Dog as the experimental model: Laboratory uses of dogs in the United States.

Dogs are the experimental model in many types of biomedical research. Each year, hundreds of publications report the use of dogs in invasive biomedical procedures, often without sufficient explanation of the purpose and justification for selecting dog as the experimental model. The European Union requires detailed reporting of animal use that includes research purpose, but animal use reporting in the United States, overseen by the USDA, does not require this information. The ability to replace dogs with alternative models begins by understanding how they are used. Therefore, this study was undertaken to investigate the types of invasive biomedical procedures that dogs are subjected to in US laboratories. Well-defined sets of research publications and grants were accessed to obtain information on the types of biomedical research using dogs. USDA databases provided additional information. An ontology to categorize biomedical research uses of dogs identified the most common as translational studies for cardiovascular, cancer, nervous/mental, and mus-culoskeletal disorders. Information typically reported for experimental animals was sometimes missing or incomplete in publications, including the number, source, fate, species justification, and pain management of dogs, suggesting that many journals have not adopted the ARRIVE guidelines on animal use reporting. It was not possible to identify the research purpose for all dogs used by US institutions because (a) not all dog use is published and (b) animal research purpose is not required reporting in the US. These findings should be informative to future initiatives to replace, reduce, and refine the use of dogs in research.

Educators' Views on the Use of Dissection and Dissection Alternatives in American Biology Classrooms.

Animal dissection remains a common practice in American biology classrooms, despite the availability of dissection alternatives to study anatomy and physiology. Indeed, there is a growing body of evidence in the literature suggesting that the use of alternatives leads to the greater achievement of learning objectives, as compared to dissection. To better understand the current use of and attitudes toward dissection and alternatives, a nationwide survey of (mainly high-school) biology teachers (n = 2687) was conducted. Most educators believed that learning objectives related to biology subject content could be met through the use of alternatives, yet they preferred the hands-on experience of dissection. Most educators allow their students to use alternatives if requested, although few teachers ask students about their preference for using an animal specimen versus an alternative. Educators cited student engagement as the main factor driving their decision to choose between dissection specimens and alternatives, and felt that cost is the biggest barrier to implementing alternatives at their schools. Additional perspectives on dissection and alternatives were shared by survey participants. Since alternatives can be used to meet learning objectives associated with dissection, we recommend their use as replacements for traditional animal specimens, in line with the replacement, reduction and refinement of animal use in education, according to the Three Rs principles.

Part 1: Notch-sparing γ-secretase inhibitors: The identification of novel naphthyl and benzofuranyl amide analogs.

γ-Secretase is one of two proteases directly involved in the production of the amyloid ß-peptide (Aß), which is pathogenic in Alzheimer's disease. Inhibition of γ-secretase to suppress the production of Aß should not block processing of one of its alternative substrates, Notch1 receptors, as interference with Notch1 signaling leads to severe toxic effects. In the course of our studies to identify γ-secretase inhibitors with selectivity for APP over Notch, 1 [3-(benzyl(isopropyl)amino)-1-(naphthalen-2-yl)propan-1-one] was found to inhibit γ-secretase-mediated Aß production without interfering with γ-secretase-mediated Notch processing in purified enzyme assays. As 1 is chemically unstable, efforts to increase the stability of this compound led to the identification of 2 [naphthalene-2-carboxylic acid benzyl-isopropyl-amide] which showed similar biological activity to compound 1. Synthesis and evaluation of a series of amide analogs resulted in benzofuranyl amide analogs that showed promising Notch-sparing γ-secretase inhibitory effects. This class of compounds may serve as a novel lead series for further study in the development of γ-secretase inhibitors.

Part 2. Notch-sparing γ-secretase inhibitors: The study of novel γ-amino naphthyl alcohols.

One therapeutic approach for Alzheimer's disease is to inhibit the cleavage of the amyloid precursor protein (APP) by γ-secretase. At the beginning of a series of studies from our laboratories, a series of novel γ-amino alcohols (1) were found to possess γ-secretase inhibitory activity and Notch-sparing effects. A new one-pot synthesis of γ-amino alcohols and the structure-activity relationship (SAR) of these analogs will be discussed.

