MMP-7 marks severe pancreatic cancer and alters tumor cell signaling by proteolytic release of ectodomains.

Pancreatic cancer incurs the worst survival rate of the major cancers. High levels of the protease matrix metalloproteinase-7 (MMP-7) in circulation correlate with poor prognosis and limited survival of patients. MMP-7 is required for a key path of pancreatic tumorigenesis in mice and is present throughout tumor progression. Enhancements to chemotherapies are needed for increasing the number of pancreatic tumors that can be removed and for preventing relapses after surgery. With these ends in mind, selective inhibition of MMP-7 may be worth investigation. An anti-MMP-7 monoclonal antibody was recently shown to increase the susceptibility of several pancreatic cancer cell lines to chemotherapeutics, increase their apoptosis, and decrease their migration. MMP-7 activities are most apparent at the surfaces of innate immune, epithelial, and tumor cells. Proteolytic shedding of multiple protein ectodomains by MMP-7 from such cell surfaces influence apoptosis, proliferation, migration, and invasion. These activities warrant targeting of MMP-7 selectively in pancreatic cancer and other tumors of mucosal epithelia. Competitive and non-competitive modes of MMP-7 inhibition are discussed.

The BADC and BCCP subunits of chloroplast acetyl-CoA carboxylase sense the pH changes of the light-dark cycle.

Acetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions. At pH 7 in phosphate buffer, BADC1 and BADC2 gain an advantage over BCCP1 and BCCP2 in affinity for BC. At pH 8 in KCl solution, however, BCCP1 and BCCP2 had more than 10-fold higher affinity for BC than did BADC1. The pH-modulated shifts in BC preferences for BCCP and BADC partners suggest they contribute to light-dependent regulation of heteromeric ACCase. Using NMR spectroscopy, we found evidence for increased intrinsic disorder of the BADC and BCCP subunits at pH 7. We propose that this intrinsic disorder potentially promotes fast association with BC through a "fly-casting mechanism." We hypothesize that the pH effects on the BADC and BCCP subunits attenuate ACCase activity by night and enhance it by day. Consistent with this hypothesis, Arabidopsis badc1 badc3 mutant lines grown in a light-dark cycle synthesized more fatty acids in their seeds. In summary, our findings provide evidence that the BADC and BCCP subunits function as pH sensors required for light-dependent switching of heteromeric ACCase activity.

Fusion Peptide of SARS-CoV-2 Spike Rearranges into a Wedge Inserted in Bilayered Micelles.

The receptor binding and proteolysis of Spike of SARS-CoV-2 release its S2 subunit to rearrange and catalyze viral-cell fusion. This deploys the fusion peptide for insertion into the cell membranes targeted. We show that this fusion peptide transforms from intrinsic disorder in solution into a wedge-shaped structure inserted in bilayered micelles, according to chemical shifts, 15N NMR relaxation, and NOEs. The globular fold of three helices contrasts the open, extended forms of this region observed in the electron density of compact prefusion states. In the hydrophobic, narrow end of the wedge, helices 1 and 2 contact the fatty acyl chains of phospholipids, according to NOEs and proximity to a nitroxide spin label deep in the membrane mimic. The polar end of the wedge may engage and displace lipid head groups and bind Ca2+ ions for membrane fusion. Polar helix 3 protrudes from the bilayer where it might be accessible to antibodies.

NMR-based fragment screening and lead discovery accelerated by principal component analysis.

Protein-based NMR spectroscopy has proven to be a very robust method for finding fragment leads to protein targets. However, one limitation of protein-based NMR is that the data acquisition and analysis can be time consuming. In order to streamline the scoring of protein-based NMR fragment screening data and the determination of ligand affinities using 2D NMR experiments we have developed a data analysis workflow based on principal component analysis (PCA) within the TREND NMR Pro software package. We illustrate this using four different proteins and sets of ligands which interact with these proteins over a range of affinities. Also, these PCA-based methods can be successfully applied even to systems where ligand binding to target proteins is in intermediate or even slow exchange on the NMR time scale. Finally, these methods will work for scoring of fragment binding data using protein spectra that are either highly overlapped or lower in resolution.

