoride (PMSF) abolished the enhancement of the alpha-Syn aggregation by PREPL. Therefore, given the attention to POP inhibitors as potential drugs to treat synucleinopathies, the present data point to PREPL as another potential target to be explored for this purpose.

The PE and PPE multigene families, first discovered during the sequencing of M. tuberculosis H37Rv genome are responsible for antigenic variation and have been shown to induce increased humoral and cell mediated immune response in the host. Using the bioinformatics tools, we had earlier reported that the 225 amino acid residue PE-PPE domain (Pfam: PF08237) common to some PE and PPE proteins has a "serine alpha/beta hydrolase" fold and conserved Ser, Asp and His catalytic triad characteristic of lipase, esterase and cutinase activities. In order to prove experimentally that PE-PPE domain is indeed a serine hydrolase, we have cloned the full-length Rv1430 and its PE-PPE domain into pET-28a vector, expressed the proteins in E. coli and purified to homogeneity. The activity assays of both purified proteins were carried out using p-nitrophenyl esters of aliphatic carboxylic acids with varying chain length (C2-C16) to study the substrate specificity. To characterize the active site of the PE-PPE domain, we mutated the Ser199 to Ala. The activity of the protein in the presence of serine protease inhibitor- PMSF and the mutant protein were measured. Our results reveal that Rv1430 and its PE-PPE domain possess esterase activity and hydrolyse short to medium chain fatty acid esters with the highest specific activity for pNPC6 at 37 degrees C, 38 degrees C and pH 7.0, 8.0. The details of this work and the observed results are reported in this manuscript.

KFase (kynurenine formamidase), also known as arylformamidase and formylkynurenine formamidase, efficiently catalyses the hydrolysis of NFK (N-formyl-L-kynurenine) to kynurenine. KFase is the second enzyme in the kynurenine pathway of tryptophan metabolism. A number of intermediates formed in the kynurenine pathway are biologically active and implicated in an assortment of medical conditions, including cancer, schizophrenia and neurodegenerative diseases. Consequently, enzymes involved in the kynurenine pathway have been considered potential regulatory targets. In the present study, we report, for the first time, the biochemical characterization and crystal structures of Drosophila melanogaster KFase conjugated with an inhibitor, PMSF. The protein architecture of KFase reveals that it belongs to the alpha/beta hydrolase fold family. The PMSF-binding information of the solved conjugated crystal structure was used to obtain a KFase and NFK complex using molecular docking. The complex is useful for understanding the catalytic mechanism of KFase. The present study provides a molecular basis for future efforts in maintaining or regulating kynurenine metabolism through the molecular and biochemical regulation of KFase.

Acetylcholinesterase (AChE), a serine hydrolase, is potentially susceptible to inactivation by phenylmethylsulfonyl fluoride (PMSF) and benzenesulfonyl fluoride (BSF). Although BSF inhibits both mouse and Torpedo californica AChE, PMSF does not react measurably with the T. californica enzyme. To understand the residue changes responsible for the change in reactivity, we studied the inactivation of wild-type T. californica and mouse AChE and mutants of both by BSF and PMSF both in the presence and absence of substrate. The enzymes investigated were wild-type mouse AChE, wild-type T. californica AChE, wild-type mouse butyrylcholinesterase, mouse Y330F, Y330A, F288L, and F290I, and the double mutant T. californica F288L/F290V (all mutants given T. californica numbering). Inactivation rate constants for T. californica AChE confirmed previous reports that this enzyme is not inactivated by PMSF. Wild-type mouse AChE and mouse mutants Y330F and Y330A all had similar inactivation rate constants with PMSF, implying that the difference between mouse and T. californica AChE at position 330 is not responsible for their differing PMSF sensitivities. In addition, butyrylcholinesterase and mouse AChE mutants F288L and F290I had increased rate constants ( approximately 14 fold) over those of wild-type mouse AChE, indicating that these residues may be responsible for the increased sensitivity to inactivation by PMSF of butyrylcholinesterase. The double mutant T. californica AChE F288L/F290V had a rate constant nearly identical with the rate constant for the F288L and F290I mouse mutant AChEs, representing an increase of approximately 4000-fold over the T. californica wild-type enzyme. It remains unclear why these two positions have more importance for T. californica AChE than for mouse AChE.

Differential inhibition of brain versus peripheral acetylcholinesterase (AChE) by phenylmethylsulfonyl fluoride (PMSF) suggested that PMSF might preferentially inhibit different AChE molecular forms. AChE inhibition was examined after systemic and in vitro PMSF treatment. Systemic administration resulted in no overt behavioral changes but produced a 71% reduction in brain AChE; hemidiaphragm, extensor digitorum longus and soleus muscles showed 65, 50 and 41% reductions. Muscle asymmetric AChE was reduced to the greatest extent (50-80%). The tetrameric form was inhibited in brain and hemidiaphragm (60-76%) but spared in other muscles (18-22%). Monomeric AChE was spared in all tissues. When PMSF was added to a muscle homogenate all forms were inhibited equally. Purified monomer and tetramer forms were inhibited equally in vitro. These results suggest that PMSF inhibition of AChE is a consequence of a selective inhibition of membrane-associated forms and that the apparent brain selectivity is related to the greater fraction of membrane-associated AChE in brain.