Actin, Troponin C, Alzheimer Amyloid Precursor Protein and Pro ...

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Human Immunodeficiency Virus*. (Received for publication, February 22, 1991). Alfred0 G. Tomasselli, John 0. Hui, Lonnie Adams, John Chosay, David Lowery, ...
Vol . 266, No. 22, Issue of August 5, pp. 1454&14553,1991 Printed in U.S. A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Actin, TroponinC, Alzheimer Amyloid PrecursorProtein and Pro-interleukin 18 as Substrates of the Protease from Human Immunodeficiency Virus* (Received for publication, February 22, 1991)

Alfred0 G. Tomasselli, John 0.Hui, Lonnie Adams, John Chosay, David Lowery,Barry Greenberg, Anthony Yem, Martin R. Deibel, Heidi Ziircher-Neely, and RobertL. HeinriksonS From Discovery Research, Upjohn Laboratories, The Upjohn Company,Kalumazoo, Michigan 49001

We show here for the first time that actin, troponin1989; Wlodawer et al., 1989), and this feature provides an C, Alzheimer amyloid precursor protein (AAP), and attractive mechanism for regulating the activity of the viral pro-interleukin 18 (pro-IL-lo), are substrates of the protease. An active dimer implies that two polyproteins, each protease encoded by the human immunodeficiency vi- containinga single protease chain,must come into close rus (HIV) type-1. As has been seen in other non-viral enough proximity to allow dimerization and initiation of the protein substratesof the HIV protease, thepresence of maturation process. The most likely location for such an Glu residues in the Pz, position appears to play an interaction is the budded, spherical particle where the polyimportant role in substrate recognition. Three of the proteins are most concentrated. Although this mode of regufour bonds cleaved in actin, two of the three in troponin lation of retroviral protease activity is appealing and reasonC, and allof the bonds hydrolyzed in AAP and pro-ILit is lacking in experimental support. Moreover, little is 18 have a ParGlu residue. In fact, Glu residues are able, known about what happens to the protease after it has comaccommodated in allpositions fromP4to P4,surrounding thescissile bond in substratesof the HIV proteases, pleted processing duringmaturationandpost-maturation and as many as 4 adjacent Glu residues were seen in phases of the viral life cycle (Roberts and Oroszlan, 1990) one of the bonds cleaved in AAP. This study of non- and, more specifically,whether it can ever have access to nonviral protein substrates has also revealedunexpected viral proteins as substrates.This latterpossibility raises quesamino acids such as Gly, Arg, and Glu in the scissile tions with regard to ramifications of retroviral diseases which breakdown of needed host proteins, or bond itself rather than the more conventional hydro- have as their basis the generation of bioactive and, possibly, immunomodulatory phobic amino acids. The HIV-2 protease hydrolyzed actin ina manner similar to that of the HIV-1 enzyme, fragments. Despite the fact that many aspects pertaining to the actibut its cleavage of troponin C was distinct in that it split a bond adjacent to a triplet of Glu residues inPz, vationandsubsequentfate of retroviral proteases remain P3, and P4 that was refractory to the HIV-1 enzyme. conjectural, it is clear that these enzymes are indispensable Documentation of cleavage sites in the several impor-for generation of infectious virus (Kohl et al., 1988). For this tant cellular proteins noted above has extended our reason, the protease from human immunodeficiency virus understanding of the features in a substrate that are (HIV)’ has been targeted for development of inhibitors that recognized by these. multi sub-site proteases of retro- might serve in the treatment of acquired immunodeficiency viral maturation. Moreover, the present work adds to syndrome (AIDS). The recent literature contains numerous an accumulating body of evidence which demonstrates references to work in this area, and considerable success has that these enzymes can damage crucial structural and been achieved in a variety of laboratories in synthesizing regulatory cellular proteins if ever their activity is peptidomimetic compounds with potentantiviral activity expressed outsidethe viral particle itself. (McQuade et al., 1990; Meek et al., 1990; Roberts et al., 1990; Ashorn et al., 1990). Our major research effort has been directed toward questions regarding the specificity of the HIV proteaseb) and the The protease encoded within the pol gene of retroviruses is indispensable for processing of the viral gag and gaglpol susceptibility of host proteins to hydrolysis by these enzymes (Tomasselli et al., 1990a, 1990b, 1991). Documentation of polyproteins during the final stages of maturation. This matcleavage sites in non-viralproteins such as Pseudomonas uration process, in which the protease also releases itself from exotoxin (Tomasselli et al., 1990a,1990b), ribonuclease A the polyprotein format, would appear to be required to take (Hui et al., 1990), and calmodulin (Tomasselli et al., 1991) has place outside the infected cell within the newly budded imadded to the database of specificity derived from knowledge mature virus. Any premature activation of the protease prior of cleavage patterns in the retroviral polyproteins themselves. to exocytosis from the host cell could yield a particle with an This information should, in turn, translate into designof incomplete set of structural proteins andenzymes needed in better inhibitors as therapeutic agents in treatmentof AIDS. order to produce the mature, infectious virus. The retroviral A second major objective, consistent with the first, is to proteases are unique among proteolytic enzymes in that they document host proteins that can serve as substrates of the are obligate dimers (cf. Pearl and Taylor, 1987; Miller et al., HIV proteases. Shoeman et al. (1990) have shown that vimentin, desmin, and the glial fibrillary acidic protein are broken * The costs of publication of this article were defrayed in part by down by the HIV-1 protease, and Wallin et al. (1990) docuthe payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed.

