Fluorescence Imaging for Cancer Screening and Surveillance.

The advent of fluorescence imaging (FI) for cancer cell detection in the field of oncology is promising for both cancer screening and surgical resection. Particularly, FI in cancer screening and surveillance is actively being evaluated in many new clinical trials with over 30 listed on Clinical Trials.gov . While surgical resection forms the foundation of many oncologic treatments, early detection is the cornerstone for improving outcomes and reducing cancer-related morbidity and mortality. The applications of FI are twofold as it can be applied to high-risk patients in addition to those undergoing active surveillance. This technology has the promise of highlighting lesions not readily detected by conventional imaging or physical examination, allowing disease detection at an earlier stage of development. Additionally, there is a persistent need for innovative, cost-effective imaging modalities to ameliorate healthcare disparities and the global burden of cancer worldwide. In this review, we outline the current utility of FI for screening and detection in a range of cancer types.

Loss of Prkar1a leads to Bcl-2 family protein induction and cachexia in mice.

Loss of function mutations in the Prkar1a gene are the cause of most cases of Carney complex disorder. Defects in Prkar1a are thought to cause hyper-activation of PKA signalling, which drives neoplastic transformation, and Prkar1a is therefore considered to be a tumour suppressor. Here we show that loss of Prkar1a in genetically modified mice caused transcriptional activation of several proapoptotic Bcl-2 family members and thereby caused cell death. Interestingly, combined loss of Bim and Prkar1a increased colony formation of fibroblasts in culture and promoted their growth as tumours in immune-deficient mice. Apart from inducing apoptosis, systemic deletion of Prkar1a caused cachexia with muscle loss, macrophage activation and increased lipolysis as well as serum triglyceride levels. Loss of single allele of Prkar1a did not enhance tumour development in a skin cancer model, but surprisingly, when combined with the loss of Bim, caused a significant delay in tumorigenesis and this was associated with upregulation of other BH3-only proteins, PUMA and NOXA. These results show that loss of Prkar1a can only promote tumorigenesis when Prkar1a-mediated apoptosis is somehow countered.

Serine proteases and protease-activated receptor 2 mediate the proinflammatory and algesic actions of diverse stimulants.

BACKGROUND AND PURPOSE: Although serine proteases and agonists of protease-activated receptor 2 (PAR2) cause inflammation and pain, the spectrum of proteases that are activated by proinflammatory and algesic stimuli and their contribution to inflammatory pain are uncertain. EXPERIMENTAL APPROACH: Enzymic assays and selective inhibitors were used to characterize protease activity in mice after intraplantar injections of formalin, bradykinin, PAR2 activating peptide (AP) or vehicle. The capacity of these proteases and of recombinant mouse trypsin 4 to cleave fragments of PAR2 and to activate PAR2 in cell lines was determined. Protease inhibitors and par2 (-/-) mice were used to assess the contributions of proteases and PAR2 to pain and inflammation. KEY RESULTS: Intraplantar injection of formalin, bradykinin or PAR2-AP led to the activation of proteases that were susceptible to the serine protease inhibitor melagatran but resistant to soybean trypsin inhibitor (SBTI). Melagatran inhibited mouse trypsin 4, which degraded SBTI. Proteases generated in inflamed tissues cleaved PAR2-derived peptides. These proteases and trypsin 4 increased [Ca(2+) ]i in PAR2-transfected but not in untransfected cells, and melagatran suppressed this activity. Melagatran or PAR2 deletion suppressed oedema and mechanical hypersensitivity induced by intraplantar formalin, bradykinin and PAR2-AP, but had no effect on capsaicin-induced pain. CONCLUSIONS AND IMPLICATIONS: Diverse proinflammatory and algesic agents activate melagatran-sensitive serine proteases that cause inflammation and pain by a PAR2-mediated mechanism. By inducing self-activating proteases, PAR2 amplifies and sustains inflammation and pain. Serine protease inhibitors can attenuate the inflammatory and algesic effects of diverse stimuli, representing a useful therapeutic strategy.

Cathepsin X-mediated beta2 integrin activation results in nanotube outgrowth.