Peripheral membrane associations of matrix metalloproteinases.

Water soluble matrix metalloproteinases (MMPs) have been regarded as diffusing freely in the extracellular matrix. Yet multiple MMPs are also observed at cell surfaces. Their membrane-proximal activities include sheddase activities, collagenolysis, bacterial killing, and intracellular trafficking reaching as far as the nucleus. The catalytic domains of MMP-7 and MMP-12 bind bilayers peripherally, each in two different orientations, by presenting positive charges and a few hydrophobic groups to the surface. Related peripheral membrane associations are predicted for other soluble MMPs. The peripheral membrane associations may support pericellular proteolysis and endocytosis. The isolated soluble domains of MT1-MMP can also associate with membranes. NMR assays suggest transient association of the hemopexin-like domains of MT1-MMP and MMP-12 with lipid bilayers. Peripheral association of soluble MMP domains with bilayers or heparin sulfate proteoglycans probably concentrates them near the membrane. This could increase the probability of forming complexes with membrane-associated proteins, such as those targeted for proteolysis. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

Tracking Equilibrium and Nonequilibrium Shifts in Data with TREND.

Principal component analysis (PCA) discovers patterns in multivariate data that include spectra, microscopy, and other biophysical measurements. Direct application of PCA to crowded spectra, images, and movies (without selecting peaks or features) was shown recently to identify their equilibrium or temporal changes. To enable the community to utilize these capabilities with a wide range of measurements, we have developed multiplatform software named TREND to Track Equilibrium and Nonequilibrium population shifts among two-dimensional Data frames. TREND can also carry this out by independent component analysis. We highlight a few examples of finding concurrent processes. TREND extracts dual phases of binding to two sites directly from the NMR spectra of the titrations. In a cardiac movie from magnetic resonance imaging, TREND resolves principal components (PCs) representing breathing and the cardiac cycle. TREND can also reconstruct the series of measurements from selected PCs, as illustrated for a biphasic, NMR-detected titration and the cardiac MRI movie. Fidelity of reconstruction of series of NMR spectra or images requires more PCs than needed to plot the largest population shifts. TREND reads spectra from many spectroscopies in the most common formats (JCAMP-DX and NMR) and multiple movie formats. The TREND package thus provides convenient tools to resolve the processes recorded by diverse biophysical methods.

Path to Collagenolysis: COLLAGEN V TRIPLE-HELIX MODEL BOUND PRODUCTIVELY AND IN ENCOUNTERS BY MATRIX METALLOPROTEINASE-12.

Collagenolysis is essential in extracellular matrix homeostasis, but its structural basis has long been shrouded in mystery. We have developed a novel docking strategy guided by paramagnetic NMR that positions a triple-helical collagen V mimic (synthesized with nitroxide spin labels) in the active site of the catalytic domain of matrix metalloproteinase-12 (MMP-12 or macrophage metalloelastase) primed for catalysis. The collagenolytically productive complex forms by utilizing seven distinct subsites that traverse the entire length of the active site. These subsites bury ∼1,080 Å(2)of surface area, over half of which is contributed by the trailing strand of the synthetic collagen V mimic, which also appears to ligate the catalytic zinc through the glycine carbonyl oxygen of its scissile G∼VV triplet. Notably, the middle strand also occupies the full length of the active site where it contributes extensive interfacial contacts with five subsites. This work identifies, for the first time, the productive and specific interactions of a collagen triple helix with an MMP catalytic site. The results uniquely demonstrate that the active site of the MMPs is wide enough to accommodate two strands from collagen triple helices. Paramagnetic relaxation enhancements also reveal an extensive array of encounter complexes that form over a large part of the catalytic domain. These transient complexes could possibly facilitate the formation of collagenolytically active complexes via directional Brownian tumbling.

Binding Isotherms and Time Courses Readily from Magnetic Resonance.

