Biometals play an important role in Alzheimer disease, and recent reports have described the development of potential therapeutic agents based on modulation of metal bioavailability. The metal ligand clioquinol (CQ) has shown promising results in animal models and small phase clinical trials; however, the actual mode of action in vivo has not been determined. We now report a novel effect of CQ on amyloid ␤-peptide (A␤) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu 2؉ or Zn 2؉ resulted in an ϳ85-90% reduction of secreted A␤-(1-40) and A␤-(1-42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted A␤ was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu 2؉ . MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu 2؉ also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of A␤-(1-40). Our findings identify an alternative mechanism of action for CQ in the reduction of A␤ deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients. Alzheimer disease (AD)4 is characterized by progressive neuronal dysfunction, reactive gliosis, and the formation of amyloid plaques in the brain. The major constituent of AD plaques is the amyloid ␤-peptide (A␤), which is cleaved from the membrane-bound amyloid precursor protein (APP) (1). Aggregated or oligomeric A␤ can induce neurotoxicity through pathways involving free radical production and increased neuronal oxidative stress (2). Among the factors capable of promoting A␤ aggregation in vivo, recent evidence supports a central role for biometals such as Cu 2ϩ and Zn 2ϩ in this process (3). An important factor in controlling A␤ accumulation in AD patients is the activity of A␤-degrading enzymes. Recent studies have identified several candidate proteases that may contribute to catabolism of A␤ in the brain. Neprilysin, insulin-degrading enzyme, angiotensin-converting enzyme, and matrix metalloproteases (MMPs) have all demonstrated A␤-degrading activity in vitro and/or in vivo (4 -6). Reduced activity of these or other A␤-degrading proteases with age may play a role in promoting accumulation and deposition of A␤ in AD patients. Development of strategies to enhance clearance of A␤ may lead to novel therapeutic treatments for AD patients.Promoting A␤ clearance may be achieved through modulating metal sequestration or metal-protein interactions. 5-Chloro-7-iodo-8-hydroxyquinoline or clioquinol (CQ), a disused antibiotic, has received considerable attention as a potential metal ligand in AD and Parkinson disease patients (7-9). Preliminary studies revealed that CQ rapidly and potently dissolved aggregates of synthetic or AD brain-derived A␤ in vitro (10). In subsequent animal studies, a 9-week oral treatment with CQ resulted in a 49% reduction of...

LipL32 is the major outer membrane protein in pathogenic Leptospira. It is highly conserved throughout pathogenic species and is expressed in vivo during human infection. While these data suggest a role in pathogenesis, a function for LipL32 has not been defined. Outer membrane proteins of gram-negative bacteria are the first line of molecular interaction with the host, and many have been shown to bind host extracellular matrix (ECM). A search for leptospiral ECM-interacting proteins identified the major outer membrane protein, LipL32. To verify this finding, recombinant LipL32 was expressed in Escherichia coli and was found to bind Matrigel ECM and individual components of ECM, including laminin, collagen I, and collagen V. Likewise, an orthologous protein found in the genome of Pseudoalteromonas tunicata strain D2 was expressed and found to be functionally similar and immunologically cross-reactive. Lastly, binding activity was mapped to the C-terminal 72 amino acids. These studies show that LipL32 and an orthologous protein in P. tunicata are immunologically cross-reactive and function as ECM-interacting proteins via a conserved C-terminal region.

Leptospira interrogans is responsible for leptospirosis, a zoonosis of worldwide distribution. LipL32 is the major outer membrane protein of pathogenic leptospires, accounting for up to 75% of total outer membrane protein. In recent times LipL32 has become the focus of intense study because of its surface location, dominance in the host immune response, and conservation among pathogenic species. In this study, an lipL32 mutant was constructed in L. interrogans using transposon mutagenesis. The lipL32 mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. Protein composition of the cell membranes was found to be largely unaffected by the loss of LipL32, with no obvious compensatory increase in other proteins. Microarray studies found no obvious stress response or upregulation of genes that may compensate for the loss of LipL32 but did suggest an association between LipL32 and the synthesis of heme and vitamin B 12 . When hamsters were inoculated by systemic and mucosal routes, the mutant caused acute severe disease manifestations that were indistinguishable from wild-type L. interrogans infection. In the rat model of chronic infection, the LipL32 mutant colonized the renal tubules as efficiently as the wild-type strain. In conclusion, this study showed that LipL32 does not play a role in either the acute or chronic models of infection. Considering the abundance and conservation of LipL32 among all pathogenic Leptospira spp. and its absence in saprophytic Leptospira, this finding is remarkable. The role of this protein in leptospiral biology and pathogenesis thus remains elusive.

The kallikrein-related peptidase (KLK) family of proteases is involved in many aspects of human health and disease. One member of this family, KLK4, has been implicated in cancer development and metastasis. Understanding mechanisms of inactivation are critical to developing selective KLK4 inhibitors. We have determined the X-ray crystal structures of KLK4 in complex with both sunflower trypsin inhibitor-1 (SFTI-1) and a rationally designed SFTI-1 derivative to atomic (~1 Å) resolution, as well as with bound nickel. These structures offer a structural rationalization for the potency and selectivity of these inhibitors, and together with MD simulation and computational analysis, reveal a dynamic pathway between the metal binding exosite and the active site, providing key details of a previously proposed allosteric mode of inhibition. Collectively, this work provides insight into both direct and indirect mechanisms of inhibition for KLK4 that have broad implications for the enzymology of the serine protease superfamily, and may potentially be exploited for the design of therapeutic inhibitors.