View source for Biology:Early 35 kDa protein

{{Short description|Anti-apoptotic viral protein}}
{{Infobox nonhuman protein
| Name = Early 35 kDa protein (AcMNPV)
| image = P35 structure by Fisher 1999, PDB 1P35.jpg
| caption = P35 structure by Fisher ''et al.'' 1999<ref name=Fisher1999>{{cite journal | vauthors = Fisher AJ, Cruz W, Zoog SJ, Schneider CL, Friesen PD | title = Crystal structure of baculovirus P35: role of a novel reactive site loop in apoptotic caspase inhibition | journal = The EMBO Journal | volume = 18 | issue = 8 | pages = 2031–9 | date = April 1999 | pmid = 10205157 | pmc = 1171287 | doi = 10.1093/emboj/18.8.2031 }}</ref>
| Organism = Autographa californica nuclear polyhedrosis virus (AcMNPV)
| TaxID = 46015
| Symbol = P35
| EntrezGene = 1403968
| PDB = 1P35
| RefSeqmRNA = NC_001623.1
| RefSeqProtein = NP_054165.1
| UniProt = P08160
| Chromosome = 0
| EntrezChromosome = NC_001623.1
| GenLoc_start = 116492
| GenLoc_end = 117391
}}
{{Infobox nonhuman protein
| Name = Early 35 kDa protein (BmNPV)
| Organism = Bombyx mori nuclear polyhedrosis virus (BmNPV)
| TaxID = 271108
| Symbol = P35
| EntrezGene = 1488744
| RefSeqmRNA = NC_001962.1
| RefSeqProtein = NP_047533.1
| UniProt = P31354
| Chromosome = 0
| EntrezChromosome = NC_001962.1
| GenLoc_start = 105923
| GenLoc_end = 106822
}}
The '''Early 35 kDa protein''', or '''P35''' in short, is a baculoviral protein that inhibits [[Biology:Apoptosis|apoptosis]] in the cells infected by the virus. Although baculoviruses infect only [[Biology:Invertebrate|invertebrate]]s in nature, [[Biology:Ectopic expression|ectopic expression]] of P35 in [[Biology:Vertebrate|vertebrate]] animals and cells also results in inhibition of apoptosis, thus indicating a universal mechanism. P35 has been shown to be a [[Biology:Caspase|caspase]] inhibitor with a very wide spectrum of activity both in regard to inhibited caspase types and to species in which the mechanism is conserved.

==Species distribution==
P35 has been found in different strains of the [[Biology:Nuclear polyhedrosis virus|nuclear polyhedrosis virus]], a species of baculovirus that infects insects. Two [[Biology:Homology#Orthology|orthologs]] of P35 that have been studied in detail are the ones from the ''[[Biology:Autographa californica|Autographa californica]]'' multicapsid nuclear polyhedrosis virus (AcMNPV) and from the ''[[Biology:Bombyx mori|Bombyx mori]]'' nuclear polyhedrosis virus (BmNPV). The P35 ortholog from AcMNPV was found to block apoptosis in mammalian cells much more efficiently as compared to the ortholog from BmNPV.<ref name=Morishima1998>{{cite journal | vauthors = Morishima N, Okano K, Shibata T, Maeda S | title = Homologous p35 proteins of baculoviruses show distinctive anti-apoptotic activities which correlate with the apoptosis-inducing activity of each virus | journal = FEBS Letters | volume = 427 | issue = 1 | pages = 144–8 | date = May 1998 | pmid = 9613616 | doi = 10.1016/S0014-5793(98)00389-5 | s2cid = 29840073 | doi-access =  }}</ref>

