Volume 391, Issue 5

Molecular chaperones are well known as facilitators of protein folding and assembly. However, in recent years multiple chaperone-assisted degradation pathways have also emerged, including CAP ( c haperone- a ssisted p roteasomal degradation), CASA ( c haperone- a ssisted s elective a utophagy), and CMA ( c haperone- m ediated a utophagy). Within these pathways chaperones facilitate the sorting of non-native proteins to the proteasome and the lysosomal compartment for disposal. Impairment of these pathways contributes to the development of cancer, myopathies, and neurodegenerative diseases. Chaperone-assisted degradation thus represents an essential aspect of cellular proteostasis, and its pharmacological modulation holds the promise to ameliorate some of the most devastating diseases of our time. Here, we discuss recent insights into molecular mechanisms underlying chaperone-assisted degradation in mammalian cells and highlight its biomedical relevance.

Biology-oriented synthesis (BIOS) represents an alternative approach for the generation of compound collections for biological applications. In BIOS, biologically relevant and prevalidated scaffold structures, such as core structures of natural products or known drugs, are employed as scaffolds for the generation of compound collections with focused diversity. In this review, we discuss the underlying concept of the BIOS approach, and its practical implementation in library design and synthesis. To highlight its relevance for chemical biology applications, we finally present examples in which compound collections generated under the BIOS principle have been used to elucidate biological questions.

Moenomycins are phosphoglycolipid antibiotics and the only known natural product inhibitors of peptidoglycan glycosytransferases (PGTs). Techniques that would allow facile diversification of the moenomycin structure would facilitate the development of novel antibiotics, which are urgently needed in the wake of multidrug resistant bacterial infections. The cloning and initial characterization of the moenomycin biosynthetic genes has already redefined the minimal moenomycin pharmacophore and now opens the door for the biocombinatorial generation of bioactive moenomycin fragments. Here, we highlight the importance of research on the genetic mechanisms that regulate moenomycin biosynthesis and that confer moenomycin resistance to bacteria in the development of novel anti-infectives based on PGT inhibition.

Tissue kallikrein ( KLK1 ) and the kallikrein-related peptidase ( KLK2–15 ) genes encode for a subgroup of 15 homologous secreted serine proteases possessing numerous physiological roles, such as the regulation of blood pressure, hormone processing and tissue remodeling. The expression of KLKs is detected in a broad spectrum of human tissues where it has been found to be regulated mainly by steroids hormones. The aberrant expression of KLKs , presented in many human malignancies, highlights the significance of this gene family for early diagnosis, prognosis and monitoring of cancer patients, as it is strongly emphasized by the routine use of PSA (KLK3) for prostate cancer management. Here, we review the presently known data regarding the role of KLKs as cancer biomarkers, giving emphasis on novel information about the subject.

Parkinson's disease (PD) is a progressive neurodegenerative disorder in which oxidative stress is implicated as a major causative factor. Mutations in the gene encoding Parkin, a ubiquitin ligase, are responsible for a familial form of PD. In a Drosophila disease model lacking Parkin ( park 25 null mutant), we tested the effect of zinc supplementation. Zinc is an essential trace metal and a component of many enzymes and transcriptional regulators. Unlike copper and iron, zinc is not redox-active and under most conditions serves as an antioxidant. We find that the condition of parkin mutants raised on zinc-supplemented food is greatly improved. At zinc concentrations where controls begin to show adverse effects as a result of the metal supplement, parkin mutants perform best, as manifested in a higher frequency of reaching adulthood, extended lifespan and improved motoric abilities.