The abbreviations usedare: HIV, human immunodeficiency virus; AAP, Alzheimer amyloid precursor.

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Non-viral Substrates Protein mented cleavage by the protease of microtubule-associated proteins. This information, coupled with that provided through our work with calmodulin (Tomasselli et al., 1991), underscores a concern that expression of protease activity outside of the viral particle could influence the course of the disease in entirely unsuspected ways. The present paper describes the course of cleavage by the HIV protease of actin, troponinC, Alzheimer amyloid precursor protein (AAP), and pro-interleukin 1p (pro-IL-lp). An indication that actin might be a substrate came from studies of HIV protease digests of mononuclear cell extracts. The other proteins were explored because of their multidomain structure and their importance as intra- and extracellular mediators in metabolic pathways. Results presented herein provide new information about protease specificity and add to the list of important regulatory proteins of the host that are susceptible to breakdown in HIV infection. MATERIALS A N D METHODS A N D RESULTS’

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TABLEIV Some unusual features of HIVprotease specificity revealed through study of non-viral protein substrates Cleavage site

Protein substrate

Actin Ser-Phe-Ile-Gly-1-Met-Glu-Ser-Ala AAP Leu-Pro-Val-Asn-1-Gly-Glu-Phe-Ser B chain (HIVT y r - L e u - V a l - C y s - ~ - G l y - G l u - A r g - GInsulin ly 2 protease only) CAM Ribonuclease A Ile-Ile-Val-Ala-1-Cys-Glu-Gly-Asn CAM

Troponin C (HIV-1 Ala-Glu-Cys-Phe-1-Arg-Ile-Phe-Asp protease only) Arg-Glu-Ala-Phe-1-Arg-Val-Phe-AspCalmodulin (HIV-1 protease only) Insulin B chain His-Leu-Val-Glu-1-Ala-Leu-Tyr-Leu V a l - G l u - V a l - A l a - ~ - G 1 u - G l u - G l u - GAAP lu pro-IL-16 Pro-Phe-Ile-Phe-1-Glu-Glu-Glu-Pro pro-IL-lp Asp-Asp-Leu-Phe-1-Phe-Glu-Ala-Asp Troponin C (HIV-2 Glu-Glu-Glu-Leu-1-Ala-Glu-Cys-Phe protease only) Troponin C Ala-Glu-Glu-Leu-j,-Ala-Glu-Ile-Phe

DISCUSSION

The objective of the present work was 2-fold to expand the data base relative to sequences recognized and cleaved by the HIV proteases, and to examine as substrates, host proteins that play distinctive roles in health and disease. With regard to the first goal, knowledge derived from inspection of cleavage sites in viral polyproteins alone (Darke et al.,1988; Henderson et al.,1988; Tozser etal., 1991)provides only a limited view of the capabilities of either the HIV-1 or the HIV-2 enzyme. Moreover, it might be argued that a narrow focus on the naturalprocessing sites introduces a bias, in that specificity for these bonds is geared to viral survival. That is, the virus has evolved to produce polyproteins which must remain stable within the host cell so that they can escape, intact,intotheimmature virion, and only then undergo processing by the HIV protease. This could be why we see so many X-Pro bonds, sites refractive to most proteolytic enzymes, in the processing sites of the viral polyproteins. We have never seen such a bond cleaved in the non-viral protein substrates. It is reasonable that the viral polyproteins are folded into a multiprotein array that would be resistant to proteolysis at regions other than those connecting each protein unit, and it could be important that these segments be inert to proteases other than the viral enzyme. Whatever the case, the processing sites reflect information having to do with folding and function above and beyond that necessarily needed to explain the specificity of the HIV protease. It is for this reason that we have pursued a more classical line of inquiry into the enzyme specificity using protein substrates where one is looking at theenzyme’spreferences for sequence/ conformation pure and simple. Unique, and heretofore unexpected aspects of enzyme specificity that have been revealed by our studies of non-viral protein substrates are highlighted in Table IV. Here we see an array of amino acids participating in bonds cleaved by the protease that would not be predicted from studies of viral polyproteins. These include glycine, glutamic acid, and Scarboxamidomethylcysteine at P1 or P18, and arginine and aspartic acid, seen thus far only at P1,.Conversion of Lys-7 of ribonuclease A to the succinyl derivative makes the bond linking it to Ala-8 susceptible to hydrolysis (Hui et al., 1990). Therefore, charged amino acids can occupy either position of Portions of this paper (including “Materials and Methods,” “Results,” Figs. 1-4 and Tables 1-111) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