== Function ==
The P35 protein inhibits apoptosis by acting as a [[Chemistry:Competitive inhibition|competitive]], [[Biology:Enzyme inhibitor#Irreversible inhibitors|irreversible inhibitor]] of caspases.<ref name="Bertin1996"/> P35 first serves as a caspase substrate and is cleaved between the amino acids D87 and G88, i.e. after the sequence DQMD in P35 from AcMNPV and after the sequence DKID in P35 from BmNPV, resulting in two polypeptide products of about 10 kDa and 25 kDa in size.<ref name=Bertin1996>{{cite journal | vauthors = Bertin J, Mendrysa SM, LaCount DJ, Gaur S, Krebs JF, Armstrong RC, Tomaselli KJ, Friesen PD | title = Apoptotic suppression by baculovirus P35 involves cleavage by and inhibition of a virus-induced CED-3/ICE-like protease | journal = Journal of Virology | volume = 70 | issue = 9 | pages = 6251–9 | date = September 1996 | doi = 10.1128/jvi.70.9.6251-6259.1996 | pmid = 8709252 | pmc = 190650 }}</ref> The cleavage site is situated in a solvent-exposed loop that extends from the protein's beta sheet core, thus ensuring good accessibility to the caspase.<ref name=Fisher1999/><ref name=Zoog1999>{{cite journal | vauthors = Zoog SJ, Bertin J, Friesen PD | title = Caspase inhibition by baculovirus P35 requires interaction between the reactive site loop and the beta-sheet core | journal = The Journal of Biological Chemistry | volume = 274 | issue = 37 | pages = 25995–6002 | date = September 1999 | pmid = 10473544 | doi = 10.1074/jbc.274.37.25995 | url = http://www.jbc.org/content/274/37/25995 | doi-access = free }}</ref> However, unlike other caspase substrate proteins, the fragments of P35 do not dissociate from the caspase after cleavage. Instead, the N-terminal, 10 kDa cleavage fragment remains bound to the caspase by a covalent, stable [[Biology:Thioester|thioester]] bond between the cleavage residue D87 of P35 and the cysteine residue at the active site of the caspase.<ref name=Xu2001>{{cite journal | vauthors = Xu G, Cirilli M, Huang Y, Rich RL, Myszka DG, Wu H | title = Covalent inhibition revealed by the crystal structure of the caspase-8/p35 complex | journal = Nature | volume = 410 | issue = 6827 | pages = 494–7 | date = March 2001 | pmid = 11260720 | doi = 10.1038/35068604 | s2cid = 279649 }}</ref>

While the formation of a thioester intermediate between the aspartate of the substrate's recognition site and the cysteine of the caspase's active site is a normal event in caspase-mediated protein cleavage, the resulting bond is normally quickly hydrolysed so that the cleaved products can detach. In the case of P35, however, the caspase-substrate complex remains stable. Cleavage of P35 triggers rapid conformational changes that reposition its N-terminus, which is normally buried in the protein's beta-sheet core, to the caspase's active site. As a consequence of this rearrangement, the N-terminal P35 residues C2 and V3 interact with the caspase's active site to displace water and prevent the hydrolysis reaction. The P35 residue C2 competes with the caspase's active site cysteine residue for binding of the P35 residue D87, keeping the reaction trapped in an equilibrium state.<ref name=Xu2001/><ref>{{cite journal | vauthors = Riedl SJ, Renatus M, Snipas SJ, Salvesen GS | title = Mechanism-based inactivation of caspases by the apoptotic suppressor p35 | journal = Biochemistry | volume = 40 | issue = 44 | pages = 13274–80 | date = November 2001 | pmid = 11683637 | doi = 10.1021/bi010574w }}</ref><ref name=Xu2003>{{cite journal | vauthors = Xu G, Rich RL, Steegborn C, Min T, Huang Y, Myszka DG, Wu H | title = Mutational analyses of the p35-caspase interaction. A bowstring kinetic model of caspase inhibition by p35 | journal = The Journal of Biological Chemistry | volume = 278 | issue = 7 | pages = 5455–61 | date = February 2003 | pmid = 12458208 | doi = 10.1074/jbc.M211607200 | doi-access = free }}</ref><ref name=Lu2006>{{cite journal | vauthors = Lu M, Min T, Eliezer D, Wu H | title = Native chemical ligation in covalent caspase inhibition by p35 | journal = Chemistry & Biology | volume = 13 | issue = 2 | pages = 117–22 | date = February 2006 | pmid = 16492559 | doi = 10.1016/j.chembiol.2005.12.007 | doi-access =  }}</ref>