Dissociation between the processivity and total activity of γ-secretase: implications for the mechanism of Alzheimer's disease-causing presenilin mutations.

The amyloid ß-peptide (Aß), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is produced from the amyloid ß-protein precursor (APP) through consecutive proteolysis by ß- and γ-secretases. The latter protease contains presenilin as the catalytic component of a membrane-embedded aspartyl protease complex. Missense mutations in presenilin are associated with early-onset familial AD, and these mutations generally both decrease Aß production and increase the ratio of the aggregation-prone 42-residue form (Aß42) to the 40-residue form (Aß40). The connection between these two effects is not understood. Besides Aß40 and Aß42, γ-secretase produces a range of Aß peptides, the result of initial cutting at the ε site to form Aß48 or Aß49 and subsequent trimming every three or four residues. Thus, γ-secretase displays both overall proteolytic activity (ε cutting) and processivity (trimming) toward its substrate APP. Here we tested whether a decrease in total activity correlates with decreased processivity using wild-type and AD-mutant presenilin-containing protease complexes. Changes in pH, temperature, and salt concentration that reduced the overall activity of the wild-type enzyme did not consistently result in increased proportions of longer Aß peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aß peptides, suggesting that the charged state of certain residues may influence processivity. Five different AD mutant complexes, representing a broad range of effects on overall activity, Aß42:Aß40 ratios, and ages of disease onset, were also tested, revealing again that changes in total activity and processivity can be dissociated. Factors that control initial proteolysis of APP at the ε site apparently differ significantly from factors affecting subsequent trimming and the distribution of Aß peptides.

Cryoelectron microscopy structure of purified gamma-secretase at 12 A resolution.

Gamma-secretase, an integral membrane protein complex, catalyzes the intramembrane cleavage of the beta-amyloid precursor protein (APP) during the neuronal production of the amyloid beta-peptide. As such, the protease has emerged as a key target for developing agents to treat and prevent Alzheimer's disease. Existing biochemical studies conflict on the oligomeric assembly state of the protease complex, and its detailed structure is not known. Here, we report that purified active human gamma-secretase in digitonin has a total molecular mass of approximately 230 kDa when measured by scanning transmission electron microscopy. This result supports a complex that is monomeric for each of the four component proteins. We further report the three-dimensional structure of the gamma-secretase complex at 12 A resolution as obtained by cryoelectron microscopy and single-particle image reconstruction. The structure reveals several domains on the extracellular side, three solvent-accessible low-density cavities, and a potential substrate-binding surface groove in the transmembrane region of the complex.

Direct and potent regulation of gamma-secretase by its lipid microenvironment.

gamma-Secretase is an unusual and ubiquitous aspartyl protease with an intramembrane catalytic site that cleaves many type-I integral membrane proteins, most notably APP and Notch. Several reports suggest that cleavage of APP to produce the Abeta peptide is regulated in part by lipids. As gamma-secretase is a multipass protein complex with 19 transmembrane domains, it is likely that the local lipid composition of the membrane can regulate gamma-activity. To determine the direct contribution of the lipid microenvironment to gamma-secretase activity, we purified the human protease from overexpressing mammalian cells, reconstituted it in vesicles of varying lipid composition, and examined the effects of individual phospholipids, sphingolipids, cholesterol, and complex lipid mixtures on substrate cleavage. A conventional gamma-activity assay was modified to include a detergent-removal step to facilitate proteoliposome formation, and this increased baseline activity over 2-fold. Proteoliposomes containing sphingolipids significantly increased gamma-secretase activity over a phosphatidylcholine-only baseline, whereas the addition of phosphatidylinositol significantly decreased activity. Addition of soluble cholesterol in the presence of phospholipids and sphingolipids robustly increased the cleavage of APP- and Notch-like substrates in a dose-dependent manner. Reconstitution of gamma-secretase in complex lipid mixtures revealed that a lipid raft-like composition supported the highest level of activity compared with other membrane compositions. Taken together, these results demonstrate that membrane lipid composition is a direct and potent modulator of gamma-secretase and that cholesterol, in particular, plays a major regulatory role.

The inactive 44-kDa processed form of membrane type 1 matrix metalloproteinase (MT1-MMP) enhances proteolytic activity via regulation of endocytosis of active MT1-MMP.

