Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L

Puzer, Luciano; Cotrin, Simone S.; Alves, Márcio Fernando Madureira; Egborge, Tobore; Araújo, Magali; Juliano, María A.; Juliano, Luiz; Brὂmme, Dieter; Carmona, Adriana K.

doi:10.1016/j.abb.2004.07.006

Cited by 60 publications

(48 citation statements)

References 30 publications

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“…The P 2 specificity observed in the microarray assay is in accordance with Maciewicz et al who found cathepsin L preferred the substrate Cbz-Phe-Arg-coumarin (62). Similarly, using a panel of FRET substrates to study cathepsin L, Puzer et al report a preference for substrates with P 2 Leu and Phe (70). At the P 3 position, basic residues were preferred, consistent with literature reports (68,70).…”

Section: Resultssupporting

confidence: 87%

“…3F), consistent with literature reports (70,72). This similarity can be attributed to the fact that both cathepsins V and L contain an Ala 205 in the S 2 pocket, allowing for accommodation of large residues at position 205, resulting in a deeper S 2 subsite pocket (72).…”

Section: Resultssupporting

confidence: 87%

“…Similarly, using a panel of FRET substrates to study cathepsin L, Puzer et al report a preference for substrates with P 2 Leu and Phe (70). At the P 3 position, basic residues were preferred, consistent with literature reports (68,70).…”

Section: Resultssupporting

confidence: 66%

“…This similarity can be attributed to the fact that both cathepsins V and L contain an Ala 205 in the S 2 pocket, allowing for accommodation of large residues at position 205, resulting in a deeper S 2 subsite pocket (72). Puzer et al reported that cathepsin V preferred Leu over Phe in the P 2 position in studies with a panel of dipeptidyl-coumarin substrate studies (70,73), showing that cathepsin V has an intermediate specificity between cathepsin L and S.…”

Section: Resultsmentioning

confidence: 99%

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High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Gosalia

Salisbury

Ellman

et al. 2005

Molecular & Cellular Proteomics

154

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Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format AcAla-X-X-(Arg/Lys)-coumarin was synthesized (X ‫؍‬ all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXa␤, factor XIa and factor ␣ XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates ؋ 24 different proteases ؋ 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P 2 preference for Leu, Val, Phe, and Tyr in both the P 1 ‫؍‬ Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage. Molecular & Cellular Proteomics 4:626 -636, 2005.Because of their critical roles in biological pathways like hormone activation, proteasomal degradation, and apoptosis, proteases are essential for cellular function and viability. Proteases regulate hormonal activation, cellular homeostasis, apoptosis, and coagulation and play an important role in the pathogenicity and progression of many diseases (1). Proteases comprise one of the largest protein families in organisms from Escherichia coli to humans (2-4). Improved understanding of proteases will provide insight into biological systems and will likely provide a number of important new therapeutic targets (1).To properly function, proteases must preferentially cleave their target substrates in the presence of other proteins. While many factors impact protease substrate selection, one of the key aspects is the complementary nature of the enzymeactive site with the residues surrounding the cleaved bond in the substrate. As such, determination of the residues that comprise the preferred cleavage site of a protease provides critical information regarding substrate selection. Furthermore, determination of substrate specificity also provides a framework for the design of potent and selective inhibitors.Here we exploit solution-phase substrate nanodroplet microarrays (5), in which fluorogenic substrates suspended in glycerol droplets are treated with aerosolized aqueous enzyme solutions, to provide protease substrate specificity profiles (6). These arrays allow high throughput characterization of the preferred residues on the P side (7) of the substrate in a highl...