Arsenic trioxide (arsenite) was the first chemotherapeutic drug to be described and is now being rediscovered in cancer treatment, including glioblastoma multiforme. Arsenite toxicity triggers autophagy in cancer cells, although final stages of the process involve executive caspases, suggesting an interplay between autophagic and apoptotic pathways that awaits to be explained at a molecular level. We evaluated the contribution of the lysosomal cathepsins (Cat) L and B, which are upregulated in glioblastomas, in the mechanism of arsenite toxicity in human glioblastoma cells. Arsenite treatment induced autophagosome formation and permeabilization of mitochondria, followed by caspase 3/7-mediated apoptosis. The autophagy inhibitor 3-methyladenine protected from arsenite toxicity, whereas bafilomycin A1 did not. Furthermore, arsenite significantly decreased CatB levels and selectively inhibited its cellular and recombinant protein activity, while not affecting CatL. However, downregulation of CatL greatly enhanced apoptosis by arsenite. Our results show that arsenite toxicity involves a complex interplay between autophagy and apoptosis in human glioblastoma cells and is associated with inhibition of CatB, and that this toxicity is highly exacerbated by simultaneous CatL inhibition. The latter points to a synergy that could be used in clinical treatment to lower the therapeutic dose, thus avoiding the toxic side effects of arsenite in glioblastoma management.

N -Acetyl- N -nitroso-tryptophan (NANT) is well known for its capacity to generate nitric oxide (NO)-releasing compounds. It is unknown, however, whether NANT can be successfully applied as a precursor of NO in a complex biological environment such as a cell culture system. NO donors can be useful to induce the transcription factor hypoxia-inducible factor 1 (HIF-1) that coordinates the protection of cells and tissues from the lack of oxygen, termed hypoxia. HIF-1 degradation is controlled by prolyl hydroxylase 2 (PHD2) which needs to be inhibited for HIF-1 accumulation. Here, the effects of NANT in inhibiting recombinant PHD2 and up-regulating of HIF-1 and HIF-1-mediated carboanhydrase-9 (CA9) mRNA expression were compared in living cells with the NO donors N -nitrosomelatonin (NOMela) and S -nitrosoglutathione (GSNO) under normoxic and hypoxic conditions. In contrast to GSNO, NANT was similar to NOMela being highly effective in inhibiting recombinant PHD2. NANT-mediated activation of HIF-1 in oxygenated cells was comparable to hypoxic activation of HIF-1 in all cases. In contrast, under hypoxia NANT was able to boost hypoxic cellular HIF-1 levels by further reducing the activity of cellular PHD2. The strong increase of HIF-dependent CA9 mRNA expression demonstrated that NANT-induced HIF-1 was transcriptionally active. Finally, the efficacy of NANT to increase both HIF-1 and CA9 mRNA did not depend on the absolute conformation of the tryptophan moiety. In conclusion, NANT appears to be an excellent NO donor for cells in culture and l -NANT should be useful for in vivo animal studies.

Antimicrobial peptides are multifunctional in innate immunity and wound repair of multicellular organisms. We were the first to discover that histatins, a family of salivary antimicrobial peptides, enhance epithelial cell migration, suggesting a role in oral wound healing. It is unknown whether histatins display innate-immunity activities, similar to other antimicrobial peptides such as LL-37. Therefore, we compared the effect of Histatin-2 and LL-37 on several activities within the context of wound healing and innate immunity. We found that Histatin-2 enhances fibroblast migration, but only weakly induces proliferation. LL-37 enhances both fibroblast migration and proliferation, but only at a narrow concentration optimum (approximately 1 μ m ). At higher concentrations LL-37 causes cell death, whereas Histatin-2 is not cytotoxic. Both peptides do not alter fibroblast-to-myofibroblast differentiation. Histatin-2 does not alter interleukin-8 (IL-8) expression and lipopolysaccharide (LPS)-elevated cytokine and chemokine expression. In contrast, LL-37 induces IL-8 expression, but dampens the LPS-induced immune response. Neither Histatin-2 nor LL-37 affects human-neutrophil migration. Histatins are, unlike other antimicrobial peptides, not cytotoxic or proinflammatory. It seems that they are important for the initial stage of wound healing in which fast wound coverage is important for healing without infection, inflammation, or fibrosis development. Interestingly, these characteristics are more typical for the mouth than for skin.