Membrane type 1 (MT1) matrix metalloproteinase (MMP-14) is a membrane-tethered MMP considered to be a major mediator of pericellular proteolysis. MT1-MMP is regulated by a complex array of mechanisms, including processing and endocytosis that determine the pool of active proteases on the plasma membrane. Autocatalytic processing of active MT1-MMP generates an inactive membrane-tethered 44-kDa product (44-MT1) lacking the catalytic domain. This form preserves all other enzyme domains and is retained at the cell surface. Paradoxically, accumulation of the 44-kDa form has been associated with increased enzymatic activity. Here we report that expression of a recombinant 44-MT1 (Gly(285)-Val(582)) in HT1080 fibrosarcoma cells results in enhanced pro-MMP-2 activation, proliferation within a three-dimensional collagen I matrix, and tumor growth and lung metastasis in mice. Stimulation of pro-MMP-2 activation and growth in collagen I was also observed in other cell systems. Expression of 44-MT1 in HT1080 cells is associated with a delay in the rate of active MT1-MMP endocytosis resulting in higher levels of active enzyme at the cell surface. Consistently, deletion of the cytosolic domain obliterates the stimulatory effects of 44-MT1 on MT1-MMP activity. In contrast, deletion of the hinge turns the 44-MT1 form into a negative regulator of enzyme function in vitro and in vivo, suggesting a key role for the hinge region in the functional relationship between active and processed MT1-MMP. Together, these results suggest a novel role for the 44-kDa form of MT1-MMP generated during autocatalytic processing in maintaining the pool of active enzyme at the cell surface.

Rapid purification of active gamma-secretase, an intramembrane protease implicated in Alzheimer's disease.

Gamma-secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid-beta precursor protein generates the amyloid-beta protein (Abeta) of Alzheimer's disease, partially inhibiting gamma-secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of gamma-secretase, but at low resolution (15 A). The limited amount of purified gamma-secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X-ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S-20) that overexpresses strikingly high levels of all four gamma-secretase components (presenilin, nicastrin, Aph-1 and Pen-2). We then used these cells to develop a rapid protocol for the high-grade purification of proteolytically active gamma-secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme.

Prostate cancer-associated membrane type 1-matrix metalloproteinase: a pivotal role in bone response and intraosseous tumor growth.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a major mediator of collagen I degradation. In human samples, we show that prostate cancer cells in skeletal metastases consistently express abundant MT1-MMP protein. Because prostate cancer bone metastasis requires remodeling of the collagen-rich bone matrix, we investigated the role of cancer cell-derived MT1-MMP in an experimental model of tumor-bone interaction. MT1-MMP-deficient LNCaP human prostate cancer cells were stably transfected with human wild-type MT1-MMP (MT1wt). Furthermore, endogenous MT1-MMP was down-regulated by small interfering RNA in DU145 human prostate cancer cells. Intratibial tumor injection in severe combined immunodeficient mice was used to simulate intraosseous growth of metastatic tumors. LNCaP-MT1wt cells produced larger osseous tumors than Neo control cells and induced osteolysis, whereas DU145 MT1-MMP-silenced transfectants induced osteogenic changes. In vitro assays showed that MT1wt overexpression enhanced collagen I degradation, whereas MT1-MMP-silencing did the opposite, suggesting that tumor-derived MT1-MMP may contribute directly to bone remodeling. LNCaP-MT1wt-derived conditioned medium stimulated in vitro multinucleated osteoclast formation. This effect was inhibited by osteoprotegerin, a decoy receptor for receptor activator of nuclear factor kappaB ligand, and by 4-[4-(methanesulfonamido) phenoxy] phenylsulfonyl methylthiirane, an MT1-MMP inhibitor. Our findings are consistent with the hypothesis that prostate cancer-associated MT1-MMP plays a direct and/or indirect role in bone matrix degradation, thus favoring intraosseous tumor expansion.

SPARC upregulates MT1-MMP expression, MMP-2 activation, and the secretion and cleavage of galectin-3 in U87MG glioma cells.