Membrane nanotubes were recently described as a new principle of cell-cell communication enabling complex and specific messaging to distant cells. Calcium fluxes, vesicles, and cell-surface components can all traffic between cells connected by nanotubes. Here we report for the first time the mechanism of membrane nanotube formation in T cells through LFA-1 (CD11a/CD18; alpha(L)beta(2)) integrin activation by the cysteine protease cathepsin X. Cathepsin X is shown to induce persistent LFA-1 activation. Cathepsin X-upregulated T cells exhibit increased homotypic aggregation and polarized, migration-associated morphology in 2D and 3D models, respectively. In these cells, extended uropods are frequently formed, which subsequently elongate to nanotubes connecting T lymphocytes. Our results demonstrate that LFA-1 activation with subsequent cytoskeletal reorganization induces signal transmission through a physically connected network of T lymphocytes for better coordination of their action at various stages of the immune response.

Specificity of aza-peptide electrophile activity-based probes of caspases.

Activity-Based Probes (ABPs) are small molecules that form stable covalent bonds with active enzymes thereby allowing detection and quantification of their activities in complex proteomes. A number of ABPs that target proteolytic enzymes have been designed based on well-characterized mechanism-based inhibitors. We describe here the evaluation of a novel series of ABPs based on the aza-aspartate inhibitory scaffold. Previous in vitro kinetic studies showed that this scaffold has a high degree of selectivity for the caspases, clan CD cysteine proteases activated during apoptotic cell death. Aza-aspartate ABPs containing either an epoxide or Michael acceptor reactive group were potent labels of executioner caspases in apoptotic cell extracts. However they were also effective labels of the clan CD protease legumain and showed unexpected crossreactivity with the clan CA protease cathepsin B. Interestingly, related aza peptides containing an acyloxymethyl ketone reactive group were relatively weak but highly selective labels of caspases. Thus azapeptide electrophiles are valuable new ABPs for both detection of a broad range of cysteine protease activities and for selective targeting of caspases. This study also highlights the importance of confirming the specificity of covalent protease inhibitors in crude proteomes using reagents such as the ABPs described here.

O-sulfonation of serine and threonine: mass spectrometric detection and characterization of a new posttranslational modification in diverse proteins throughout the eukaryotes.

Protein sulfonation on serine and threonine residues is described for the first time. This post-translational modification is shown to occur in proteins isolated from organisms representing a broad span of eukaryote evolution, including the invertebrate mollusk Lymnaea stagnalis, the unicellular malaria parasite Plasmodium falciparum, and humans. Detection and structural characterization of this novel post-translational modification was carried out using liquid chromatography coupled to electrospray tandem mass spectrometry on proteins including a neuronal intermediate filament and a myosin light chain from the snail, a cathepsin-C-like enzyme from the parasite, and the cytoplasmic domain of the human orphan receptor tyrosine kinase Ror-2. These findings suggest that sulfonation of serine and threonine may be involved in multiple functions including protein assembly and signal transduction.

Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity.

We report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 microM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 microM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.

Proteasome inhibitors: complex tools for a complex enzyme.

As the dominant protease dedicated to protein turnover, the proteasome shapes the cellular protein repertoire. Our knowledge of proteasome regulation and activity has improved considerably over the past decade. Novel inhibitors, in particular, have helped to advance our understanding of proteasome biology. They range from small peptide-based structures that can be modified to vary target specificity, to large macromolecular inhibitors that include proteins. While these reagents have played an important role in establishing our current knowledge of the proteasome's catalytic mechanism, many questions remain. Rapid advances in the synthesis and identification of new classes of proteasome inhibitors over the last 10 years serve as a positive indicator that many of these questions will soon be resolved. The future lies in designing compounds that can function as drugs to target processes involved in disease progression. It may only be a short while before the products of such research have safe application in a practical setting. Structural and combinatorial chemistry approaches are powerful techniques that will bring us closer to these goals.

Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense.

Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei. With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S2, S1 and S' subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S2 prevents rhodesain from cleaving substrates in which P2 is arginine.

Lysine 188 substitutions convert the pattern of proteasome activation by REGgamma to that of REGs alpha and beta.

