Modulation of the Activity of Mycobacterium tuberculosis LipY by Its PE Domain.
Mycobacterium tuberculosis harbors over 160 genes encoding PE/PPE proteins, several of which have roles in the pathogen's virulence. A number of PE/PPE proteins are secreted via Type VII secretion systems known as the ESX secretion systems. One PE protein, LipY, has a triglyceride lipase domain in addition to its PE domain. LipY can regulate intracellular triglyceride levels and is also exported to the cell wall by one of the ESX family members, ESX-5. Upon export, LipY's PE domain is removed by proteolytic cleavage. Studies using cells and crude extracts suggest that LipY's PE domain not only directs its secretion by ESX-5, but also functions to inhibit its enzymatic activity. Here, we attempt to further elucidate the role of LipY's PE domain in the regulation of its enzymatic activity. First, we established an improved purification method for several LipY variants using detergent micelles. We then used enzymatic assays to confirm that the PE domain down-regulates LipY activity. The PE domain must be attached to LipY in order to effectively inhibit it. Finally, we determined that full length LipY and the mature lipase lacking the PE domain (LipYΔPE) have similar melting temperatures. Based on our improved purification strategy and activity-based approach, we concluded that LipY's PE domain down-regulates its enzymatic activity but does not impact the thermal stability of the enzyme.
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America.
Christopher K
CK
Garrett
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America.
Lindsey J
LJ
Broadwell
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America.
Cassandra K
CK
Hayne
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America.
Saskia B
SB
Neher
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D058947:Bacterial Secretion Systems / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D014280:Triglycerides / Q000235:genetics / Q000378:metabolism; D037521:Virulence Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D014280:Triglycerides; D037521:Virulence Factors; D002265:Carboxylic Ester Hydrolases; C000594149:LipY protein, Mycobacterium tuberculosis