The role of ClpXP-mediated proteolysis in resculpting the proteome after DNA damage

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Includes bibliographical references (p. 120-136).When faced with environmental assaults, E. coli can take extreme measures to survive. For example, starving bacteria consume their own proteins, and bacteria with severe DNA damage introduce mutations into their genomes. These survival tactics require restructuring of the bacterial proteomic landscape. To reshape the proteome, bacteria alter both protein synthesis and protein degradation. For important regulatory proteins and proteins potentially harmful to the cell under non-stress conditions, these changes must be environmentally responsive and specific. This thesis explores the role of the ClpXP protease in the response to DNA damage. First, we determine how DNA damage affects substrate selection by ClpXP. These experiments combine quantitative proteomics and use of an inactive variant of ClpP to "trap" cellular ClpXP substrates and compare their relative levels with and without DNA damage. Analysis of trapped substrates reveals that cellular stress can result in dramatic changes in protease substrate selection. Next, we explore a specific mechanism that allows coupling of an environmental signal to a change in proteolysis. When the cell senses DNA damage, it triggers autocleavage of the LexA repressor. Autocleavage creates new signals for ClpXP recognition, ensuring the timely degradation of the LexA cleavage products. The mechanism of LexA recognition became a model for cleavage-dependent recognition of other substrates. Finally, we determined the mechanism of ClpX recognition of a known, damage-inducible substrate, UmuD'. We find that UmuD directs UmuD' degradation in an SspB-like manner. These experiments show how, with the right sequence motif, an interacting partner can become a ClpXP delivery factor.(cont.) This thesis work contributes to the idea that the bacterial cell has an imperative to degrade certain stress response proteins. Substrate priorities may change throughout the stress response and cellular proteases have devised a variety of strategies to ensure selection of the right substrate at the right time with respect to cellular conditions. This allows the cell to put its best proteome forward as it meets repeated cycles of environmental stress.by Saskia B. Neher.Ph.D

Purification, cellular levels, and functional domains of lipase maturation factor 1

Over a third of the US adult population has hypertriglyceridemia, resulting in an increased risk of atherosclerosis, pancreatitis, and metabolic syndrome. Lipoprotein lipase (LPL)1, a dimeric enzyme, is the main lipase responsible for TG clearance from the blood after food intake. LPL requires an endoplasmic reticulum (ER)-resident, transmembrane protein known as lipase maturation factor 1 (LMF1) for secretion and enzymatic activity. LMF1 is believed to act as a client specific chaperone for dimeric lipases, but the precise mechanism by which LMF1 functions is not understood. Here, we examine which domains of LMF1 contribute to dimeric lipase maturation by assessing the function of truncation variants. N-terminal truncations of LMF1 show that all the domains are necessary for LPL maturation. Fluorescence microscopy and protease protection assays confirmed that these variants were properly oriented in the ER. We measured cellular levels of LMF1 and found that it is expressed at low levels and each molecule of LMF1 promotes the maturation of 50 or more molecules of LPL. Thus we provide evidence for the critical role of the N-terminus of LMF1 for the maturation of LPL and relevant ratio of chaperone to substrate

Angiopoietin-like Protein 4 Inhibition of Lipoprotein Lipase: EVIDENCE FOR REVERSIBLE COMPLEX FORMATION

Elevated triglycerides are associated with an increased risk of cardiovascular disease, and lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglycerides from circulating lipoproteins. The N-terminal domain of angiopoietin-like protein 4 (ANGPTL4) inhibits LPL activity. ANGPTL4 was previously described as an unfolding molecular chaperone of LPL that catalytically converts active LPL dimers into inactive monomers. Our studies show that ANGPTL4 is more accurately described as a reversible, noncompetitive inhibitor of LPL. We find that inhibited LPL is in a complex with ANGPTL4, and upon dissociation, LPL regains lipase activity. Furthermore, we have generated a variant of ANGPTL4 that is dependent on divalent cations for its ability to inhibit LPL. We show that LPL inactivation by this regulatable variant of ANGPTL4 is fully reversible after treatment with a chelator

Structure of dimeric lipoprotein lipase reveals a pore adjacent to the active site

