- Email: [email protected]
Human T-cell Leukemia Virus Type I (HTLV-I) Retropepsin
Clan AA (A2) | 49. Human T-cell Leukemia Virus Type I (HTLV-I) Retropepsin
[9]
[10]
[11]
[12]
[13]
[14]
binding pockets of the Rous sarcoma virus and human immunodeficiency virus type 1 proteases. J. Biol. Chem. 268, 1171111720. Ridky, T.W., Cameron, C.E., Cameron, J., Leis, J., Copeland, T., Wlodawer, A., Weber, I.T., Harrison, R.W. (1996). Human immunodeficiency virus, type 1 protease substrate specificity is limited by interactions between substrate amino acids bound in adjacent enzyme subsites. J. Biol. Chem. 271, 47094717. Schatz, G., Pichova, I., Vogt, V.M. (1997). Analysis of cleavage site mutations between the NC and PR Gag domains of Rous sarcoma virus. J. Virol. 71, 444450. Schatz, G.W., Reinking, J., Zippin, J., Nicholson, L.K., Vogt, V.M. (2001). Importance of the N terminus of Rous sarcoma virus protease for structure and enzymatic function. J. Virol. 75, 47614770. Grinde, B., Cameron, C.E., Leis, J., Weber, I.T., Wlodawer, A., Burstein, H., Bizub, D., Skalka, A.M. (1992). Mutations that alter the activity of the Rous sarcoma virus protease. J. Biol. Chem. 267, 94819490. Sedlacek, J., Fabry, M., Coward, J.E., Horejsi, M., Strop, P., Luftig, R.B. (1993). Myeloblastosis associated virus (MAV) proteinase site-mutated to be HIV-like has a higher activity and allows production of infectious but morphologically altered virus. Virology 192, 667672. Cameron, C.E., Ridky, T.W., Shulenin, S., Leis, J., Weber, I.T., Copeland, T., Wlodawer, A., Burstein, H., Bizub-Bender, D., Skalka, A.M. (1994). Mutational analysis of the substrate binding pockets of the Rous sarcoma virus and human immunodeficiency virus-1 proteases. J. Biol. Chem. 269, 1117011177.
213
[15] Ridky, T.W., Bizub-Bender, D., Cameron, C.E., Weber, I.T., Wlodawer, A., Copeland, T., Skalka, A.M., Leis, J. (1996). Programming the Rous sarcoma virus protease to cleave new substrate sequences. J. Biol. Chem. 271, 1053810544. [16] Ridky, T., Leis, J. (1995). Development of drug resistance to HIV1 protease inhibitors. J. Biol. Chem. 270, 2962129623. [17] Bizub, D., Weber, I.T., Cameron, C.E., Leis, J.P., Skalka, A.M. (1991). A range of catalytic efficiencies with avian retroviral protease subunits genetically linked to form single polypeptide chains. J. Biol. Chem. 266, 49514958. [18] Xiang, Y., Ridky, T.W., Krishna, N.K., Leis, J. (1997). Altered Rous sarcoma virus Gag polyprotein processing and its effects on particle formation. J. Virol. 71, 20832091. [19] Miller, M., Jaskolski, M., Rao, J.K., Leis, J., Wlodawer, A. (1989). Crystal structure of a retroviral protease proves relationship to aspartic protease family. Nature 337, 576579. [20] Jaskolski, M., Miller, M., Rao, J.K., Leis, J., Wlodawer, A. (1990). Structure of the aspartic protease from Rous sarcoma retrovirus refined at 2-A resolution. Biochemistry 29, 58895898. [21] Wu, J., Adomat, J.M., Ridky, T.W., Louis, J.M., Leis, J., Harrison, R.W., Weber, I.T. (1998). Structural basis for specificity of retroviral proteases. Biochemistry 37, 45184526. [22] Alexander, F., Leis, J., Soltis, D.A., Crowl, R.M., Danho, W., Poonian, M.S., Pan, Y.C., Skalka, A.M. (1987). Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain. J. Virol. 61, 534542. [23] Skalka, A.M. (1989). Retroviral proteases: first glimpses at the anatomy of a processing machine. Cell 56, 911913.
Todd W. Ridky Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Jonathan Leis Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. Email: [email protected] This article is reproduced from the previous edition, Volume 1, pp. 163166, r 2004, Elsevier Ltd., with revisions made by the Editors. Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00048-X
Chapter 49
Human T-cell Leukemia Virus Type I (HTLV-I) Retropepsin DATABANKS MEROPS name: retropepsin (human T-cell leukemia virus) MEROPS classification: clan AA, family A2, subfamily A2A, peptidase A02.012
Tertiary structure: Available Species distribution: known only from human T-cell lymphotropic virus type I Reference sequence from: human T-cell lymphotropic virus type I (UniProt: P14074)
214
Clan AA (A2) | 49. Human T-cell Leukemia Virus Type I (HTLV-I) Retropepsin
Name and History A set of symptoms and infections associated with human T-cell lymphotropic/leukemia virus type 1 (HTLV-I or HTLV-1) was described in 1977 [1]. In 1980, the HTLVI virus was isolated as the pathological cause and became the first identified human retrovirus [2]. In 1983, the entire proviral genome of HTLV-I was sequenced [3]. In 1988, HTLV-I protease (or HTLV-I retropepsin) was identified as a viral aspartic proteolytic enzyme that plays a critical role in HTLV-I replication [4]. The protease was expressed by vaccinia virus recombinants in 1988 [4], by Escherichia coli recombinants in 1991 [5], and the protease was produced by chemical synthesis in 1997 [6]. In 2005, the X-ray diffraction crystal structure of a des(117-125)-[Ile40]HTLV-I protease in complex with a moderate potency nonapeptidic inhibitor was reported [7], and in 2010, the inhibitorprotease crystal structure complexes of several synthetic potent tetrapeptidic [Ile40]HTLV-I protease inhibitors (molecular weight ,800 g mol21; IC50 ,10 nM) were solved [8]. HTLV-I infection can aggravate to T-cell lymphoma and T-cell leukemia. Hence, depending on authors, the ‘L’ in ‘HTLV-I’ can stand for ‘lymphotropic’ or ‘leukemia’ or both. There are four known types of HTLV, of which HTLV-I and HTLV-II are the most prevalent worldwide. Beware that prior to 1986, the human immunodeficiency virus (HIV), referring to both HIV-1 (Chapter 44) and HIV-2 (Chapter 45), was named HTLV-III. Moreover, HIV-2 used to be called HTLV-IV. In the current nomenclature, HIV-1, HIV-2, HTLV-III and HTLV-IV are different viral species.