Evidence is presented that binding isotherms, simple or biphasic, can be extracted directly from noninterpreted, complex 2D NMR spectra using principal component analysis (PCA) to reveal the largest trend(s) across the series. This approach renders peak picking unnecessary for tracking population changes. In 1:1 binding, the first principal component captures the binding isotherm from NMR-detected titrations in fast, slow, and even intermediate and mixed exchange regimes, as illustrated for phospholigand associations with proteins. Although the sigmoidal shifts and line broadening of intermediate exchange distorts binding isotherms constructed conventionally, applying PCA directly to these spectra along with Pareto scaling overcomes the distortion. Applying PCA to time-domain NMR data also yields binding isotherms from titrations in fast or slow exchange. The algorithm readily extracts from magnetic resonance imaging movie time courses such as breathing and heart rate in chest imaging. Similarly, two-step binding processes detected by NMR are easily captured by principal components 1 and 2. PCA obviates the customary focus on specific peaks or regions of images. Applying it directly to a series of complex data will easily delineate binding isotherms, equilibrium shifts, and time courses of reactions or fluctuations.

Phosphorylation in the catalytic cleft stabilizes and attracts domains of a phosphohexomutase.

Phosphorylation can modulate the activities of enzymes. The phosphoryl donor in the catalytic cleft of α-D-phosphohexomutases is transiently dephosphorylated while the reaction intermediate completes a 180° reorientation within the cleft. The phosphorylated form of 52 kDa bacterial phosphomannomutase/phosphoglucomutase is less accessible to dye or protease, more stable to chemical denaturation, and widely stabilized against NMR-detected hydrogen exchange across the core of domain 3 to juxtaposed domain 4 (each by ≥ 1.3 kcal/mol) and parts of domains 1 and 2. However, phosphorylation accelerates hydrogen exchange in specific regions of domains 1 and 2, including a metal-binding residue in the active site. Electrostatic field lines reveal attraction across the catalytic cleft between phosphorylated Ser-108 and domain 4, but repulsion when Ser-108 is dephosphorylated. Molecular dynamics (MD) simulated the dephosphorylated form to be expanded due to enhanced rotational freedom of domain 4. The contacts and fluctuations of the MD trajectories enabled correct simulation of more than 80% of sites that undergo either protection or deprotection from hydrogen exchange due to phosphorylation. Electrostatic attraction in the phosphorylated enzyme accounts for 1) domain 4 drawing closer to domains 1 and 3; 2) decreased accessibility; and 3) increased stability within these domains. The electrostriction due to phosphorylation may help capture substrate, whereas the opening of the cleft upon transient dephosphorylation allows rotation of the intermediate. The long-range effects of phosphorylation on hydrogen exchange parallel reports on protein kinases, suggesting a conceptual link among these multidomain, phosphoryl transfer enzymes.

The role of collagen charge clusters in the modulation of matrix metalloproteinase activity.

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-L-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, kcat/Km values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P23-P23' subsites of collagenous substrates.

SARS-CoV-2 fusion peptide sculpting of a membrane with insertion of charged and polar groups.

The fusion peptide of SARS-CoV-2 spike is essential for infection. How this charged and hydrophobic domain occupies and affects membranes needs clarification. Its depth in zwitterionic, bilayered micelles at pH 5 (resembling late endosomes) was measured by paramagnetic NMR relaxation enhancements used to bias molecular dynamics simulations. Asp830 inserted deeply, along with Lys825 or Lys835. Protonation of Asp830 appeared to enhance agreement of simulated and NMR-measured depths. While the fusion peptide occupied a leaflet of the DMPC bilayer, the opposite leaflet invaginated with influx of water and choline head groups in around Asp830 and bilayer-inserted polar side chains. NMR-detected hydrogen exchange found corroborating hydration of the backbone of Thr827-Phe833 inserted deeply in bicelles. Pinching of the membrane at the inserted charge and the intramembrane hydration of polar groups agree with theory. Formation of corridors of hydrated, inward-turned head groups was accompanied by flip-flop of head groups. Potential roles of the defects are discussed.