the actual cleavage site without seriously altering the rate of hydrolysis. This is in marked contrast to the situation with viral polyprotein substrates in which the natural cleavage positions usually have aromatic or aliphatic amino acids in P1 and P18(Henderson et al., 1988). Looking further out in either direction from the scissile bond, we find a preponderance of Glu residues, sometimes in pairsor even in triplets adjacent to the site of cleavage. Inspection of the crystalstructure of the HIV-1 protease reveals positive charges that can accommodate the negative charges of Glu residues, but not sufficient to explain binding of substrates with multiple glutamic acids. Nor can pHeffects alone account for this preference for Glu residues. It is noteworthy that thepolyGlu site cleaved in AAP at pH 5.5 is not cleaved at pH 7.0. However, such was not the case for a similar bond in pro-IL-lp (Table I). Therefore, the lack of cleavage of AAP at pH 7.0 is probably due to a conformational change in this range of pH which leads to a folding pattern no longer recognized by the protease. A notable exception among the charged residues tolerated by the protease at PI and P18is the otherwise abundant amino acid lysine. Lysine seems to be abhorred in any position from Ppto P,, and although a PBLys has been noted in one of the viral polyprotein cleavage sites (Henderson et al., 1988), and it has been inferred to exist at both P2 and Pp,relative to bonds cleaved in the nucleocapsid proteins(Robertsand Oroszlan, 1990), we have never seen a lysine within 4 residues of the bond hydrolyzed. Interestingly, Partin et al. (1990) reported that substitution of lysine for either the P2 or Ps, residue surrounding the scissile Tyr-Pro bond at thegag MA/ CA junction (. . . SQNYPIVQ . . .) prevented cleavage. Clearly, the results generated thus far with non-viral protein substrates present a more complex picture of specificity for these aspartyl proteases which display a multipoint attachment in their binding of substrates.Further work is needed to show whether the observations reported herein can be incorporated into a strategyfor design of inhibitors of the HIV protease which may find therapeutic application in the treatment of AIDS. However, a statistical analysis of all the sites of cleavage documented thus far both in viral and nonviral proteins has been undertaken in the companion article to this paper (Poormanet al.,1991) with the aim of being able to understand in greater detail the preferences shown by the HIV-1 and -2 proteases for amino acids in each of the eight sites from P4to P4,which flank the scissile bond. The second goal of the research described in this paper has