Abstract Lipoprotein lipase (LPL) hydrolyzes triglycerides from circulating lipoproteins, releasing free fatty acids. Active LPL is needed to prevent hypertriglyceridemia, which is a risk factor for cardiovascular disease (CVD). Using cryogenic electron microscopy (cryoEM), we determined the structure of an active LPL dimer at 3.9 Å resolution. This structure reveals an open hydrophobic pore adjacent to the active site residues. Using modeling, we demonstrate that this pore can accommodate an acyl chain from a triglyceride. Known LPL mutations that lead to hypertriglyceridemia localize to the end of the pore and cause defective substrate hydrolysis. The pore may provide additional substrate specificity and/or allow unidirectional acyl chain release from LPL. This structure also revises previous models on how LPL dimerizes, revealing a C-terminal to C-terminal interface. We hypothesize that this active C-terminal to C-terminal conformation is adopted by LPL when associated with lipoproteins in capillaries

Latent ClpX-recognition signals ensure LexA destruction after DNA damage

The DNA-damage response genes in bacteria are up-regulated when LexA repressor undergoes autocatalytic cleavage stimulated by activated RecA protein. Intact LexA is stable to intracellular degradation but its auto-cleavage fragments are degraded rapidly. Here, both fragments of LexA are shown to be substrates for the ClpXP protease. ClpXP recognizes these fragments using sequence motifs that flank the auto-cleavage site but are dormant in intact LexA. Furthermore, ClpXP degradation of the LexA-DNA-binding fragment is important to cell survival after DNA damage. These results demonstrate how one protein-processing event can activate latent protease recognition signals, triggering a cascade of protein turnover in response to environmental stress

Biochemical Analysis of the Lipoprotein Lipase Truncation Variant, LPLS447X, Reveals Increased Lipoprotein Uptake

Lipoprotein lipase (LPL) is responsible for the hydrolysis of triglycerides from circulating lipoproteins. Whereas most identified mutations in the LPL gene are deleterious, one mutation, LPLS447X, causes a gain of function. This mutation truncates two amino acids from LPL's C-terminus. Carriers of LPLS447X have decreased VLDL levels and increased HDL levels, a cardioprotective phenotype. LPLS447X is used in Alipogene tiparvovec, the gene therapy product for individuals with familial LPL deficiency. It is unclear why LPLS447X results in a serum lipid profile more favorable than that of LPL. In vitro reports vary as to whether LPLS447X is more active than LPL. We report a comprehensive, biochemical comparison of purified LPLS447X and LPL dimers. We found no difference in specific activity on synthetic and natural substrates. We also did not observe a difference in the Ki for ANGPTL4 inhibition of LPLS447X relative to that of LPL. Finally, we analyzed LPL-mediated uptake of fluorescently labeled lipoprotein particles and found that LPLS447X enhanced lipoprotein uptake to a greater degree than LPL did. An LPL structural model suggests that the LPLS447X truncation exposes residues implicated in LPL binding to uptake receptors

Modulation of the Activity of <i>Mycobacterium tuberculosis</i> LipY by Its PE Domain

<div><p><i>Mycobacterium tuberculosis</i> 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.</p></div

LipY and LipYΔPE share similar thermal unfolding profiles.

<p>CD temperature scans of LipY (black) and LipYΔPE (gray) monitored at 222 nm between 25 and 95°C. Data points were collected every 1°C.</p

There are more active LipY molecules in peak 1 compared to peak 2.

<p>(A) Equal concentrations of <i>total</i> LipY Peak 1 and Peak 2 were incubated with a 1.5 molar excess of TAMRA-FP serine hydrolase probe for 30 minutes at room temperature. A standard was created using the probe alone. The amount of active Peak 1 and Peak 2 were calculated based on the standard curve. (B) Bar graph showing the V_max and K_m from Michaelis-Menten curves comparing equal amounts of <i>total</i> LipY from Peak 1 and Peak 2 using the DGGR substrate. Error bars represent the standard error of the mean of 6 independent measurements[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135447#pone.0135447.ref032" target="_blank">32</a>].</p