11S REGs (PA28s) are multimeric rings that bind proteasomes and stimulate peptide hydrolysis. Whereas REGalpha activates proteasomal hydrolysis of peptides with hydrophobic, acidic or basic residues in the P1 position, REGgamma only activates cleavage after basic residues. We have isolated REGgamma mutants capable of activating the hydrolysis of fluorogenic peptides diagnostic for all three active proteasome beta subunits. The most robust REGgamma specificity mutants involve substitution of Glu or Asp for Lys188. REGgamma(K188E/D) variants are virtually identical to REGalpha in proteasome activation but assemble into less stable heptamers/hexamers. Based on the REGalpha crystal structure, Lys188 of REGgamma faces the aqueous channel through the heptamer, raising the possibility that REG channels function as substrate-selective gates. However, covalent modification of proteasome chymotrypsin-like subunits by 125I-YL3-VS demonstrates that REGgamma(K188E)'s activation of all three proteasome active sites is not due to relaxed gating. We propose that decreased stability of REGgamma(K188E) heptamers allows them to change conformation upon proteasome binding, thus relieving inhibition of the CT and PGPH sites normally imposed by the wild-type REGgamma molecule.

Defining a link between gap junction communication, proteolysis, and cataract formation.

Disruption of the connexin alpha 3 (Cx46) gene (alpha 3 (-/-)) in mice results in severe cataracts within the nuclear portion of the lens. These cataracts are associated with proteolytic processing of the abundant lens protein gamma-crystallin, leading to its aggregation and subsequent opacification of the lens. The general cysteine protease inhibitor, E-64, blocked cataract formation and gamma-crystallin cleavage in alpha 3 (-/-) lenses. Using a new class of activity-based cysteine protease affinity probes, we identified the calcium-dependent proteases, m-calpain and Lp82, as the primary targets of E-64 in the lens. Profiling changes in protease activities throughout cataractogenesis indicated that Lp82 activity was dramatically increased in alpha 3 (-/-) lenses and correlated both spatially and temporally with cataract formation. Increased Lp82 activity was due to calcium accumulation as a result of increased influx and decreased outflux of calcium ions in alpha 3 (-/-) lenses. These data establish a role for alpha 3 gap junctions in maintaining calcium homeostasis that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key initiator of the process of cataractogenesis.

Global analysis of proteasomal substrate specificity using positional-scanning libraries of covalent inhibitors.

The proteasome is a large protease complex consisting of multiple catalytic subunits that function simultaneously to digest protein substrates. This complexity has made deciphering the role each subunit plays in the generation of specific protein fragments difficult. Positional scanning libraries of peptide vinyl sulfones were generated in which the amino acid located directly at the site of hydrolysis (P1 residue) was held constant and sequences distal to that residue (P2, P3, and P4 positions) were varied across all natural amino acids (except cysteine and methionine). Binding information for each of the individual catalytic subunits was obtained for each library under a variety of different conditions. The resulting specificity profiles indicated that substrate positions distal to P1 are critical for directing substrates to active subunits in the complex. Furthermore, specificity profiles of IFN-gamma-regulated subunits closely matched those of their noninducible counterparts, suggesting that subunit swapping may modulate substrate processing by a mechanism that does require a change in the primary sequence specificity of individual catalytic subunits in the complex. Finally, specificity profiles were used to design specific inhibitors of a single active site in the complex. These reagents can be used to further establish the role of each subunit in substrate processing by the proteasome.

Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools.

BACKGROUND: Analysis of global changes in gene transcription and translation by systems-based genomics and proteomics approaches provides only indirect information about protein function. In many cases, enzymatic activity fails to correlate with transcription or translation levels. Therefore, a direct method for broadly determining activities of an entire class of enzymes on a genome-wide scale would be of great utility. RESULTS: We have engineered chemical probes that can be used to broadly track activity of cysteine proteases. The structure of the general cysteine protease inhibitor E-64 was used as a scaffold. Analogs were synthesized by varying the core peptide recognition portion while adding affinity tags (biotin and radio-iodine) at distal sites. The resulting probes containing a P2 leucine residue (DCG-03 and DCG-04) targeted the same broad set of cysteine proteases as E-64 and were used to profile these proteases during the progression of a normal skin cell to a carcinoma. A library of DCG-04 derivatives was constructed in which the leucine residue was replaced with all natural amino acids. This library was used to obtain inhibitor activity profiles for multiple protease targets in crude cellular extracts. Finally, the affinity tag of DCG-04 allowed purification of modified proteases and identification by mass spectrometry. CONCLUSIONS: We have created a simple and flexible method for functionally identifying cysteine proteases while simultaneously tracking their relative activity levels in crude protein mixtures. These probes were used to determine relative activities of multiple proteases throughout a defined model system for cancer progression. Furthermore, information obtained from libraries of affinity probes provides a rapid method for obtaining detailed functional information without the need for prior purification/identification of targets.