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to do with discovery of proteins related to cell function or disease which are subject to hydrolysis by the protease. Our earlier demonstration that calmodulin is a substrate of the HIV proteases (Tomasselli et ai., 1991) is now bolstered by a list of important structural and regulatory proteins that are susceptible to hydrolysis by these enzymes. These findings have causedus toexpress concern about the consequencesof premature activation of the HIV protease within the host cell. Experimental support for such an idea is now beginning to emerge. Kaplan and Swanstrom (1991) have demonstrated that the HIV protease can process viral polyproteins within the cytoplasm of the infected cell, and they concluded that the enzyme may well cleave cellular proteins and thus contribute to c~otoxicity.Moreover, RiviGre et ai. (1991) have reported the processing bythe HIV-1 protease of the precursors of NF-KB,a host DNA-binding protein, in acutely infected human T cells. These results add an important dimension to the findings reported herein that proteolysis of actin, troponin C, AAP, and pro-interleukin I@could also take place and constitute a serious assault on the host cell and organism. Considered together, these observations make the protease an even more attractive target for HIV therapy. Acknowledgments-We thank Drs. Charles S. Craik and Diane L. De Camp of the University of California, San Francisco for the HIV2 protease, Drs. Don Carter and Che-Shen C. Tomich of Upjohn Laboratories for their role in providing recombinant HIV-1 protease, IL-lP and pro-IL-lp,and P. Gonzalez-DeWhitt for the AAP derivatives. We are also indebted to Paula Lupina and Peg Kornacker for help in preparation of the manuscript. REFERENCES Adams, L. D. (1987) in Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl,K., eds) pp. 10.3.1-10.3.12, John Wiley & Sons, New York Arai, H., Lee, V. M.-Y., Greenberg, B. D., Lowery, D. E., Sharma, S. K., Schmidt, M. L., and Trojanowski, J. Q. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2249-2253 Ashorn, P.,McQuade, T. J., Thaisrivongs, S., Tomasselli, A. C., Tarpley, W. G., and Moss, B. (1990) Proc. Natl. h a d . Sei. U. S. A. 87,7472-7476 Carter, D. B., Curry, K. A., Tomich, C.-S. C., Yem, A. W., Deibel, M. R., Tracey, D.E., Paslay, 3. W., Carter, J. B., Theriault, N.Y., Harris, P. K. W., Reardon, I. M., Ziircher-Neely, H. A., Heinrikson, R.L., Clancy, 1,. L., Muchmore, S. W., Watenpaugh, K. D., and Einspahr, H. M. (1988) Proteins Struct. Funct. Genet. 3, 121-129 Collins, J. H., and Elzinga, M. (1975) J. Biol. Chem.250,5915-5920 Darke, P. L., Nutt, R. F., Brady, S. F., Garsky, V. M., Ciccarone, T. M., Leu, C.-T., Lumma, P. K., Freidinger, R. M., Veber, D. F., and Sigal, I. S. (1988) Biochem. Biophys. Res. Commun. 156,297-303 Hanauer, A., Levin, M., Heilig, R., Daegelen, D., Kahn, A., and Mandel, d. L. (1983) Nucleic Acid Res. 11, 3503-3516 Hazuda, D. J., Webb, R. L., Simon, P., and Young, P. (1989) J. Biol. Chem. 264,1689-1693 Henderson, L. E., Benveniste, R. E., Sowder, R., Copeland, T. D., Schultz, A. M., and Oroszlan, S. (1988) J. Virol. 6 2 , 2587-2595 Hui, J. O., Tomasselli, A. G., Zurcher-Neely, H. A., and Heinrikson, R. L.(1990) J. Biol. Chern. 265,21386-21389 Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K.-H., Multhaup, G., Beyreuther, K., and Muller-

