S. Gaylen Bradley

Meprin A metalloproteases enhance renal damage and bladder inflammation after LPS challenge

Meprin metalloproteases, composed of alpha and/or beta subunits, consist of membrane-bound and secreted forms that are abundantly expressed in proximal tubules of the kidney as well as secreted into the urinary tract. Previous studies indicated that meprin metalloproteases play a role in pathological conditions such as ischemic acute renal failure and urinary tract infection. The aim of this work was to examine the role of meprins in endotoxemic acute renal failure using meprin alpha knockout (alphaKO), meprin beta knockout (betaKO), and wild-type (WT) mice. Differences among the responses of the genotypes were observed as early as 1 h after challenge with 2.5 mg/kg ip Escherichia coli LPS, establishing roles for meprins in the endotoxemic response. Meprin alphaKO mice displayed lower blood urea nitrogen levels and decreased nitric oxide levels, indicative of a decreased systemic response to LPS compared with WT and meprin betaKO mice. Serum cytokine profiles showed lower levels of IL-1beta and TNF-alpha in the meprin alphaKO mice within 3 h after LPS challenge and confirmed a role for meprins in the early phases of the host response. Meprin alphaKO mice were also hyporesponsive to LPS administered to the bladder, exhibiting significantly less bladder edema, leukocyte infiltration, and bladder permeability than WT mice. These data indicate that meprin A contributes to the renal and urogenital pathogenesis of endotoxicity.

Balance of meprin A and B in mice affects the progression of experimental inflammatory bowel disease

MEP1A, which encodes the α subunit of meprin metalloproteinases, is a susceptibility gene for inflammatory bowel disease (IBD), and decreased intestinal meprin-α expression is associated with enhanced IBD in humans. Mice lacking meprin α (α knockout, αKO) have more severe colitis induced by dextran sulfate sodium (DSS) than wild-type (WT) mice, indicating an anti-inflammatory role for meprin A. Previous studies and those herein indicate the meprin B has proinflammatory activities. Therefore, mice lacking both meprin A and B (dKO mice) were generated to determine how their combined absence alters the inflammatory response to DSS. Unchallenged dKO mice grow and reproduce normally and have no obvious abnormal phenotype, except for a slightly elevated plasma albumin in both males and females and a lower urine creatinine level in dKO males. Upon oral administration of 3.5% DSS, the dKO mice have more severe colitis than the WT and βKO mice but significantly less than the αKO mice. The dKO mice lose more weight and have elevated MPO and IL-6 activities in the colon compared with WT mice. Systemic inflammation, monitored by plasma nitric oxide levels, is absent in DSS-treated dKO mice, unlike WT mice. The severity of experimental IBD in dKO mice is intermediate between αKO and WT mice. The data indicate that the absence of meprin A aggravates chronic inflammation and the lack of meprin B affords some protection from injury. Manipulation of the expression of meprin gene products may have therapeutic potential.

Delta‐9‐Tetrahydrocannabinol (THC), the Major Psychoactive Component of Marijuana, Exacerbates Brain Infection by Acanthamoeba

The genus Acanthamoeba consists of free-living amebae associated with human infections. Acanthamoeba spp. are the causative agents of Granulomatous Amebic Encephalitis (GAE), a progressive disease of the central nervous system (CNS) which often is diagnosed postmortem. An increase in the number of GAE cases has been reported world-wide [9,10]. GAE occurs primarily in hosts whose immune systems have been suppressed from cancer chemotherapy, steroid therapy, HIV infection, or other debilitating diseases [6,9]. Antibodies to Acanthamoeba are found frequently in human serum suggesting that exposure is common [8]. Marijuana has been advocated for therapeutic application to individuals suffering from debilitating disease characterized by cachexia, chronic pain, or nausea. However, marijuana also has been reported to exert deleterious effects on the immune system [2]. The majority of these effects have been attributed to delta-9-tetrahydrocannabinol (THC), its major psychoactive component. THC has been shown to increase the susceptibility of experimental animals to infection with viruses and bacteria [1–4,7,13]. Thus, individuals who utilize marijuana or its psychoactive component, THC, and have compromised immune systems, such as AIDS patients, could be at greater risk of infection with Acanthamoeba. Thus, the goal of this study was to examine the effect of THC on susceptibility to infection with Acanthamoeba, an opportunistic pathogen of the central nervous system (CNS). A murine in vivo model of CNS infection with Acanthamoeba was employed to assess effects of THC on the host response.

Meprin A impairs epithelial barrier function, enhances monocyte migration, and cleaves the tight junction protein occludin

Meprin metalloproteases are highly expressed at the luminal interface of the intestine and kidney and in certain leukocytes. Meprins cleave a variety of substrates in vitro, including extracellular matrix proteins, adherens junction proteins, and cytokines, and have been implicated in a number of inflammatory diseases. The linkage between results in vitro and pathogenesis, however, has not been elucidated. The present study aimed to determine whether meprins are determinative factors in disrupting the barrier function of the epithelium. Active meprin A or meprin B applied to Madin-Darby canine kidney (MDCK) cell monolayers increased permeability to fluorescein isothiocyanate-dextran and disrupted immunostaining of the tight junction protein occludin but not claudin-4. Meprin A, but not meprin B, cleaved occludin in MDCK monolayers. Experiments with recombinant occludin demonstrated that meprin A cleaves the protein between Gly(100) and Ser(101) on the first extracellular loop. In vivo experiments demonstrated that meprin A infused into the mouse bladder increased the epithelium permeability to sodium fluorescein. Furthermore, monocytes from meprin knockout mice on a C57BL/6 background were less able to migrate through an MDCK monolayer than monocytes from their wild-type counterparts. These results demonstrate the capability of meprin A to disrupt epithelial barriers and implicate occludin as one of the important targets of meprin A that may modulate inflammation.