Solution NMR of a 463-residue phosphohexomutase: domain 4 mobility, substates, and phosphoryl transfer defect.

Phosphomannomutase/phosphoglucomutase contributes to the infectivity of Pseudomonas aeruginosa, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. Nuclear magnetic resonance (NMR) spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. (13)C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 α-helices, C-capping motifs, and 20 of the 22 ß-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These can be attributed to the phosphorylation state and possibly to conformational interconversions. The S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose 1-phosphate substrate by impairing k(cat). Addition of the glucose 1,6-bisphosphate intermediate accelerated this reaction by 2-3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft while preserving the secondary structure in solution. Diminished peak heights and faster (15)N relaxation suggest line broadening and millisecond fluctuations within four loops that can contact phosphosugars. (15)N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1-3, and with a different principal axis of diffusion. This adds to the crystallographic evidence of domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of the intermediate, substrate binding, and product release.

Entropy increases from different sources support the high-affinity binding of the N-terminal inhibitory domains of tissue inhibitors of metalloproteinases to the catalytic domains of matrix metalloproteinases-1 and -3.

The avid binding of tissue inhibitors of metalloproteinases (TIMPs) to matrix metalloproteinases (MMPs) is crucial for the regulation of pericellular and extracellular proteolysis. The interactions of the catalytic domain (cd) of MMP-1 with the inhibitory domains of TIMP-1 and TIMP-2 (N-TIMPs) and MMP-3cd with N-TIMP-2 have been characterized by isothermal titration calorimetry and compared with published data for the N-TIMP-1/MMP-3cd interaction. All interactions are largely driven by increases in entropy but there are significant differences in the profiles for the interactions of both N-TIMPs with MMP-1cd as compared with MMP-3cd; the enthalpy change ranges from small for MMP-1cd to highly unfavorable for MMP-3cd (-0.1 ± 0.7 versus 6.0 ± 0.5 kcal mol(-1)). The heat capacity change (ΔC(p)) of binding to MMP-1cd (temperature dependence of ΔH) is large and negative (-210 ± 20 cal K(-1) mol(-1)), indicating a large hydrophobic contribution, whereas the ΔC(p) values for the binding to MMP-3cd are much smaller (-53 ± 3 cal K(-1) mol(-1)), and some of the entropy increase may arise from increased conformational entropy. Apart from differences in ionization effects, it appears that the properties of the MMP may have a predominant influence in the thermodynamic profiles for these N-TIMP/MMP interactions.

Development of a Therapeutic Peptide for Cachexia Suggests a Platform Approach for Drug-like Peptides.

During the development of a melanocortin (MC) peptide drug to treat the condition of cachexia (a hypermetabolic state producing lean body mass wasting), we were confronted with the need for peptide transport across the blood-brain barrier (BBB): the MC-4 receptors (MC4Rs) for metabolic rate control are located in the hypothalamus, i.e., behind the BBB. Using the term "peptides with BBB transport", we screened the medical literature like a peptide library. This revealed numerous "hits"-peptides with BBB transport and/or oral activity. We noted several features common to most peptides in this class, including a dipeptide sequence of nonpolar residues, primary structure cyclization (whole or partial), and a Pro-aromatic motif usually within the cyclized region. Based on this, we designed an MC4R antagonist peptide, TCMCB07, that successfully treated many forms of cachexia. As part of our pharmacokinetic characterization of TCMCB07, we discovered that hepatobiliary extraction from blood accounted for a majority of the circulating peptide's excretion. Further screening of the literature revealed that TCMCB07 is a member of a long-forgotten peptide class, showing active transport by a multi-specific bile salt carrier. Bile salt transport peptides have predictable pharmacokinetics, including BBB transport, but rapid hepatic clearance inhibited their development as drugs. TCMCB07 shares the general characteristics of the bile salt peptide class but with a much longer half-life of hours, not minutes. A change in its C-terminal amino acid sequence slows hepatic clearance. This modification is transferable to other peptides in this class, suggesting a platform approach for producing drug-like peptides.