== Interactions ==

In insect cells, P35 inhibits an enzyme called [[Biology:Sf caspase-1|Sf caspase-1]], which was identified as a structural and functional ortholog of human CASP3 (CPP32) and CASP7 (MCH3).<ref name=Ahmad1997>{{cite journal | vauthors = Ahmad M, Srinivasula SM, Wang L, Litwack G, Fernandes-Alnemri T, Alnemri ES | title = Spodoptera frugiperda caspase-1, a novel insect death protease that cleaves the nuclear immunophilin FKBP46, is the target of the baculovirus antiapoptotic protein p35 | journal = The Journal of Biological Chemistry | volume = 272 | issue = 3 | pages = 1421–4 | date = January 1997 | pmid = 8999805 | doi = 10.1074/jbc.272.3.1421 | doi-access = free }}</ref> Studies using purified human caspases in ''vitro'' found that the protein is able to also inhibit several of these, including CASP1, CASP3, CASP6, CASP7, CASP8, and CASP10.<ref name=Zhou1998>{{cite journal | vauthors = Zhou Q, Krebs JF, Snipas SJ, Price A, Alnemri ES, Tomaselli KJ, Salvesen GS | title = Interaction of the baculovirus anti-apoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex | journal = Biochemistry | volume = 37 | issue = 30 | pages = 10757–65 | date = July 1998 | pmid = 9692966 | doi = 10.1021/bi980893w }}</ref>

==Clinical significance==
Since baculoviridae infect only insects and not humans, the function of P35 in the immune evasion of infected cells is not clinically relevant. However, P35 has been considered as a potential tool in [[Biology:Gene therapy|gene therapy]] to suppress apoptosis where it is not wanted, such as in the protection of [[Medicine:Organ transplantation|transplanted tissue]] from immune rejection or in the killing of [[Biology:Bystander effect (radiobiology)|bystander cells]] in cancer therapy; such methods are still far from clinical application though.<ref>{{cite journal | vauthors = Doloff JC, Su T, Waxman DJ | title = Adenoviral delivery of pan-caspase inhibitor p35 enhances bystander killing by P450 gene-directed enzyme prodrug therapy using cyclophosphamide+ | journal = BMC Cancer | volume = 10 | pages = 487 | date = September 2010 | pmid = 20836875 | pmc = 2946310 | doi = 10.1186/1471-2407-10-487 | doi-access = free }}</ref>

==History and discovery==
The role of P35 in the inhibition of apoptosis was first described by Rollie J. Clem in the research group of Lois K. Miller at the Department of Genetics at the [[Organization:University of Georgia|University of Georgia]] in 1991.<ref name=Clem1991>{{cite journal | vauthors = Clem RJ, Fechheimer M, Miller LK | title = Prevention of apoptosis by a baculovirus gene during infection of insect cells | journal = Science | volume = 254 | issue = 5036 | pages = 1388–90 | date = November 1991 | pmid = 1962198 | doi = 10.1126/science.1962198 | bibcode = 1991Sci...254.1388C }}</ref> Four years later, in 1995, the reason for apoptosis inhibition by P35 was identified as its ability to bind and inhibit caspases (then still called ''ICE homologs'') by Nancy J. Bump and co-workers at the BASF Bioresearch Corporation in Worcester, Massachusetts.<ref name=Bump1998>{{cite journal | vauthors = Bump NJ, Hackett M, Hugunin M, Seshagiri S, Brady K, Chen P, Ferenz C, Franklin S, Ghayur T, Li P | title = Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35 | journal = Science | volume = 269 | issue = 5232 | pages = 1885–8 | date = September 1995 | pmid = 7569933 | doi = 10.1126/science.7569933 | bibcode = 1995Sci...269.1885B }}</ref> The mechanism of caspase inhibition was discovered by Guozhou Xu in the team of Hao Wu at the Department of Biochemistry at Weill Cornell Medical College in 2001.<ref name=Xu2001/>

== References ==
{{reflist|32em}}


[[Category:Apoptosis]]

{{Sourceattribution|Early 35 kDa protein}}