Activity and Specificity HTLV-I protease is an autoprocessing protein encoded within a single stranded positive sense viral RNA genome which is flanked by long terminal repeats [9]. The genome describes gag (group antigen), pro (protease), pol (polymerase), env (envelope) and short regulatory genes (rex, tax and others) with overlaps between the first four genes. There are three open reading frames as a result of reading frameshift: gag is encoded in reading frame 0; pro, env and rex/tax are found on reading frame -1; and pol is read from reading frame -2. The genes are reverse transcribed, transcribed and translated to three precursor proteins (or precursor polyproteins) Gag, Gag-Pro and Gag-Pro-Pol, respectively [10]. From the Gag precursor protein, a viral matrix (MA), capsid (CA) and nucleocapsid (NC) are produced by HTLV-I protease cleavage. The Gag-Pro precursor protein is processed by the protease to afford MA, CA, TF1 (transframe 1, a C-terminal
truncated NC), another HTLV-I protease (PR), and two short peptides with unidentified functions (p1 and p2 at 1 and 2 kDa, respectively). HTLV-I protease hydrolysis of the Gag-Pro-Pol precursor protein results in MA, CA, TF1, PR, p1, reverse transcriptase (RT), ribonuclease H (RH) and integrase (IN). Note that the proteolysis between CA/NC and CA/TF1 represents the same cleavage site on different precursor proteins. Cleavage has been assayed using synthetic peptides incorporating a chromogenic reporter group p-nitrophenylalanine (Nph) at the P10 [11] or P1 position [12], or using the full-length Gag precursor protein [4,13]. The addition of a Lys([7methoxycoumarin4yl]acetyl) fluorophore as the P6 residue to a P10 Nph chromophore containing substrate further enhances detection [8]. The pH optimum is within 55.5 [5,9,14] with 13 M salt [9,12,14]. Be aware that the literature is full of inconsistent functional assay results, even from the same enzyme and substrate, because the proteases are obtained from different sources, mutated, truncated, refolded under different conditions, prone to autolysis, or different in purity, and because of differences in substrate and other assay conditions [14]. Of the seven common cleavage sites, six have Leu as the P1 residue and four have Pro as the P10 residue. A substrate study, where each 20 natural amino acids was substituted at the P1 and P10 positions, indicates cleavage efficiency follows the order of Leu 5 Met . Phe 5 Cys for the P1 position, and Pro is clearly preferred as the P10 residue [15]. In the design of peptidic HTLV-I protease inhibitors, the P1 residue or P1P10 unit is a non-cleavable moiety that mimics the transition-state formed during amide hydrolysis by the protease, and is also structurally similar to Leu or Phe (Figure 49.1) [79,16,17]. At the hydrophobic S2 subsite, substrate [15,18] and inhibitor [16] studies show Ile is preferred as the P2 residue over Val and Leu which are in turn better tolerated than Gly, Phe, Glu and Gln (Figure 49.2). Similarly, Ile is favored as the P20 residue over Val and Leu with lower regard for Thr [16]. The S3 subsite prefers Ile and tolerates Ala, Val, Leu, Phe, Lys and Gln, with lower tolerance for Gly, Asp and Glu [15,16,18]. On the other hand, the S30 subsite favors Met and Gln over Val, Leu, Ile and Phe [16]. The S4 subsite can accommodate a variety of residues, including Ala, Val, Leu, Ile, Pro, Thr, Glu and Gln [15,18]. The S5 and S50 subsites are located on the surface of the protease, and consequently, can even tolerate hydrophilic residues [7]. Generally, subsites that are more distant from the catalytic S1S10 subsites are more tolerant to changes in the substrate or inhibitor [7,16,19]. HTLV-I protease displays a higher degree of specificity for substrates and inhibitors over that of HIV-1 protease [17,18]. This means HTLV-I protease inhibitors generally also exhibit potent HIV-1 protease inhibition potency,
Clan AA (A2) | 49. Human T-cell Leukemia Virus Type I (HTLV-I) Retropepsin
215
FIGURE 49.1 The possible hydrogen bond and cation-π interactions of [Ile40]HTLV-I protease inhibitor KNI-10562 (IC50 5 7 nM) in complex with a des-(117-125)-[Ile40]HTLV-I ˚ protease as defined by X-ray analysis at 2.0 A [8]. For simplicity, inter- and intramolecular hydrogen bond interactions between the two protease chains are not illustrated.
FIGURE 49.2 Cross-section view of [Ile40]HTLV-I protease inhibitor KNI-10562 in complex with a des-(117-125)-[Ile40]HTLV-I protease.
whereas compounds with potent HIV-1 protease inhibition do not necessarily potently inhibit HTLV-I protease. The smallest reported potent tetrapeptidic [Ileu40]HTLVI protease inhibitor (IC50 5 13 nM) has interactions covering the S3 to S20 pockets [20].