Isolation and characterization of an intracellular serine protease from Rhodococcus erythropolis

Abstract An intracellular serine protease from the bacterium Rhodococcus erythropolis was partially purified and characterized. The enzyme, which is present in the 100,000g supernatant fraction of disrupted cells, was purified 160-fold by ammonium sulfate fractionation, and chromatography on DEAE-cellulose, Sephadex G-150, hydroxyapatite, and benzamidine-glycylglycine-Sepharose. The molecular weight of the protease estimated by gel filtration was 82,000. The subunit molecular weight of the protein was estimated to be 90,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, the enzyme appears to be a monomer. The purified enzyme preparation hydrolyzed several arginine-containing synthetic substrates; benzoyl-arginine-4-methyl-7-coumarylamide was routinely used as substrate. Hemoglobin, casein, and azocasein were also hydrolyzed. No carboxypeptidase activity was detected. The pH optimum of the enzyme was 7.0–7.2. The protease was not affected by inhibitors of cysteine proteases (iodoacetate), aspartic proteases (pepstatin), or metallo-proteases (EDTA). Inhibition was observed with the serine protease inhibitor diisopropylfluorophosphate. Several trypsin inhibitors (tosyl-lysine chloromethyl ketone, antipain, leupeptin, 4-aminobenzamidine, ovomucoid, and gramicidin S) inhibited the Rhodococcus protease, but others did not (phenylmethylsulfonylfluoride, lima bean trypsin inhibitor, aprotinin, α-1-antitrypsin). Salts with monovalent cations (e.g., NaCl, KCl) activated the protease two-to threefold at ionic strengths of 0.2 to 0.5. MgCl2 stimulated activity fourfold at ionic strengths of 0.05 to 0.15; CaCl2 stimulated activity (1.5-fold) maximally at an ionic strength of 0.05 and inhibited above 0.15.

A cysteine metalloproteinase from mouse liver cytosol.

Abstract A cysteine metalloproteinase that degrades 125I-insulin B chain at neutral pH values was isolated from C3H mouse liver. The enzyme was partially purified from the 100,000g supernatant fraction by ammonium sulfate precipitation, DEAE-cellulose chromatography, and fast protein liquid chromatography. The molecular weight of the proteinase was estimated to be 190,000 by gel filtration on Sephadex G-200. Degradation of 125I-insulin B chain by the proteinase was inhibited by p-hydroxymercuribenzoate (PHMB) and iodoacetate (cysteine proteinase inhibitors) and by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (metalloproteinase inhibitors). The proteinase also degraded 125I-glucagon but did not hydrolyze 125I-insulin, leucine-2-naphthylamide, or several large proteins. Equivalent levels of EDTA- and PHMB-inhibitable 125I-insulin B chain-degrading activity were observed in the 100,000g supernatant fractions of brain, liver, lung, kidney, heart, and spleen from four mouse strains (C3H/HeN, CBA/J, ICR, and C57BL/6). High levels of 125I-insulin B chain-degrading activity were found in the particulate fraction of kidneys and lungs from these four mouse strains; these activities were inhibited by EDTA but not by PHMB. The activity of the soluble liver cysteine metalloproteinase was not altered in C3H mice treated ip with metal chelators, bacterial endotoxin, phenobarbital, dexamethasone, or insulin. Starvation for 24 or 48 hr and alloxan-induced diabetes diminished total activity of this enzyme in liver by about 50 and 30%, respectively. This soluble polypeptide-degrading enzyme appears to be ubiquitous in mice and to be regulated by nutritional conditions.

Proteases in the Mammalian Digestive System

The digestive system works in concert with the other organ systems of the body, and with the external environment, in particular ingested material and microorganisms. The pathway from the point of ingestion to the point of excretion is long and tortuous, and is made up of dozens of niches with specialized physiologic roles and constellations of proteases. In each of these niches, there is a characteristic array of molecules from the lumen to the surface of the cells lining the alimentary tract (Hopfer 2011). The cellular level is a heterogeneous mosaic of resident and transient cells with special functions and arrays of proteases. This chapter will focus on the mammalian proteases found in the lumen and on the cell surface of the alimentary tract, both membrane-bound and secreted proteases. The proteases of the intestinal bacteria, mainly in the lower intestine, are dealt with in separate chapters, but structures and products of bacteria will be addressed as substrates and as factors that affect protease expression and activity. Similarly, the proteases of immune cells, which are transient members of the digestive system, are dealt with separately in other chapters, but their roles in the alimentary tract are inextricably enmeshed in normal physiologic processes of digestion and pathologic responses to injury. Accordingly, this review of proteases in the digestive system will focus on the human system beginning anatomically at the oral cavity, and proceed to the esophagus, stomach, and the niches of the intestinal tract, ending with the colon. Representative protease functions in intestinal pathobiology are discussed in the anatomical sections and in separate sections at the end of the chapter. There is detailed information on the many proteases mentioned in this chapter in the Handbook of Proteolytic Enzymes, second Edition (Barrett et al. 2004) and the third Edition (Rawlings and Salvesen 2013), and in the MEROPs database (http://www.merops.sanger.ac.uk).