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity.

Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.

Release of signal peptide fragments into the cytosol requires cleavage in the transmembrane region by a protease activity that is specifically blocked by a novel cysteine protease inhibitor.

Signal peptides of secretory and membrane proteins are generated by proteolytic processing of precursor proteins after insertion into the endoplasmic reticulum membrane. Liberated signal peptides can be further processed, and the resulting N-terminal fragments are released toward the cytosol, where they may interact with target proteins like calmodulin. We show here that the processing of signal peptides requires a protease activity distinct from signal peptidase. This activity is inhibited specifically with a newly developed cysteine protease inhibitor, 1, 3-di-(N-carboxybenzoyl-l-leucyl-l-leucyl)amino acetone ((Z-LL)(2) ketone). Inhibitor studies revealed that the final, (Z-LL)(2) ketone-sensitive cleavage event occurs within the hydrophobic transmembrane region of the signal peptide, thus promoting the release of an N-terminal fragment into the cytosol.

Identification of a cDNA encoding an active asparaginyl endopeptidase of Schistosoma mansoni and its expression in Pichia pastoris.

Asparaginyl endopeptidases, or legumains, are a recently identified family of cysteine-class endopeptidases. A single gene encoding a Schistosoma mansoni asparaginyl endopeptidase (a.k.a. Sm32 or schistosome legumain) has been reported, but by sequence homology it would be expected to yield an inactive product as the active site C197 had been replaced by N. We now describe a new S. mansoni gene in which C197 is present. Both gene products were expressed in Pichia pastoris. Autocatalytic processing to fully active C197 Sm32 occurred at acid pH. In contrast, N197 Sm32 was not processed and this is consistent with the hypothesis that C197 is essential for catalysis. This was confirmed by mutation of N197 to C and re-expression in Pichia. The availability of recombinant active Sm32 allows detailed analysis of its catalytic mechanism and its function(s) in the biology of this important human parasite.

Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs.

BACKGROUND: The lysosomal cysteine proteases of the papain family are some of the best studied proteolytic enzymes. Small-molecule inhibitors and fluorogenic substrate mimics have been used to probe the physiological roles of these proteases. A high degree of homology between family members and overlap in substrate specificity have made elucidating individual protease function, expression and activity difficult. RESULTS: Using peptide vinyl sulfones and epoxide as templates, we have generated probes that can be tagged with radioactive iodine. The resulting compounds covalently label various cathepsins and several unidentified polypeptides likely to be proteases. MB-074 was found to be a highly selective probe of cathepsin B activity. Probes that labeled several cathepsins were used to examine the specificity and cell permeability of the CA-074 family of inhibitors. Although CA-074 reportedly acts in vivo, we find it is unable to penetrate cells. Esterifying CA-074 resulted in a cell-permeable inhibitor with dramatically reduced activity and specificity for cathepsin B. The probes were also used to monitor protease activity in primary human tumor tissue and cells derived from human placenta. CONCLUSIONS: We have generated a highly selective cathepsin B probe and several less specific reagents for the study of cathepsin biology. The reagents have several advantages over commonly used fluorogenic substrates, allowing inhibitor targets to be identified in a pool of total cellular enzymes. We have used the probes to show that cathepsin activity is regulated in tumor tissues and during differentiation of placental-derived cytotrophoblasts to invasive cells required for establishing blood circulation in a developing embryo.

How an inhibitor of the HIV-I protease modulates proteasome activity.

The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years. Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease. In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 microM). In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced. In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome. Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor(125)I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir. At low concentrations of Ritonavir (5 microM) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 microM) protein degradation was affected, and the cell cycle was arrested in the G(1)/S phase. Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded. Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response.

Cysteine protease inhibitors as chemotherapy: lessons from a parasite target.

Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.