Structural Chemistry The fold of HTLV-I protease grossly structurally resembles HIV-1 (Chapter 44), HIV-2 (Chapter 45), simian immunodeficiency virus (Chapter 46), equine infectious anemia virus (Chapter 47), Rous sarcoma virus (Chapter 48) and feline immunodeficiency virus (Chapter 54) with differences found largely in the external surface loops [7]. Mature HTLV-I protease is a small C2 symmetry homodimer of 125-residue peptide chains: PVIPLDPARR10-PVIKAQVDTQ20-TSHPKTIEAL30LDTGADMTVL40-PIALFSSNTP50-LKNTSVLGAG60GQTQDHFKLT70-SLPVLIRLPF80-RTTPIVLTSC90LVDTKNNWAI100-IGRDALQQCQ110GVLYLPEAKG120-PPVIL125
Deletional mutanogenesis of the mature protease reveals the first five N-terminal residues are not essential for enzymatic activity [13]. Likewise, the last nine or ten Cterminal residues (116125 or 117125) are not critical for proteolytic activity [7,14,21], despite a conflicting report [22]. About half of the HTLV-I protease expression protein readily autodigests between Leu40 and Pro41 during isolation and purification, because of the similarity to the processing sequence (Val, Ile or Gln)-LeukPro-(Val or Ile) at several cleavage sites [12]. Autolysis is preventable by a Leu40 to Ile40 mutation without greatly affecting enzyme kinetic parameters. Cys90 and Cys109 residues are not involved in the formation of structurally important disulfide bonds, and the [L40I,C90A,C109A]mutation protease is considered more stable because of lower risks for undesired intramolecular disulfide bond formations. The crystal structures of the des-(117-125)[Ile40]HTLV-I protease dimer in complex with a moderately potent nonapeptidic inhibitor or a selection of moderatehigh potency tetrapeptidic inhibitors have been solved [8]. There are four subsites, from S2 to S20 , inside the cylindrical active site which is flanked by partly solvent-exposed S3/S30 , solvent-exposed S4/S40 and surface S5/S50 subsites (Figure 49.2). Direct or water-mediated hydrogen bond interactions along the backbone of the inhibitor involve Arg10, Asp32, Gly34, Asp36, Leu57, Ala59 and Arg103 from both chains (Figure 49.1). At the critical S1S10 subsites, one water molecule mediates interactions with Ala59 on each flap, and the hydroxyl group of the P1 inhibitory unit mimics the catalytic water molecule involved with each Asp32 during peptide hydrolysis. The amino acid layout ranging from the S3 to S30 pockets of HTLV-I protease suggests interactions with the side chains of the inhibitor are predominantly hydrophobic [7]. However, the guanidinyl group of Arg10 in the S3 pocket can form either a cation-π interaction with
216
Clan AA (A2) | 49. Human T-cell Leukemia Virus Type I (HTLV-I) Retropepsin
the P3 L-phenylglycine residue of the inhibitor or a watermediated interaction with the P3 Gln [8,20].
Preparation An active protease can be expressed either from a precursor protein construct or as the mature protein coding sequence in eukaryotic cells from a vaccinia virus recombinant [4]. Recombinant HTLV-I protease can be expressed and purified from E. coli on milligram scale, where the protease accumulates in inclusion bodies [5]. The recombinant protease is then denatured and refolded. The recovery and hydrolytic activity of the protease is dependent on the refolding protocol [14]. The enzyme can also be chemically synthesized by segment condensation from solid-phase synthesis [6,14]. The protease is commonly obtained by E. coli recombinant or chemical synthesis. Modified HTLV-I proteases are often prepared for stability reasons, because the wild full-length protease can undergo autolysis, aggregation and side-reactions during isolation, purification and storage.
Biological Aspects The etiology of HTLV-I associated diseases is still under investigation. HTLV-I infection causes HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) from T-cellmediated chronic inflammatory processes in the spinal cord targeting HTLV-I infected T-cells leading to bystander neurological degeneration [23]. HTLV-I is also the only known retrovirus to cause adult T-cell lymphoma/leukemia (ATL) from oncogenic induction by Tax protein and oncogenic process support by HTLV-I basic leucine zipper factor (HBZ) which is encoded from the minus strand of the proviral genome [24]. HAM/TSP and ATL are generally mutually exclusive, and only a few cases with both disorders have been described. If the virus infiltrates the skin and brain, several inflammatory diseases, such as alveolitis, rheumatoid arthritis, infective dermatitis, myositis, uveitis and opportunistic infections from Staphyloccocus aureus and Strongyloides stercoralis may also be present. HTLV-I protease is not known as a target for immune attack in immunocompetent-infected individuals, probably because of HTLV-I protease sequestration within the virion capsid. As a result of this lack of immune selection, narrow substrate specificity and lower in vivo replication rate in HTLV-I than HIV-1, the protease sequence for HTLV-I protease is highly conserved between isolates. The HTLV-II type is associated with encephalomyelopathy with symptoms similar to HAM/TSP, although it does not cause leukemia. The HTLV-III and HTLV-IV types are not clearly linked to any illness.
Distinguishing Features Rabbit polyclonal antisera specific to HTLV-I protease was raised to the C-terminal 123140 [13] and N-terminal 3854 residues [25]. A rabbit antiserum to the protease precursor peptide 221234 was described [13]. Three monoclonal antibodies directed against HTLV-I protease fused to a maltose binding protein were developed, with two directed against the N-terminal 116 and one was specific to residues 3544 [26]. None are commercially available.
Related Peptidases Of the retroviral proteases, the bovine leukemia virus (BLV) protease is most closely related to HTLV-I protease with 32% amino acid identity (Chapter 50). BLV and HTLV-I/II/III/IV are members of the deltaretrovirus genus of the Retroviridae family. Although BLV protease showed broader substrate specificity than HTLV-I protease, the specificity of individual subsites of BLV protease resembles that of HTLV-I protease more closely than HIV-1 protease (lentivirus genus) [10].