Remote exosites of the catalytic domain of matrix metalloproteinase-12 enhance elastin degradation.

How does matrix metalloproteinase-12 (MMP-12 or metalloelastase) degrade elastin with high specific activity? Nuclear magnetic resonance suggested soluble elastin covers surfaces of MMP-12 far from its active site. Two of these surfaces have been found, by mutagenesis guided by the BINDSIght approach, to affect degradation and affinity for elastin substrates but not a small peptide substrate. Main exosite 1 has been extended to Asp124 that binds calcium. Novel exosite 2 comprises residues from the II-III loop and ß-strand I near the back of the catalytic domain. The high degree of exposure of these distal exosites may make them accessible to elastin made more flexible by partial hydrolysis. Importantly, the combination of one lesion each at exosites 1 and 2 and the active site decreased the catalytic competence toward soluble elastin by 13-18-fold to the level of MMP-3, homologue and poor elastase. Double-mutant cycle analysis of conservative mutations of Met156 (exosite 2) and either Asp124 (exosite 1) or Ile180 (active site) showed they had additive effects. Compared to polar substitutions observed in other MMPs, Met156 enhanced affinity and Ile180 the k(cat) for soluble elastin. Both residues detracted from the higher folding stability with polar mutations. This resembles the trend in enzymes of an inverse relationship between folding stability and activity. Restoring Asp124 from combination mutants enhanced the k(cat) for soluble elastin. In elastin degradation, exosites 1 and 2 contributed in a manner independent of each other and Ile180 at the active site, but with partial coupling to Ala182 near the active site. The concept of weak, separated interactions coalescing somewhat independently can be extended to this proteolytic digestion of a protein from fibrils.

NMR and bioinformatics discovery of exosites that tune metalloelastase specificity for solubilized elastin and collagen triple helices.

The catalytic domain of metalloelastase (matrix metalloproteinase-12 or MMP-12) is unique among MMPs in exerting high proteolytic activity upon fibrils that resist hydrolysis, especially elastin from lungs afflicted with chronic obstructive pulmonary disease or arteries with aneurysms. How does the MMP-12 catalytic domain achieve this specificity? NMR interface mapping suggests that α-elastin species cover the primed subsites, a strip across the ß-sheet from ß-strand IV to the II-III loop, and a broad bowl from helix A to helix C. The many contacts may account for the comparatively high affinity, as well as embedding of MMP-12 in damaged elastin fibrils in vivo. We developed a strategy called BINDSIght, for bioinformatics and NMR discovery of specificity of interactions, to evaluate MMP-12 specificity without a structure of a complex. BINDSIght integration of the interface mapping with other ambiguous information from sequences guided choice mutations in binding regions nearer the active site. Single substitutions at each of ten locations impair specific activity toward solubilized elastin. Five of them impair release of peptides from intact elastin fibrils. Eight lesions also impair specific activity toward triple helices from collagen IV or V. Eight sites map to the "primed" side in the III-IV, V-B, and S1' specificity loops. Two map to the "unprimed" side in the IV-V and B-C loops. The ten key residues circumscribe the catalytic cleft, form an exosite, and are distinctive features available for targeting by new diagnostics or therapeutics.

Solubilized elastin substrate for continuous fluorimetric assay of kinetics of elastases.

Elastolysis is central to progression of emphysema and aortic aneurysms. Characterization of steady-state enzyme kinetics of elastolysis is fettered by the insolubility of mature elastin and the polydispersity of solubilized elastin. We prepared a fluor-tagged, 100-kDa fraction (fEln-100) from commercial α-elastin. It is soluble, less heterogeneous in mass, cross-linked like mature elastin, and likely to retain the capacity of α-elastin to self-assemble. fEln-100 has introduced the ability to compare quantitatively the apparent k(cat) and K(m) of elastases. For example, metalloelastase (MMP-12) displays higher apparent affinity for fEln-100, while MMP-2 displays faster catalytic turnover.