Secreted protein acidic and rich in cysteine (SPARC) is highly expressed in human gliomas and promotes glioma invasion. We have shown by cDNA array analysis that SPARC upregulates membrane type 1-matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) transcripts. To confirm these findings at the protein level and determine whether SPARC expression correlates with increased MMP activity, we used Western blot to assess the levels of MT1-MMP, and gelatin zymography to assess MMP-2 levels and activity. We also examined the expression, secretion, and cleavage of galectin-3, a target of MT1-MMP and MMP-2. Our data confirm that SPARC upregulates MT1-MMP levels and MMP-2 activity. There was also an increase in secreted galectin-3, as well as an increase in the proteolytically processed form of galectin-3. Previous studies have demonstrated that MT1-MMP, MMP-2, and galectin-3 are increased in gliomas. Our results suggest that their upregulation and activation may be a consequence of increased SPARC expression. These data provide a provisional mechanism whereby SPARC contributes to brain tumor invasion.

Mutational and structural analyses of the hinge region of membrane type 1-matrix metalloproteinase and enzyme processing.

Membrane type 1 (MT1)-matrix metalloproteinase (MMP) is a major mediator of collagen degradation in the pericellular space in both physiological and pathological conditions. Previous evidence has shown that on the cell surface, active MT1-MMP undergoes autocatalytic processing to a major membrane-tethered 44-kDa product lacking the catalytic domain and displaying Gly285 at its N terminus, which is at the beginning of the hinge domain. However, the importance of this site and the hinge region in MT1-MMP processing is unknown. In the current study, we generated mutations and deletions in the hinge of MT1-MMP and followed their effect on processing. These studies established Gly284-Gly285 as the main cleavage site involved in the formation of the 44-kDa species. However, alterations at this site did not prevent processing. Instead, they forced downstream cleavages within the stretch of residues flanked by Gln296 and Ser304 in the hinge region, as determined by the processing profile of various hinge deletion mutants. Also, replacement of the hinge of MT1-MMP with the longer MT3-MMP hinge did not prevent processing of MT1-MMP. Molecular dynamic studies using a computational model of MT1-MMP revealed that the hinge region is a highly motile element that undergoes significant motion in the highly exposed loop formed by Pro295-Arg302 consistent with being a prime target for proteolysis, in agreement with the mutational data. These studies suggest that the hinge of MT1-MMP evolved to facilitate processing, a promiscuous but compulsory event in the destiny of MT1-MMP, which may play a key role in the control of pericellular proteolysis.

Cleavage at the stem region releases an active ectodomain of the membrane type 1 matrix metalloproteinase.

MT1-MMP (membrane type 1 matrix metalloproteinase) is a membrane-anchored MMP that can be shed to the extracellular milieu. In the present study we report the primary structure and activity of the major soluble form of MT1-MMP. MS analysis of the purified 50-kDa soluble MT1-MMP form shows that the enzyme extends from Tyr112 to Val524, indicating that formation of this species requires a proteolytic cleavage within the stem region. In agreement, deletion of the entire stem region of MT1-MMP inhibited shedding of the 50-kDa species. A recombinant 50-kDa species (Tyr112-Val524) expressed in cells exhibited enzymatic activity against pro-MMP-2 and galectin-3, and thus this species is a competent protease. The recombinant 50-kDa soluble form also decreased the level of surface-associated TIMP-2 (tissue inhibitor of metalloproteinase 2) when administered to cells expressing wild-type membrane-anchored MT1-MMP, suggesting that ectodomain shedding of MT1-MMP can alter the MMP/TIMP balance on the cell surface. A approximately 53-kDa species of MT1-MMP was also isolated from a non-detergent extract of human breast carcinoma tissue and was found to lack the cytosolic tail, as determined with specific MT1-MMP domain antibodies. Together, these data show that MT1-MMP ectodomain shedding is a physiological process that may broaden MT1-MMP activity to the pericellular space.

Processing, shedding, and endocytosis of membrane type 1-matrix metalloproteinase (MT1-MMP).