Further Reading The substrate specificity of HTLV-I and several retroviral proteases was compared by To¨zse´r [10]. The structural X-ray crystal models of HTLV-I and other retroviral proteases were superposed by Li et al. [7]. The amino acid specificity at each subsite can be summarized from the work by Ka´das et al. [18], Bang et al. [15] and Kimura et al. [16]. The refined X-ray crystal structures of the protease in complex with several inhibitors were solved by Satoh et al. [8].
References [1] Uchiyama, T., Yodoi, J., Sagawa, K., Takatsuki, K., Uchino, H. (1977). Adult T-cell leukemia: clinical and hematologic features of 16 cases. Blood 50(3), 481492. [2] Poiesz, B.J., Ruscetti, F.W., Gazdar, A.F., Bunn, P.A., Minna, J.D., Gallo, R.C. (1980). Detection and isolation of type C retrovirus particles from fresh and cocultured lymphotcytes of a patient with cutaneous T cell lymphoma. Proc. Natl. Acad. Sci. USA 77(12), 74157419. [3] Seiki, M., Hattori, S., Hirayama, Y., Yoshida, M. (1983). Human adult T-cell leukemia virus: complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA. Proc. Natl. Acad. Sci. USA 80(12), 36183622. [4] Nam, S.H., Kidokoro, M., Shida, H., Hatanaka, M. (1988). Processing of gag precursor polyprotein of human T-cell leukemia virus type 1 by virus-encoded protease. J. Virol. 62(10), 37183728. [5] Kobayashi, M., Ohi, Y., Asano, T., Hayakawa, T., Kato, K., Kakinuma, A., Hatanaka, M. (1991). Purification and
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characterization of human T-cell leukemia virus type 1 protease produced in Escherichia coli. FEBS Lett. 293(12), 106110. Hruskova´-Heidingsfeldova´, O., Bla´ha, I., Urban, J., Strop, P., Pichova´, I. (1997). Substrates and inhibitors of human T-cell leukemia virus type 1 (HTLV-1) proteinase. Leukemia 11(Suppl.3), 4546. Li, M., Laco, G.S., Jaskolski, M., Rozycki, J., Alexandratos, J., Wlodawer, A., Gustchina, A. (2005). Crystal structure of human T cell leukemia virus protease, a novel target for anticancer drug design. Proc. Natl. Acad. Sci. USA 102(51), 1833218337. Satoh, T., Li, M., Nguyen, J.-T., Kiso, Y., Gustchina, A., Wlodawer, A. (2010). Crystal structures of inhibitor complexes of human T-cell leukemia virus (HTLV-1) protease. J. Mol. Biol. 401(4), 626641. Ding, Y.S., Rich, D.H., Ikeda, R.A. (1998). Substrates and inhibitors of human T-cell leukemia virus type I protease. Biochemistry 37(50), 1751417518. To¨zse´r, J. (2010). Comparative studies on retroviral proteases: Substrate specificity. Viruses 2(1), 147165. Daenke, S., Schramm, H.J., Bangham, C.R.M. (1994). Analysis of substrate cleavage by recombinant protease of human T cell leukaemia virus type 1 reveals preference and specificity of binding. J. Gen. Virol. 75(9), 22332239. Louis, J.M., Oroszlan, S., To¨sze´r, J. (1999). Stabilization from autoproteolysis and kinetic characterization of the human T-cell leukemia virus type 1 proteinase. J. Biol. Chem. 274(10), 66606666. Hayakama, T., Misumi, Y., Kobayashi, M, Ohi, Y., Yamamoto, Y., Fujisawa, Y. (1992). Requirement of N- and C-terminal regions for enzymatic activity of human T-cell leukemia virus type I protease. Eur. J. Biochem. 206(3), 919925. Li, C., Li, X., Lu, W. (2010). Total chemical synthesis of human T-cell leukemia virus type 1 protease via native chemical ligation. Biopolymers (Pept. Sci.) 94(4), 487494. Bang, J.K., Teruya, K., Aimoto, S., Konno, H., Nosaka, K., Tatsumi, T., Akaji, K. (2007). Studies on substrate specificity at PR/p3 cleavage site of HTLV-1 protease. Int. J. Pept. Res. Ther. 13(12), 173179. Kimura, T., Nguyen, J.-T., Maegawa, H., Nishiyama, K., Arii, Y., Matsui, Y., Hayashi, Y., Kiso, Y. (2007). Chipping at large, potent human T-cell leukemia virus type 1 protease inhibitors to uncover smaller, equipotent inhibitors. Bioorg. Med. Chem. Lett. 17(12), 32763280.