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Hill, B. (1987) Nature 325, 733-736 Kaplan, A., and Swanstrom, R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,4528-4532 Kiaguchi, N.,Takahashi, Y., Tokushima, Y., Shiojiri, S., and Ito, H. (1988) Nature 331,530-532 Kohl, N. E., Emini, E. A., Schieif, W. A., Davis, L. J., Heimbach, J. C., Dixon, R. A. F., Scolnick, E. M., and Sigal, 1. S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4686-4690 Laemmli, U. K. (1970) Nature 227,680-685 Lowery, D. E., Gonzalez-DeWhitt, P. A., Tomich, C.-S. C., Altmen, R. A., and Greenberg, B. D. (1991) J. Biol. Chem., in press Lu, R. C., and Elzinga, M. (1977) Biochemistry 16,5801-5806 March, C. J., Mosley, B., Larsen, A., Cerretti, D. P., Braedt, G., Price, V., Cillis, S., Henney, C. S., Krohneim, S. R., Grabstein, K., Conlon, P. J., Hopp, T. P., and Cosman, D. (1985) Nature 315,641-647 McQuade, T. J., Tomasselli, A. G., Liu, L., Karacostas, V., Moss, B., Sawyer, T. K., Heinrikson, R. L., and Tarpley, W. G. (1990)Science 247,454-456 Meek, T. D., Lambert, D.M., Dreyer, G. B., Carr, J. J., Tomaszek, T. A., Jr., Moore, M. L., Strickler, J. E., Debouck, C., Hyland, L. J., Matthews, T. J., Metcalf, B. W., and Petteway, S. R. (1990) Nature 343,90-92 Miller, M., Jaskolski, M., Rao, J. K. M., Leis, J., and Wlodawer, A. (1989) Nature 337, 576-579 Partin, K., Krausslich, H.-G., Ehrlich, L., Wimmer, E., and Carter, C. (1990) J. Virol. 64, 3938-3947 Pearl, L. H., and Taylor, W. R. (1987) Nature 329,351-354 Pichuantes, S., Babb, L. M., Barr, P. J., Decamp, D. L., and Craik, C. S. (1990) J. Biol. Chem. 265, 13890-13898 Poorman, R. A., and KBzdy, F. J. (1991) Nature 350,625-626 Poorman, R. A., Tomasselli, A. G., Heinrikson, R. L., and Kbzdy, F. J. (1991) J. Biol. Chem. 265,14554-14561 RiviGre,Y., Blank, V., Kourilsky, P., and Israel, A. (1991) Nature 350,625-626 Roberts, M. M., and Oroszlan, S. (1990)in Retroviral Proteases(Pearl, L. H., ed) pp. 131-139, Macmillan Press Ltd., London Roberts, N. A., Martin, J. A., K i n c h i n ~ o nD., , ~roadhurst,A.V., Craig, J. C., Duncan, I. B., Galpin, S. A., Handa, B. K., Kay, J., Krohn, A., Lambert, R. W., Merrett, J. H., Mills, J. S., Parkes, K. E. B., Redshaw, S., Ritchie, A. J., Taylor, D. L., Thomas, G. J., and Machin, P. J. (1990) Science 248,358-361 Shoeman, R. L., Honer, B., Stoller, T. J., Kesselmeier, C., Miedel, M. C., Traub, P., and Graves, M. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87,6336-6340 Tomasselli, A. G., Hui, J. O., Sawyer, T. K., Staples, D. J., Bannow, C., Reardon, I. M., Howe, W. J., Decamp, D. L., Craik, C. S., and Heinrikson, R. L. (1990a) J. Biol. Chem. 265, 14675-14683 Tomasselli, A. G., Hui, 3. O., Sawyer, T. K., Staples, D. J., FitzGerald, D. J., Chaudhary, V. K., Pastan, I., and Heinrikson, R. L. (199Ob) J. Biol. Chem. 265,408-413 Tomasselli, A. G., Olsen, M. K., Hui, J., Staples, D. J., Sawyer, T. K., Heinrikson, R. L., and Tomich, C.-S. C. (1990~)Biochem~~ry 29,264-269 Tomasselli, A. G., Howe, W. J,, Hui, J. O., Sawyer, T. K., Reardon, I. M., Decamp, D. L., Craik, C. S., and Heinrikson, R. L. (1991) Proteins Struct. Funct. Genet., in press Tozser, J., Blaha, I., Copeland, T. D., Wondrak, E. M., and Oroszlan, S. (1991) FEBS Lett. 281, 77-80 Vandekerckhove, J., and Weber, K. (1978) Eur. J. Bwchem. 90,451462 Wallin, M., Deinum, J., Goobar, L., and Danielson, U.H. (1990) J. Gen. Virol. 71, 1985-1991 Watterson, D. M., Sharief, F., and Vanaman, T. C. (1980) J. Biol. Chem. 255,962-975 Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B. K., Baldwin, E., Weber, I. T., Selk, L. M., Clawson, L., Schneider, J., and Kent, S. B. H. (1989) Science 245,616-621 Yem, A. W., Richard, K.A., Staite, N. D., and Deibel, M. R., Jr. ~ ~ 7,85-92 ne (1988) ~ y m ~ h o Res.

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Non-viral Protein Substrates of HIV Protease SUPPLEMENTAL MATERIALTO: ACTIN,TROPONIN C. ALZHEIMER AMYLOID PRECURSORPROTEIN AND PRO-INTERLEUKIN SUBSTRATESOF THE PROTEASE FROM HUMAN IMMUNODEFICIENCYVIRUS

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by Alfred0 G Tomarrella. John 0 Hul. Lonnle Adam$, John Choray. Davld Lowery. Barry Greenberg. Anthony Yem. Man," R. Oelbel, Held, Zurcher-Neely. and Robert L Helnrlkson MATERIALSANDMETHODS

mer and roteln substrates- Recombonant HlWl and HIW2 protearer were exprerred m €scher;chya ro1iand:eart. rerpemvely. they were purlfled. refolded and characterized ar dercrlbed earlier (Tomarre118 e t al.. 1990~.Pichuanter e1 d l . . 1990) Rabblt muscle a m n and troponin were En2

purchased from Sqma Chemlcal Co.. St. Lou#$. MO The nomenclature employed wnh reference to a d m fragments is that taken from the sequence a n a l y ~byCallhnr and Elnnga 11975) wrth the ~ n r e r t ~ oof n a serine a t 234 t h a t w a r r h o w n later to be present by both proteln (Lu and Elzmga. 1977, Vandekerckhave and Weber. 1978) and