Bilayer Membrane Modulation of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Structure and Proteolytic Activity.

Cell surface proteolysis is an integral yet poorly understood physiological process. The present study has examined how the pericellular collagenase membrane-type 1 matrix metalloproteinase (MT1-MMP) and membrane-mimicking environments interplay in substrate binding and processing. NMR derived structural models indicate that MT1-MMP transiently associates with bicelles and cells through distinct residues in blades III and IV of its hemopexin-like domain, while binding of collagen-like triple-helices occurs within blades I and II of this domain. Examination of simultaneous membrane interaction and triple-helix binding revealed a possible regulation of proteolysis due to steric effects of the membrane. At bicelle concentrations of 1%, enzymatic activity towards triple-helices was increased 1.5-fold. A single mutation in the putative membrane interaction region of MT1-MMP (Ser466Pro) resulted in lower enzyme activation by bicelles. An initial structural framework has thus been developed to define the role(s) of cell membranes in modulating proteolysis.

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.

Divergent sequence tunes ligand sensitivity in phospholipid-regulated hormone receptors.

The members of the NR5A subfamily of nuclear receptors (NRs) are important regulators of pluripotency, lipid and glucose homeostasis, and steroidogenesis. Liver receptor homologue 1 (LRH-1; NR5A2) and steroidogenic factor 1 (SF-1; NR5A1) have therapeutic potential for the treatment of metabolic and neoplastic disease; however, a poor understanding of their ligand regulation has hampered the pursuit of these proteins as pharmaceutical targets. In this study, we dissect how sequence variation among LRH-1 orthologs affects phospholipid (PL) binding and regulation. Both human LRH-1 (hLRH-1) and mouse LRH-1 (mLRH-1) respond to newly discovered medium chain PL agonists to modulate lipid and glucose homeostasis. These PLs activate hLRH-1 by altering receptor dynamics in a newly identified alternate activation function region. Mouse and Drosophila orthologs contain divergent sequences in this region potentially altering PL-driven activation. Structural evidence suggests that these sequence differences in mLRH-1 and Drosophila FTZ-f1 (dmFTZ-f1) confer at least partial ligand independence, making them poor models for hLRH-1 studies; however, the mechanisms of ligand independence remain untested. We show using structural and biochemical methods that the recent evolutionary divergence of the mLRH-1 stabilizes the active conformation in the absence of ligand, yet does not abrogate PL-dependent activation. We also show by mass spectrometry and biochemical assays that FTZ-f1 is incapable of PL binding. This work provides a structural mechanism for the differential tuning of PL sensitivity in NR5A orthologs and supports the use of mice as viable therapeutic models for LRH-1-dependent diseases.

Application of Collagen-Model Triple-Helical Peptide-Amphiphiles for CD44-Targeted Drug Delivery Systems.

Cancer treatment by chemotherapy is typically accompanied by deleterious side effects, attributed to the toxic action of chemotherapeutics on proliferating cells from nontumor tissues. The cell surface proteoglycan CD44 has been recognized as a cancer stem cell marker. The present study has examined CD44 targeting as a way to selectively deliver therapeutic agents encapsulated inside colloidal delivery systems. CD44/chondroitin sulfate proteoglycan binds to a triple-helical sequence derived from type IV collagen, α1(IV)1263-1277. We have assembled a peptide-amphiphile (PA) in which α1(IV)1263-1277 was sandwiched between 4 repeats of Gly-Pro-4-hydroxyproline and conjugated to palmitic acid. The PA was incorporated into liposomes composed of DSPG, DSPC, cholesterol, and DSPE-PEG-2000 (1 : 4 : 5 : 0.5). Doxorubicin-(DOX-)loaded liposomes with and without 10% α1(IV)1263-1277 PA were found to exhibit similar stability profiles. Incubation of DOX-loaded targeted liposomes with metastatic melanoma M14#5 and M15#11 cells and BJ fibroblasts resulted in IC(50) values of 9.8, 9.3, and >100 µM, respectively. Nontargeted liposomes were considerably less efficacious for M14#5 cells. In the CD44(+) B16F10 mouse melanoma model, CD44-targeted liposomes reduced the tumor size to 60% of that of the untreated control, whereas nontargeted liposomes were ineffective. These results suggest that PA targeted liposomes may represent a new class of nanotechnology-based drug delivery systems.