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[17] Nguyen, J.-T., Kiso, Y. (2009). Rational drug design of HTLV-I protease inhibitors, in: Viral Proteases and Antiviral Protease Inhibitor Therapy, Proteases in Biology and Disease, Vol. 8, Lendeckel, U., Hooper, N.M., eds., Dordretch: Springer Science, pp. 83100. [18] Ka´das, J., Weber, I.T., Bagossi, P., Miklo´ssy, G., Boross, P., Oroszlan, S., To¨zse´r, J. (2004). Narrow substrate specificity and sensitivity toward ligand-binding site mutations of human T-cell leukemia virus type 1 protease. J. Biol. Chem. 279(26), 2714827157. [19] To¨zse´r, J., Zahuczky, G., Bagossi, P., Louis, J.M., Copeland, T.D., Oroszlan, S., Harrison, R.W., Weber, I.T. (2000). Comparison of the substrate specificity of the human T-cell leukemia virus and human immunodeficiency virus proteinases. Eur. J. Biochem. 267(20), 62876295. [20] Nguyen, J.-T., Kato, K., Kumada, H.-O., Hidaka, K., Kimura, T., Kiso, Y. (2011). Maintaining potent HTLV-I protease inhibition without the P3-cap moiety in small tetrapeptidic inhibitors. Bioorg. Med. Chem. Lett. 21(6), 18321837. [21] Herger, B.E., Mariani, V.L., Dennison, K.J., Shuker, S.B. (2004). The 10 C-terminal residues of HTLV-I protease are not necessary for enzymatic activity. Biochem. Biophys. Res. Commun. 320(4), 13061308. [22] Ka´das, J., Boross, P., Weber, I.T., Bagossi, P., Matu´z, K., T To¨zse´r, J. (2008). C-Terminal residues of mature human T-lymphotropic virus type 1 protease are critical for dimerization and catalytic activity. Biochem. J. 416(3), 357364. [23] Izumo, S. (2010). Neuropathology of HTLV-1-associated meylopathy (HAM/TSP). Neuropathology 30(5), 480485. [24] Kannian, P., Green, P.L. (2010). Human T lymphotropic virus type 1 (HTLV-1): Molecular biology and oncogenesis. Viruses 2(9), 20372077. [25] Hayakawa, T., Misumi, Y., Kobayashi, M., Ohi, Y., Fujisawa, Y., Kakinuma, A., Hatanaka, M. (1991). Expression of human T-cell leukemia virus type 1 protease in Escherichia coli. Biochem. Biophys. Res. Commun. 181(3), 12811287. [26] Mamoun, R.Z., Dye, D., Rebeyrotte, N., Bouamr, Fadila, Cerutti, M., Desgranges, C. (1997). Mouse monoclonal antibodies directed against the HTLV-I protease recognize epitopes internal to the dimer. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 14(2), 184188.
Jeffrey-Tri Nguyen Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan. Email: [email protected]
Yoshiaki Kiso Laboratory of Peptide Science, Nagahama Institute of Bio-Science & Technology, Nagahama, Shiga 526-0829, Japan. Email: [email protected] This article is a revision of the previous edition article by S. Daenke, Volume 1, pp. 166169, r 2004, Elsevier Ltd. Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00049-1
[9]
[10]
[11]
[12]
[13]
[14]
binding pockets of the Rous sarcoma virus and human immunodeficiency virus type 1 proteases. J. Biol. Chem. 268, 1171111720. Ridky, T.W., Cameron, C.E., Cameron, J., Leis, J., Copeland, T., Wlodawer, A., Weber, I.T., Harrison, R.W. (1996). Human immunodeficiency virus, type 1 protease substrate specificity is limited by interactions between substrate amino acids bound in adjacent enzyme subsites. J. Biol. Chem. 271, 47094717. Schatz, G., Pichova, I., Vogt, V.M. (1997). Analysis of cleavage site mutations between the NC and PR Gag domains of Rous sarcoma virus. J. Virol. 71, 444450. Schatz, G.W., Reinking, J., Zippin, J., Nicholson, L.K., Vogt, V.M. (2001). Importance of the N terminus of Rous sarcoma virus protease for structure and enzymatic function. J. Virol. 75, 47614770. Grinde, B., Cameron, C.E., Leis, J., Weber, I.T., Wlodawer, A., Burstein, H., Bizub, D., Skalka, A.M. (1992). Mutations that alter the activity of the Rous sarcoma virus protease. J. Biol. Chem. 267, 94819490. Sedlacek, J., Fabry, M., Coward, J.E., Horejsi, M., Strop, P., Luftig, R.B. (1993). Myeloblastosis associated virus (MAV) proteinase site-mutated to be HIV-like has a higher activity and allows production of infectious but morphologically altered virus. Virology 192, 667672. Cameron, C.E., Ridky, T.W., Shulenin, S., Leis, J., Weber, I.T., Copeland, T., Wlodawer, A., Burstein, H., Bizub-Bender, D., Skalka, A.M. (1994). Mutational analysis of the substrate binding pockets of the Rous sarcoma virus and human immunodeficiency virus-1 proteases. J. Biol. Chem. 269, 1117011177.
213
[15] Ridky, T.W., Bizub-Bender, D., Cameron, C.E., Weber, I.T., Wlodawer, A., Copeland, T., Skalka, A.M., Leis, J. (1996). Programming the Rous sarcoma virus protease to cleave new substrate sequences. J. Biol. Chem. 271, 1053810544. [16] Ridky, T., Leis, J. (1995). Development of drug resistance to HIV1 protease inhibitors. J. Biol. Chem. 270, 2962129623. [17] Bizub, D., Weber, I.T., Cameron, C.E., Leis, J.P., Skalka, A.M. (1991). A range of catalytic efficiencies with avian retroviral protease subunits genetically linked to form single polypeptide chains. J. Biol. Chem. 266, 49514958. [18] Xiang, Y., Ridky, T.W., Krishna, N.K., Leis, J. (1997). Altered Rous sarcoma virus Gag polyprotein processing and its effects on particle formation. J. Virol. 71, 20832091. [19] Miller, M., Jaskolski, M., Rao, J.K., Leis, J., Wlodawer, A. (1989). Crystal structure of a retroviral protease proves relationship to aspartic protease family. Nature 337, 576579. [20] Jaskolski, M., Miller, M., Rao, J.K., Leis, J., Wlodawer, A. (1990). Structure of the aspartic protease from Rous sarcoma retrovirus refined at 2-A resolution. Biochemistry 29, 58895898. [21] Wu, J., Adomat, J.M., Ridky, T.W., Louis, J.M., Leis, J., Harrison, R.W., Weber, I.T. (1998). Structural basis for specificity of retroviral proteases. Biochemistry 37, 45184526. [22] Alexander, F., Leis, J., Soltis, D.A., Crowl, R.M., Danho, W., Poonian, M.S., Pan, Y.C., Skalka, A.M. (1987). Proteolytic processing of avian sarcoma and leukosis viruses pol-endo recombinant proteins reveals another pol gene domain. J. Virol. 61, 534542. [23] Skalka, A.M. (1989). Retroviral proteases: first glimpses at the anatomy of a processing machine. Cell 56, 911913.