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Section: Resultssupporting

confidence: 87%

Section: Resultssupporting

confidence: 87%

Section: Resultssupporting

confidence: 66%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Gosalia

Salisbury

Ellman

et al. 2005

Molecular & Cellular Proteomics

154

View full text Add to dashboard Cite

show abstract

“…Importantly, the L208/ K209 motif is consistent with a known cathepsin L cleavage preference consisting of a hydrophobic amino acid in the P-2 position and a basic residue in P-1 (49), with cleavage occurring between P-1 and P-1=. In this case, cleavage would be between amino acids 209 and 210.…”

Section: Resultssupporting

confidence: 75%

Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

Corry

Johnson

Cornwell

et al. 2016

J Virol

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All live attenuated respiratory syncytial virus (RSV) vaccines that have advanced to clinical trials have been produced in Vero cells. The attachment (G) glycoprotein in virions produced in these cells is smaller than that produced in other immortalized cells due to cleavage. These virions are 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cell cultures. Because HAE cells are isolated directly from human airways, Vero cell-grown vaccine virus would very likely be similarly inefficient at initiating infection of the nasal epithelium following vaccination, and therefore, a larger inoculum would be required for effective vaccination. We hypothesized that Vero cell-derived virus containing an intact G protein would be more infectious for HAE cell cultures. Using protease inhibitors with increasing specificity, we identified cathepsin L to be the protease responsible for cleavage. Our evidence suggests that cleavage occurs in the late endosome or lysosome during endocytic recycling. Cathepsin L activity was 100-fold greater in Vero cells than in HeLa cells. In addition, cathepsin L was able to cleave the G protein in Vero cell-grown virions but not in HeLa cell-grown virions, suggesting a difference in G-protein posttranslational modification in the two cell lines. We identified by mutagenesis amino acids important for cleavage, and these amino acids included a likely cathepsin L cleavage site. Virus containing a modified, noncleavable G protein produced in Vero cells was 5-fold more infectious for HAE cells in culture, confirming our hypothesis and indicating the value of including such a mutation in future live attenuated RSV vaccines. IMPORTANCEWorldwide, RSV is the second leading infectious cause of infant death, but no vaccine is available. Experimental live attenuated RSV vaccines are grown in Vero cells, but during production the virion attachment (G) glycoprotein is cleaved. Virions containing a cleaved G protein are less infectious for primary airway epithelial cells, the natural RSV target. In the study described here we identified the protease responsible, located the cleavage site, and demonstrated that cleavage likely occurs during endocytic recycling. Moreover, we showed that the infectivity of Vero cell-derived virus for primary airway epithelial cells is increased 5-fold if the virus contains a mutation in the G protein that prevents cleavage. The blocking of cleavage should improve RSV vaccine yield, consequently reducing production costs. Posttranslational cleavage of the fusion glycoprotein of many viruses plays an essential role in activation; however, cleavage of the RSV G protein is a novel example of a detrimental effect of cleavage on virus infectivity. In adults and teenagers, most respiratory syncytial virus (RSV) infections produce upper respiratory tract infection and symptoms. However, in infants, the immunocompromised, and the elderly, RSV has the potential to cause life-threatening lower respiratory tract infection (1-4). Worldwide, in 2010 ...

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LC–MS Reveals Isomeric Inhibition of Proteolysis by Lysosomal Cathepsins

Pandey

Julian

2022

Analysis & Sensing

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Defects in autophagy are implicated in many age‐related diseases that cause neurodegeneration including both Alzheimer's and Parkinson's. Within autophagy, the lysosome plays a crucial role by enabling the breakdown and recycling of a wide range of biomolecular species. Herein, the effects of isomerization of aspartic acid (Asp) on substrate recognition and degradation are investigated for a collection of lysosomal cathepsins using liquid chromatography coupled to mass spectrometry. By examining a series of synthetic peptides with sequences derived from long‐lived proteins known to undergo Asp isomerization, we demonstrate that isomerized forms of Asp significantly perturb cathepsin activity by impeding digestion and shifting preferential sites of proteolysis. Although the sensitivity to isomerization varies for each cathepsin, none of the cathepsins were capable of digesting sites within several residues of the C‐terminal side of the isomerized Asp. Under physiological conditions, the peptide fragments left behind after such incomplete digestion would not be suitable substrates for transporter recognition and could precipitate autophagic malfunction in the form of lysosomal storage.

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Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L

Cited by 60 publications

References 30 publications

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

LC–MS Reveals Isomeric Inhibition of Proteolysis by Lysosomal Cathepsins

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