Matrix metalloproteinases (MMPs) are multidomain zinc-dependent proteolytic enzymes that play pivotal roles in many normal and pathological processes. Some members of the MMP family are anchored to the plasma membrane via specialized domains and thus are perfectly suited for pericellular proteolysis. Membrane-anchoring also confers the membrane type-MMPs (MT-MMPs) a unique and complex array of regulatory processes that endow cells with the ability to control MT-MMP-dependent proteolytic activity independently of the levels of endogenous protease inhibitors. Emerging evidence indicates that mechanisms as diverse as autocatalytic processing, ectodomain shedding, homodimerization and internalization can all contribute to the modulation of MT-MMP activity on the cell surface. How these distinct processes interact to attain the optimal level of enzyme activity in a particular setting and the molecular signals that trigger them constitute a new paradigm in MMP regulation. This review will discuss the recent findings concerning these diverse regulatory mechanisms in the context of MT1-MMP (MMP-14).

Differential inhibition of membrane type 3 (MT3)-matrix metalloproteinase (MMP) and MT1-MMP by tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3 rgulates pro-MMP-2 activation.

The membrane type (MT)-matrix metalloproteinases (MMPs) constitute a subgroup of membrane-anchored MMPs that are major mediators of pericellular proteolysis and physiological activators of pro-MMP-2. The MT-MMPs also exhibit differential inhibition by members of the tissue inhibitor of metalloproteinase (TIMP) family. Here we investigated the processing, catalytic activity, and TIMP inhibition of MT3-MMP (MMP-16). Inhibitor profile and mutant enzyme studies indicated that MT3-MMP is regulated on the cell surface by autocatalytic processing and ectodomain shedding. Inhibition kinetic studies showed that TIMP-3 is a high affinity inhibitor of MT3-MMP when compared with MT1-MMP (K(i) = 0.008 nm for MT3-MMP versus K(i) = 0.16 nm for MT1-MMP). In contrast, TIMP-2 is a better inhibitor of MT1-MMP. MT3-MMP requires TIMP-2 to accomplish full pro-MMP-2 activation and this process is enhanced in marimastatpretreated cells, consistent with regulation of active enzyme turnover by synthetic MMP inhibitors. TIMP-3 also enhances the activation of pro-MMP-2 by MT3-MMP but not by MT1-MMP. TIMP-4, in contrast, cannot support pro-MMP-2 activation with either enzyme. Affinity chromatography experiments demonstrated that pro-MMP-2 can assemble trimolecular complexes with a catalytic domain of MT3-MMP and TIMP-2 or TIMP-3 suggesting that pro-MMP-2 activation by MT3-MMP involves ternary complex formation on the cell surface. These results demonstrate that TIMP-3 is a major regulator of MT3-MMP activity and further underscores the unique interactions of TIMPs with MT-MMPs in the control of pericellular proteolysis.

Complex pattern of membrane type 1 matrix metalloproteinase shedding. Regulation by autocatalytic cells surface inactivation of active enzyme.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a type I transmembrane MMP shown to play a critical role in normal development and in malignant processes. Emerging evidence indicates that MT1-MMP is regulated by a process of ectodomain shedding. Active MT1-MMP undergoes autocatalytic processing on the cell surface, leading to the formation of an inactive 44-kDa fragment and release of the entire catalytic domain. Analysis of the released MT1-MMP forms in various cell types revealed a complex pattern of shedding involving two major fragments of 50 and 18 kDa and two minor species of 56 and 31-35 kDa. Protease inhibitor studies and a catalytically inactive MT1-MMP mutant revealed both autocatalytic (18 kDa) and non-autocatalytic (56, 50, and 31-35 kDa) shedding mechanisms. Purification and sequencing of the 18-kDa fragment indicated that it extends from Tyr(112) to Ala(255). Structural and sequencing data indicate that shedding of the 18-kDa fragment is initiated at the Gly(284)-Gly(285) site, followed by cleavage between the conserved Ala(255) and Ile(256) residues near the conserved methionine turn, a structural feature of the catalytic domain of all MMPs. Consistently, a recombinant 18-kDa fragment had no catalytic activity and did not bind TIMP-2. Thus, autocatalytic shedding evolved as a specific mechanism to terminate MT1-MMP activity on the cell surface by disrupting enzyme integrity at a vital structural site. In contrast, functional data suggest that the non-autocatalytic shedding generates soluble active MT1-MMP species capable of binding TIMP-2. These studies suggest that ectodomain shedding regulates the pericellular and extracellular activities of MT1-MMP through a delicate balance of active and inactive enzyme-soluble fragments.