Todd W. Ridky Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Jonathan Leis Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA. Email: [email protected] This article is reproduced from the previous edition, Volume 1, pp. 163166, r 2004, Elsevier Ltd., with revisions made by the Editors. Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00048-X
Chapter 49
Human T-cell Leukemia Virus Type I (HTLV-I) Retropepsin DATABANKS MEROPS name: retropepsin (human T-cell leukemia virus) MEROPS classification: clan AA, family A2, subfamily A2A, peptidase A02.012
Tertiary structure: Available Species distribution: known only from human T-cell lymphotropic virus type I Reference sequence from: human T-cell lymphotropic virus type I (UniProt: P14074)
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Clan AA (A2) | 49. Human T-cell Leukemia Virus Type I (HTLV-I) Retropepsin
Name and History A set of symptoms and infections associated with human T-cell lymphotropic/leukemia virus type 1 (HTLV-I or HTLV-1) was described in 1977 [1]. In 1980, the HTLVI virus was isolated as the pathological cause and became the first identified human retrovirus [2]. In 1983, the entire proviral genome of HTLV-I was sequenced [3]. In 1988, HTLV-I protease (or HTLV-I retropepsin) was identified as a viral aspartic proteolytic enzyme that plays a critical role in HTLV-I replication [4]. The protease was expressed by vaccinia virus recombinants in 1988 [4], by Escherichia coli recombinants in 1991 [5], and the protease was produced by chemical synthesis in 1997 [6]. In 2005, the X-ray diffraction crystal structure of a des(117-125)-[Ile40]HTLV-I protease in complex with a moderate potency nonapeptidic inhibitor was reported [7], and in 2010, the inhibitorprotease crystal structure complexes of several synthetic potent tetrapeptidic [Ile40]HTLV-I protease inhibitors (molecular weight ,800 g mol21; IC50 ,10 nM) were solved [8]. HTLV-I infection can aggravate to T-cell lymphoma and T-cell leukemia. Hence, depending on authors, the ‘L’ in ‘HTLV-I’ can stand for ‘lymphotropic’ or ‘leukemia’ or both. There are four known types of HTLV, of which HTLV-I and HTLV-II are the most prevalent worldwide. Beware that prior to 1986, the human immunodeficiency virus (HIV), referring to both HIV-1 (Chapter 44) and HIV-2 (Chapter 45), was named HTLV-III. Moreover, HIV-2 used to be called HTLV-IV. In the current nomenclature, HIV-1, HIV-2, HTLV-III and HTLV-IV are different viral species.
Activity and Specificity HTLV-I protease is an autoprocessing protein encoded within a single stranded positive sense viral RNA genome which is flanked by long terminal repeats [9]. The genome describes gag (group antigen), pro (protease), pol (polymerase), env (envelope) and short regulatory genes (rex, tax and others) with overlaps between the first four genes. There are three open reading frames as a result of reading frameshift: gag is encoded in reading frame 0; pro, env and rex/tax are found on reading frame -1; and pol is read from reading frame -2. The genes are reverse transcribed, transcribed and translated to three precursor proteins (or precursor polyproteins) Gag, Gag-Pro and Gag-Pro-Pol, respectively [10]. From the Gag precursor protein, a viral matrix (MA), capsid (CA) and nucleocapsid (NC) are produced by HTLV-I protease cleavage. The Gag-Pro precursor protein is processed by the protease to afford MA, CA, TF1 (transframe 1, a C-terminal
truncated NC), another HTLV-I protease (PR), and two short peptides with unidentified functions (p1 and p2 at 1 and 2 kDa, respectively). HTLV-I protease hydrolysis of the Gag-Pro-Pol precursor protein results in MA, CA, TF1, PR, p1, reverse transcriptase (RT), ribonuclease H (RH) and integrase (IN). Note that the proteolysis between CA/NC and CA/TF1 represents the same cleavage site on different precursor proteins. Cleavage has been assayed using synthetic peptides incorporating a chromogenic reporter group p-nitrophenylalanine (Nph) at the P10 [11] or P1 position [12], or using the full-length Gag precursor protein [4,13]. The addition of a Lys([7methoxycoumarin4yl]acetyl) fluorophore as the P6 residue to a P10 Nph chromophore containing substrate further enhances detection [8]. The pH optimum is within 55.5 [5,9,14] with 13 M salt [9,12,14]. Be aware that the literature is full of inconsistent functional assay results, even from the same enzyme and substrate, because the proteases are obtained from different sources, mutated, truncated, refolded under different conditions, prone to autolysis, or different in purity, and because of differences in substrate and other assay conditions [14]. Of the seven common cleavage sites, six have Leu as the P1 residue and four have Pro as the P10 residue. A substrate study, where each 20 natural amino acids was substituted at the P1 and P10 positions, indicates cleavage efficiency follows the order of Leu 5 Met . Phe 5 Cys for the P1 position, and Pro is clearly preferred as the P10 residue [15]. In the design of peptidic HTLV-I protease inhibitors, the P1 residue or P1P10 unit is a non-cleavable moiety that mimics the transition-state formed during amide hydrolysis by the protease, and is also structurally similar to Leu or Phe (Figure 49.1) [79,16,17]. At the hydrophobic S2 subsite, substrate [15,18] and inhibitor [16] studies show Ile is preferred as the P2 residue over Val and Leu which are in turn better tolerated than Gly, Phe, Glu and Gln (Figure 49.2). Similarly, Ile is favored as the P20 residue over Val and Leu with lower regard for Thr [16]. The S3 subsite prefers Ile and tolerates Ala, Val, Leu, Phe, Lys and Gln, with lower tolerance for Gly, Asp and Glu [15,16,18]. On the other hand, the S30 subsite favors Met and Gln over Val, Leu, Ile and Phe [16]. The S4 subsite can accommodate a variety of residues, including Ala, Val, Leu, Ile, Pro, Thr, Glu and Gln [15,18]. The S5 and S50 subsites are located on the surface of the protease, and consequently, can even tolerate hydrophilic residues [7]. Generally, subsites that are more distant from the catalytic S1S10 subsites are more tolerant to changes in the substrate or inhibitor [7,16,19]. HTLV-I protease displays a higher degree of specificity for substrates and inhibitors over that of HIV-1 protease [17,18]. This means HTLV-I protease inhibitors generally also exhibit potent HIV-1 protease inhibition potency,
Clan AA (A2) | 49. Human T-cell Leukemia Virus Type I (HTLV-I) Retropepsin
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FIGURE 49.1 The possible hydrogen bond and cation-π interactions of [Ile40]HTLV-I protease inhibitor KNI-10562 (IC50 5 7 nM) in complex with a des-(117-125)-[Ile40]HTLV-I ˚ protease as defined by X-ray analysis at 2.0 A [8]. For simplicity, inter- and intramolecular hydrogen bond interactions between the two protease chains are not illustrated.