HDX workbench: software for the analysis of H/D exchange MS data.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an established method for the interrogation of protein conformation and dynamics. While the data analysis challenge of HDX-MS has been addressed by a number of software packages, new computational tools are needed to keep pace with the improved methods and throughput of this technique. To address these needs, we report an integrated desktop program titled HDX Workbench, which facilitates automation, management, visualization, and statistical cross-comparison of large HDX data sets. Using the software, validated data analysis can be achieved at the rate of generation. The application is available at the project home page http://hdx.florida.scripps.edu .

Antidiabetic phospholipid-nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation.

The human nuclear receptor liver receptor homolog-1 (LRH-1) has an important role in controlling lipid and cholesterol homeostasis and is a potential target for the treatment of diabetes and hepatic diseases. LRH-1 is known to bind phospholipids, but the role of phospholipids in controlling LRH-1 activation remains highly debated. Here we describe the structure of both apo LRH-1 and LRH-1 in complex with the antidiabetic phospholipid dilauroylphosphatidylcholine (DLPC). Together with hydrogen-deuterium exchange MS and functional data, our studies show that DLPC binding is a dynamic process that alters co-regulator selectivity. We show that the lipid-free receptor undergoes previously unrecognized structural fluctuations, allowing it to interact with widely expressed co-repressors. These observations enhance our understanding of LRH-1 regulation and highlight its importance as a new therapeutic target for controlling diabetes.

Identification of SR2211: a potent synthetic RORγ-selective modulator.

Nuclear receptors (NRs) are ligand-regulated transcription factors that display canonical domain structure with highly conserved DNA-binding and ligand-binding domains. The identification of the endogenous ligands for several receptors remains elusive or is controversial, and thus these receptors are classified as orphans. One such orphan receptor is the retinoic acid receptor-related orphan receptor γ (RORγ). An isoform of RORγ, RORγt, has been shown to be essential for the expression of Interleukin 17 (IL-17) and the differentiation of Th17 cells. Th17 cells have been implicated in the pathology of several autoimmune diseases, including multiple sclerosis (MS) and rheumatoid arthritis (RA). Genetic ablation of RORγ alone or in combination with RORα in mice led to impaired Th17 differentiation and protected the mice from development of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Here we describe SR2211, a selective RORγ modulator that potently inhibits production of IL-17 in cells.

Structural basis for matrix metalloproteinase 1-catalyzed collagenolysis.

The proteolysis of collagen triple-helical structure (collagenolysis) is a poorly understood yet critical physiological process. Presently, matrix metalloproteinase 1 (MMP-1) and collagen triple-helical peptide models have been utilized to characterize the events and calculate the energetics of collagenolysis via NMR spectroscopic analysis of 12 enzyme-substrate complexes. The triple-helix is bound initially by the MMP-1 hemopexin-like (HPX) domain via a four amino acid stretch (analogous to type I collagen residues 782-785). The triple-helix is then presented to the MMP-1 catalytic (CAT) domain in a distinct orientation. The HPX and CAT domains are rotated with respect to one another compared with the X-ray "closed" conformation of MMP-1. Back-rotation of the CAT and HPX domains to the X-ray closed conformation releases one chain out of the triple-helix, and this chain is properly positioned in the CAT domain active site for subsequent hydrolysis. The aforementioned steps provide a detailed, experimentally derived, and energetically favorable collagenolytic mechanism, as well as significant insight into the roles of distinct domains in extracellular protease function.

Suppression of TH17 differentiation and autoimmunity by a synthetic ROR ligand.

T-helper cells that produce interleukin-17 (T(H)17 cells) are a recently identified CD4(+) T-cell subset with characterized pathological roles in autoimmune diseases. The nuclear receptors retinoic-acid-receptor-related orphan receptors α and γt (RORα and RORγt, respectively) have indispensible roles in the development of this cell type. Here we present SR1001, a high-affinity synthetic ligand-the first in a new class of compound-that is specific to both RORα and RORγt and which inhibits T(H)17 cell differentiation and function. SR1001 binds specifically to the ligand-binding domains of RORα and RORγt, inducing a conformational change within the ligand-binding domain that encompasses the repositioning of helix 12 and leads to diminished affinity for co-activators and increased affinity for co-repressors, resulting in suppression of the receptors' transcriptional activity. SR1001 inhibited the development of murine T(H)17 cells, as demonstrated by inhibition of interleukin-17A gene expression and protein production. Furthermore, SR1001 inhibited the expression of cytokines when added to differentiated murine or human T(H)17 cells. Finally, SR1001 effectively suppressed the clinical severity of autoimmune disease in mice. Our data demonstrate the feasibility of targeting the orphan receptors RORα and RORγt to inhibit specifically T(H)17 cell differentiation and function, and indicate that this novel class of compound has potential utility in the treatment of autoimmune diseases.