Apparent tradeoff of higher activity in MMP-12 for enhanced stability and flexibility in MMP-3.

The greater activity of MMP-12 than MMP-3 toward substrates from protein fibrils has been quantified. Why is MMP-12 the more active protease? We looked for behaviors associated with the higher activity of MMP-12 than MMP-3, using nuclear magnetic resonance to monitor backbone dynamics and residue-specific stabilities of their catalytic domain. The proteolytic activities are likely to play important roles in inflammatory diseases of arteries, lungs, joints, and intestines. Nuclear magnetic resonance line broadening indicates that regions surrounding the active sites of both proteases sample conformational substates within milliseconds. The more extensive line broadening in MMP-3 suggests greater sampling of conformational substates, affecting the full length of helix B and beta-strand IV forming the active site, and more remote sites. This could suggest more excursions to functionally incompetent substates. MMP-3 also has enhanced subnanosecond fluctuations in helix A, in the beta-hairpin of strands IV and V, and before and including helix C. Hydrogen exchange protection in the EX2 regime suggests that MMP-3 possesses 2.8 kcal/mol higher folding stability than MMP-12(E219A). The beta-sheet of MMP-3 appears to be stabilized still more. The higher stability of MMP-3 relative to MMP-12 coincides with the former's considerably lower proteolytic activity. This relationship is consistent with the hypothesis that enzymes often trade stability for higher activity.

An examination of the proteolytic activity for bovine pregnancy-associated glycoproteins 2 and 12.

The pregnancy-associated glycoproteins (PAGs) represent a complex group of putative aspartic peptidases expressed exclusively in the placentas of species in the Artiodactyla order. The ruminant PAGs segregate into two classes: the 'ancient' and 'modern' PAGs. Some of the modern PAGs possess alterations in the catalytic center that are predicted to preclude their ability to act as peptidases. The ancient ruminant PAGs in contrast are thought to be peptidases, although no proteolytic activity has been described for these members. The aim of the present study was to investigate (1) if the ancient bovine PAGs (PAG-2 and PAG-12) have proteolytic activity, and (2) if there are any differences in activity between these two closely related members. Recombinant bovine PAG-2 and PAG-12 were expressed in a baculovirus expression system and the purified proteins were analyzed for proteolytic activity against a synthetic fluorescent cathepsin D/E substrate. Both proteins exhibited proteolytic activity with acidic pH optima. The k(cat)/K(m) for bovine PAG-2 was 2.7x10(5) m(-1) s(-1) and for boPAG-12 it was 6.8x10(4) m(-1) s(-1). The enzymes were inhibited by pepstatin A with a K(i) of 0.56 and 7.5 nm for boPAG-2 and boPAG-12, respectively. This is the first report describing proteolytic activity in PAGs from ruminant ungulates.

Docking of acetyl-CoA carboxylase to the plastid envelope membrane attenuates fatty acid production in plants.

In plants, light-dependent activation of de novo fatty acid synthesis (FAS) is partially mediated by acetyl-CoA carboxylase (ACCase), the first committed step for this pathway. However, it is not fully understood how plants control light-dependent FAS regulation to meet the cellular demand for acyl chains. We report here the identification of a gene family encoding for three small plastidial proteins of the envelope membrane that interact with the α-carboxyltransferase (α-CT) subunit of ACCase and participate in an original mechanism restraining FAS in the light. Light enhances the interaction between carboxyltransferase interactors (CTIs) and α-CT, which in turn attenuates carbon flux into FAS. Knockouts for CTI exhibit higher rates of FAS and marked increase in absolute triacylglycerol levels in leaves, more than 4-fold higher than in wild-type plants. Furthermore, WRINKLED1, a master transcriptional regulator of FAS, positively regulates CTI1 expression by direct binding to its promoter. This study reveals that in addition to light-dependent activation, "envelope docking" of ACCase permits fine-tuning of fatty acid supply during the plant life cycle.