FIGURE 49.2 Cross-section view of [Ile40]HTLV-I protease inhibitor KNI-10562 in complex with a des-(117-125)-[Ile40]HTLV-I protease.
whereas compounds with potent HIV-1 protease inhibition do not necessarily potently inhibit HTLV-I protease. The smallest reported potent tetrapeptidic [Ileu40]HTLVI protease inhibitor (IC50 5 13 nM) has interactions covering the S3 to S20 pockets [20].
Structural Chemistry The fold of HTLV-I protease grossly structurally resembles HIV-1 (Chapter 44), HIV-2 (Chapter 45), simian immunodeficiency virus (Chapter 46), equine infectious anemia virus (Chapter 47), Rous sarcoma virus (Chapter 48) and feline immunodeficiency virus (Chapter 54) with differences found largely in the external surface loops [7]. Mature HTLV-I protease is a small C2 symmetry homodimer of 125-residue peptide chains: PVIPLDPARR10-PVIKAQVDTQ20-TSHPKTIEAL30LDTGADMTVL40-PIALFSSNTP50-LKNTSVLGAG60GQTQDHFKLT70-SLPVLIRLPF80-RTTPIVLTSC90LVDTKNNWAI100-IGRDALQQCQ110GVLYLPEAKG120-PPVIL125
Deletional mutanogenesis of the mature protease reveals the first five N-terminal residues are not essential for enzymatic activity [13]. Likewise, the last nine or ten Cterminal residues (116125 or 117125) are not critical for proteolytic activity [7,14,21], despite a conflicting report [22]. About half of the HTLV-I protease expression protein readily autodigests between Leu40 and Pro41 during isolation and purification, because of the similarity to the processing sequence (Val, Ile or Gln)-LeukPro-(Val or Ile) at several cleavage sites [12]. Autolysis is preventable by a Leu40 to Ile40 mutation without greatly affecting enzyme kinetic parameters. Cys90 and Cys109 residues are not involved in the formation of structurally important disulfide bonds, and the [L40I,C90A,C109A]mutation protease is considered more stable because of lower risks for undesired intramolecular disulfide bond formations. The crystal structures of the des-(117-125)[Ile40]HTLV-I protease dimer in complex with a moderately potent nonapeptidic inhibitor or a selection of moderatehigh potency tetrapeptidic inhibitors have been solved [8]. There are four subsites, from S2 to S20 , inside the cylindrical active site which is flanked by partly solvent-exposed S3/S30 , solvent-exposed S4/S40 and surface S5/S50 subsites (Figure 49.2). Direct or water-mediated hydrogen bond interactions along the backbone of the inhibitor involve Arg10, Asp32, Gly34, Asp36, Leu57, Ala59 and Arg103 from both chains (Figure 49.1). At the critical S1S10 subsites, one water molecule mediates interactions with Ala59 on each flap, and the hydroxyl group of the P1 inhibitory unit mimics the catalytic water molecule involved with each Asp32 during peptide hydrolysis. The amino acid layout ranging from the S3 to S30 pockets of HTLV-I protease suggests interactions with the side chains of the inhibitor are predominantly hydrophobic [7]. However, the guanidinyl group of Arg10 in the S3 pocket can form either a cation-π interaction with
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the P3 L-phenylglycine residue of the inhibitor or a watermediated interaction with the P3 Gln [8,20].
Preparation An active protease can be expressed either from a precursor protein construct or as the mature protein coding sequence in eukaryotic cells from a vaccinia virus recombinant [4]. Recombinant HTLV-I protease can be expressed and purified from E. coli on milligram scale, where the protease accumulates in inclusion bodies [5]. The recombinant protease is then denatured and refolded. The recovery and hydrolytic activity of the protease is dependent on the refolding protocol [14]. The enzyme can also be chemically synthesized by segment condensation from solid-phase synthesis [6,14]. The protease is commonly obtained by E. coli recombinant or chemical synthesis. Modified HTLV-I proteases are often prepared for stability reasons, because the wild full-length protease can undergo autolysis, aggregation and side-reactions during isolation, purification and storage.