Flaviviral NS2B/NS3 heterocomplex serine proteases are a primary target for anti-flavivirus drug discovery. To gain insights into the enzymatic properties and molecular determinants of flaviviral NS2B/NS3 protease substrate specificity in host cells, we developed and applied a novel series of membrane-anchored red-shifted fluorescent protein substrates to detect West Nile virus (WNV) NS2B/NS3 endoproteolytic activity in human cells. The substrate consists of a fluorescent reporter group (DsRed) tethered to the endoplasmic reticulum membrane by a membrane-anchoring domain. Between the two domains is a specific peptide linker that corresponds to the NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 protein junctions within the WNV polyprotein precursor. When the protease cleaves the peptide linker, the DsRed reporter group is released, changing its localization in the cell from membrane-bound punctate perinuclear to diffuse cytoplasmic. This change in protein location can be monitored by fluorescent microscopy, and cleavage products can be quantified by Western blotting. Our data demonstrate the robustness of our trans -cleavage fluorescence assay to capture single-cell imaging of membrane-associated WNV NS2B/NS3 endoproteolytic activity and to perform in-cell selectivity profiling of the NS2B/NS3 protease. Our study is the first to provide cellular insights into the biological and enzymatic properties of a prime target for inhibitors of WNV replication.

Plasminogen is a glycoprotein implicated in angiogenesis and fibrin clot degradation associated with the release of angiostatin and plasmin activation, respectively. We have recently reported that cathepsin V, but not cathepsins L, B, and K, can release angiostatin-like fragments from plasminogen. Here, we extended the investigation to cathepsin S which has been implicated in angiogenesis and tumor cell proliferation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasminogen hydrolysis by cathepsin S revealed generation of two fragments (60 and 38 kDa). Amino-terminal sequencing indicated that cleavage occurs at the Leu469-Leu470 peptide bond. In contrast to cathepsin V, which possesses antiangiogenic activity, cathepsin S plasminogen cleavage products were not capable of inhibiting angiogenesis on endothelial cells. Moreover, we explored the different selectivities presented by cathepsins V and S towards plasminogen and synthesized fluorescence resonance energy transfer peptides encompassing the hydrolyzed peptide bonds by both enzymes. The peptide Abz-VLFEKKQ-EDDnp (Abz= ortho -aminobenzoic acid; EDDnp= N-[2,4-dinitrophenyl]ethylenediamine), hydrolyzed by cath-epsin V at the Phe-Glu bond, is a selective substrate for the enzyme when compared with cathepsins B, L, and S, whereas Abz-VLFEKKVYLQ-EDDnp is an efficient cathepsin L inhibitor. The demonstrated importance of the S 3 ′-P 3 ′ interaction indicates the significance of the extended subsites for enzyme specificity and affinity.

Cysteine cathepsins and matrix metalloproteases are considered to play important roles in the development of arthritic diseases. Their accumulation in synovial fluid of primarily rheumatoid arthritis patients is also well documented. However, a detailed comparison between the protease levels and activities between rheumatoid arthritis samples and osteoarthritis samples has never been made. Here, we report that both cysteine cathepsins B and S and matrix metalloproteases-1, -3 and -13 are detected in patient synovial fluid samples with significantly higher levels detected in rheumatoid arthritis patients. Among the proteases, cathepsin S was found to be significantly elevated, consistent with its critical role in the immune response. These results suggest that cysteine cathepsins have a major role in inflammation at least in rheumatoid arthritis. In addition to proteases, interleukin-6 was detected at significant levels in most samples, suggesting that proinflammatory cytokines might be in-volved in the stimulation of expression of these proteases during inflammation.

Human kallikrein-related peptidases (KLKs) are 15 homologous serine proteases involved in several (patho)physiological processes, including cancer. Secreted as precursors, they are activated upon proteolytic release of a short pro-peptide. We searched for interconnection of KLKs within extracellular proteolytic networks leading to activation of protease zymogens and found that (i) pro-KLK activation by other KLKs is scarce, with the exception of pro-KLK11, which is efficiently activated by KLK4 and 5; (ii) pro-KLK4 is activated by matrix metalloproteinase 3; and (iii) trypsin-like KLKs efficiently activate the serine protease urokinase. Our observations provide new insights into the regulation of these important tumor-associated proteases.