Comparison of metalloproteinase protein and activity profiling.

Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The current study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer cell lines and 1 normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media (in both amount and variety). TIMP-1 was produced in all cell lines. MMP activity assays with three different fluorescence resonance energy transfer (FRET) substrates were then used to determine whether protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was confirmed only in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays.

Variability in melanoma metalloproteinase expression profiling.

The proteolytic activities of a disintegrin and metalloproteinase (ADAM); a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and matrix metalloproteinase (MMP) families play important roles in normal and multiple pathological conditions. These metalloproteases have potential roles in the degradation of the ECM and in the processing of bioactive molecules. In the present study, RNA was isolated from multiple normal fibroblast and metastatic melanoma cell lines, as well as the isogenic normal tissue and tumor samples, and the gene expression levels of six ADAMs, eight MMPs, and four ADAMTSs were analyzed by real-time PCR. This approach allowed for detected changes in mRNA expression of the individual metalloproteinase genes to be compared between normal and metastatic states and also between tissue and cultured cells. Increased gene expression of several ADAM and MMP family members (MMP1, MMP8, MMP15, and ADAM15) occurred in melanoma tissue and was replicated in tissue cultures. In general, the level of ADAM and MMP mRNA expression was several-fold higher in cultured cells compared with the isogenic tissue from which they were derived. Passage-dependent expression patterns were observed for MMP8 and MMP9 in in-house-derived metastatic melanoma cell lines. This reiterates earlier suggestions that experiments using cells that have been maintained in culture should be interpreted with great care.

Local conformation and dynamics of isoleucine in the collagenase cleavage site provide a recognition signal for matrix metalloproteinases.

The mechanism by which enzymes recognize the "uniform" collagen triple helix is not well understood. Matrix metalloproteinases (MMPs) cleave collagen after the Gly residue of the triplet sequence Gly∼[Ile/Leu]-[Ala/Leu] at a single, unique, position along the peptide chain. Sequence analysis of types I-III collagen has revealed a 5-triplet sequence pattern in which the natural cleavage triplets are always flanked by a specific distribution of imino acids. NMR and MMP kinetic studies of a series of homotrimer peptides that model type III collagen have been performed to correlate conformation and dynamics at, and near, the cleavage site to collagenolytic activity. A peptide that models the natural cleavage site is significantly more active than a peptide that models a potential but non-cleavable site just 2-triplets away and NMR studies show clearly that the Ile in the leading chain of the cleavage peptide is more exposed to solvent and less locally stable than the Ile in the middle and lagging chains. We propose that the unique local instability of Ile at the cleavage site in part arises from the placement of the conserved Pro at the P(3) subsite. NMR studies of peptides with Pro substitutions indicate that the local dynamics of the three chains are directly modulated by their proximity to Pro. Correlation of peptide activity to NMR data shows that a single locally unstable chain at the cleavage site, rather than two or three labile chains, is more favorable for cleavage by MMP-1 and may be the determining factor for collagen recognition.

Determination of penetratin secondary structure in live cells with Raman microscopy.

Cell penetrating peptides (CPPs) have attracted recent interest as drug delivery tools, although the mechanisms by which CPPs are internalized by cells are not well-defined. Here, we report a new experimental approach for the detection and secondary structure determination of CPPs in live cells using Raman microscopy with heavy isotope labeling of the peptide. As a first demonstration of principle, penetratin, a 16-residue CPP derived from the Antennapedia homeodomain protein of Drosophila, was measured in single, living melanoma cells. Carbon-13 labeling of the Phe residue of penetratin was used to shift the intense aromatic ring-breathing vibrational mode from 1003 to 967 cm(-1), thereby enabling the peptide to be traced in cells. Difference spectroscopy and principal components analysis (PCA) were used independently to resolve the Raman spectrum of the peptide from the background cellular Raman signals. On the basis of the position of the amide I vibrational band in the Raman spectra, the secondary structure of the peptide was found to be mainly random coil and beta-strand in the cytoplasm, and possibly assembling as beta-sheets in the nucleus. The rapid entry and almost uniform cellular distribution of the peptide, as well as the lack of correlation between peptide and lipid Raman signatures, indicated that the mechanism of internalization under the conditions of study was probably nonendocytotic. This experimental approach can be used to study a wide variety of CPPs as well as other classes of peptides in living cells.