Biological Aspects The etiology of HTLV-I associated diseases is still under investigation. HTLV-I infection causes HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) from T-cellmediated chronic inflammatory processes in the spinal cord targeting HTLV-I infected T-cells leading to bystander neurological degeneration [23]. HTLV-I is also the only known retrovirus to cause adult T-cell lymphoma/leukemia (ATL) from oncogenic induction by Tax protein and oncogenic process support by HTLV-I basic leucine zipper factor (HBZ) which is encoded from the minus strand of the proviral genome [24]. HAM/TSP and ATL are generally mutually exclusive, and only a few cases with both disorders have been described. If the virus infiltrates the skin and brain, several inflammatory diseases, such as alveolitis, rheumatoid arthritis, infective dermatitis, myositis, uveitis and opportunistic infections from Staphyloccocus aureus and Strongyloides stercoralis may also be present. HTLV-I protease is not known as a target for immune attack in immunocompetent-infected individuals, probably because of HTLV-I protease sequestration within the virion capsid. As a result of this lack of immune selection, narrow substrate specificity and lower in vivo replication rate in HTLV-I than HIV-1, the protease sequence for HTLV-I protease is highly conserved between isolates. The HTLV-II type is associated with encephalomyelopathy with symptoms similar to HAM/TSP, although it does not cause leukemia. The HTLV-III and HTLV-IV types are not clearly linked to any illness.
Distinguishing Features Rabbit polyclonal antisera specific to HTLV-I protease was raised to the C-terminal 123140 [13] and N-terminal 3854 residues [25]. A rabbit antiserum to the protease precursor peptide 221234 was described [13]. Three monoclonal antibodies directed against HTLV-I protease fused to a maltose binding protein were developed, with two directed against the N-terminal 116 and one was specific to residues 3544 [26]. None are commercially available.
Related Peptidases Of the retroviral proteases, the bovine leukemia virus (BLV) protease is most closely related to HTLV-I protease with 32% amino acid identity (Chapter 50). BLV and HTLV-I/II/III/IV are members of the deltaretrovirus genus of the Retroviridae family. Although BLV protease showed broader substrate specificity than HTLV-I protease, the specificity of individual subsites of BLV protease resembles that of HTLV-I protease more closely than HIV-1 protease (lentivirus genus) [10].
Further Reading The substrate specificity of HTLV-I and several retroviral proteases was compared by To¨zse´r [10]. The structural X-ray crystal models of HTLV-I and other retroviral proteases were superposed by Li et al. [7]. The amino acid specificity at each subsite can be summarized from the work by Ka´das et al. [18], Bang et al. [15] and Kimura et al. [16]. The refined X-ray crystal structures of the protease in complex with several inhibitors were solved by Satoh et al. [8].
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[17] Nguyen, J.-T., Kiso, Y. (2009). Rational drug design of HTLV-I protease inhibitors, in: Viral Proteases and Antiviral Protease Inhibitor Therapy, Proteases in Biology and Disease, Vol. 8, Lendeckel, U., Hooper, N.M., eds., Dordretch: Springer Science, pp. 83100. [18] Ka´das, J., Weber, I.T., Bagossi, P., Miklo´ssy, G., Boross, P., Oroszlan, S., To¨zse´r, J. (2004). Narrow substrate specificity and sensitivity toward ligand-binding site mutations of human T-cell leukemia virus type 1 protease. J. Biol. Chem. 279(26), 2714827157. [19] To¨zse´r, J., Zahuczky, G., Bagossi, P., Louis, J.M., Copeland, T.D., Oroszlan, S., Harrison, R.W., Weber, I.T. (2000). Comparison of the substrate specificity of the human T-cell leukemia virus and human immunodeficiency virus proteinases. Eur. J. Biochem. 267(20), 62876295. [20] Nguyen, J.-T., Kato, K., Kumada, H.-O., Hidaka, K., Kimura, T., Kiso, Y. (2011). Maintaining potent HTLV-I protease inhibition without the P3-cap moiety in small tetrapeptidic inhibitors. Bioorg. Med. Chem. Lett. 21(6), 18321837. [21] Herger, B.E., Mariani, V.L., Dennison, K.J., Shuker, S.B. (2004). The 10 C-terminal residues of HTLV-I protease are not necessary for enzymatic activity. Biochem. Biophys. Res. Commun. 320(4), 13061308. [22] Ka´das, J., Boross, P., Weber, I.T., Bagossi, P., Matu´z, K., T To¨zse´r, J. (2008). C-Terminal residues of mature human T-lymphotropic virus type 1 protease are critical for dimerization and catalytic activity. Biochem. J. 416(3), 357364. [23] Izumo, S. (2010). Neuropathology of HTLV-1-associated meylopathy (HAM/TSP). Neuropathology 30(5), 480485. [24] Kannian, P., Green, P.L. (2010). Human T lymphotropic virus type 1 (HTLV-1): Molecular biology and oncogenesis. Viruses 2(9), 20372077. [25] Hayakawa, T., Misumi, Y., Kobayashi, M., Ohi, Y., Fujisawa, Y., Kakinuma, A., Hatanaka, M. (1991). Expression of human T-cell leukemia virus type 1 protease in Escherichia coli. Biochem. Biophys. Res. Commun. 181(3), 12811287. [26] Mamoun, R.Z., Dye, D., Rebeyrotte, N., Bouamr, Fadila, Cerutti, M., Desgranges, C. (1997). Mouse monoclonal antibodies directed against the HTLV-I protease recognize epitopes internal to the dimer. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 14(2), 184188.
Jeffrey-Tri Nguyen Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan. Email: [email protected]
Yoshiaki Kiso Laboratory of Peptide Science, Nagahama Institute of Bio-Science & Technology, Nagahama, Shiga 526-0829, Japan. Email: [email protected] This article is a revision of the previous edition article by S. Daenke, Volume 1, pp. 166169, r 2004, Elsevier Ltd. Handbook of Proteolytic Enzymes, 3rd Edn ISBN: 978-0-12-382219-2
© 2013 Elsevier Ltd. All rights reserved. DOI: http://dx.doi.org/10.1016/B978-0-12-382219-2.00049-1