The Early 35 kDa protein, or P35 in short, is a
baculoviral protein that inhibits
apoptosis
Apoptosis (from grc, ἀπόπτωσις, apóptōsis, 'falling off') is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes incl ...
in the cells infected by the virus. Although baculoviruses infect only
invertebrate
Invertebrates are a paraphyletic group of animals that neither possess nor develop a vertebral column (commonly known as a ''backbone'' or ''spine''), derived from the notochord. This is a grouping including all animals apart from the chordate ...
s in nature,
ectopic expression Ectopic is a word used with a prefix, ecto, meaning “out of place.” Ectopic expression is an abnormal gene expression in a cell type, tissue type, or developmental stage in which the gene is not usually expressed. The term ectopic expression is ...
of P35 in
vertebrate
Vertebrates () comprise all animal taxa within the subphylum Vertebrata () ( chordates with backbones), including all mammals, birds, reptiles, amphibians, and fish. Vertebrates represent the overwhelming majority of the phylum Chordata, ...
animals and cells also results in inhibition of apoptosis, thus indicating a universal mechanism. P35 has been shown to be a
caspase
Caspases (cysteine-aspartic proteases, cysteine aspartases or cysteine-dependent aspartate-directed proteases) are a family of protease enzymes playing essential roles in programmed cell death. They are named caspases due to their specific cystei ...
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
nuclear polyhedrosis virus, a species of baculovirus that infects insects. Two
orthologs
Sequence homology is the biological homology between DNA, RNA, or protein sequences, defined in terms of shared ancestry in the evolutionary history of life. Two segments of DNA can have shared ancestry because of three phenomena: either a spec ...
of P35 that have been studied in detail are the ones from the ''
Autographa californica
''Autographa californica'', the alfalfa looper, is a moth of the family Noctuidae. The species was first described by Adolph Speyer in 1875. It is found in western North America from southern British Columbia to Baja California and to Manitoba, ...
'' multicapsid nuclear polyhedrosis virus (AcMNPV) and from the ''
Bombyx mori
The domestic silk moth (''Bombyx mori''), is an insect from the moth family Bombycidae. It is the closest relative of ''Bombyx mandarina'', the wild silk moth. The silkworm is the larva or caterpillar of a silk moth. It is an economically imp ...
'' 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.
Function
The P35 protein inhibits apoptosis by acting as a
competitive
Competition is a rivalry where two or more parties strive for a common goal which cannot be shared: where one's gain is the other's loss (an example of which is a zero-sum game). Competition can arise between entities such as organisms, indivi ...
,
irreversible inhibitor
An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity. Enzymes are proteins that speed up chemical reactions necessary for life, in which substrate molecules are converted into products. An enzyme facilitates a sp ...
of
caspases
Caspases (cysteine-aspartic proteases, cysteine aspartases or cysteine-dependent aspartate-directed proteases) are a family of protease enzymes playing essential roles in programmed cell death. They are named caspases due to their specific cystei ...
.
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.
The cleavage site is situated in a solvent-exposed loop that extends from the protein's
beta sheet
The beta sheet, (β-sheet) (also β-pleated sheet) is a common motif of the regular protein secondary structure. Beta sheets consist of beta strands (β-strands) connected laterally by at least two or three backbone hydrogen bonds, forming a g ...
core, thus ensuring good accessibility to the caspase.
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 thioester
In organic chemistry, thioesters are organosulfur compounds with the functional group . They are analogous to carboxylate esters () with the sulfur in the thioester playing the role of the linking oxygen in the carboxylate ester, as implied by t ...
bond between the cleavage residue D87 of P35 and the cysteine residue at the active site of the caspase.
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.
Interactions
In insect cells, P35 inhibits an enzyme called Sf caspase-1, which was identified as a structural and functional ortholog of human CASP3
Caspase-3 is a caspase protein that interacts with caspase-8 and caspase-9. It is encoded by the ''CASP3'' gene. ''CASP3'' orthologs have been identified in numerous mammals for which complete genome data are available. Unique orthologs are also p ...
(CPP32) and CASP7 (MCH3). Studies using purified human caspases in ''vitro'' found that the protein is able to also inhibit several of these, including CASP1
Caspase-1/Interleukin-1 converting enzyme (ICE) is an evolutionarily conserved enzyme that proteolytically cleaves other proteins, such as the precursors of the inflammatory cytokines interleukin 1β and interleukin 18 as well as the pyroptosis ...
, CASP3, CASP6, CASP7, CASP8
Caspase-8 is a caspase protein, encoded by the ''CASP8'' gene. It most likely acts upon caspase-3.
''CASP8'' orthologs have been identified in numerous mammals for which complete genome data are available. These unique orthologs are also present ...
, and CASP10
Caspase-10 is an enzyme that, in humans, is encoded by the ''CASP10'' gene.
This gene encodes a protein that is a member of the cysteine-aspartic acid protease (caspase) family. Sequential activation of caspases plays a central role in the execut ...
.
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 gene therapy
Gene therapy is a medical field which focuses on the genetic modification of cells to produce a therapeutic effect or the treatment of disease by repairing or reconstructing defective genetic material. The first attempt at modifying human DN ...
to suppress apoptosis where it is not wanted, such as in the protection of transplanted tissue from immune rejection
Transplant rejection occurs when transplanted tissue is rejected by the recipient's immune system, which destroys the transplanted tissue. Transplant rejection can be lessened by determining the molecular similitude between donor and recipient a ...
or in the killing of bystander cells in cancer therapy; such methods are still far from clinical application though.
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 University of Georgia
, mottoeng = "To teach, to serve, and to inquire into the nature of things.""To serve" was later added to the motto without changing the seal; the Latin motto directly translates as "To teach and to inquire into the nature of things."
, establ ...
in 1991. 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
Worcester ( , ) is a city and county seat of Worcester County, Massachusetts, United States. Named after Worcester, England, the city's population was 206,518 at the 2020 United States census, 2020 census, making it the second-List of cities i ...
. 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
The Joan & Sanford I. Weill Medical College of Cornell University is Cornell University's biomedical research unit and medical school located in Upper East Side, Manhattan, New York City, New York.
Weill Cornell Medicine is affiliated with NewY ...
in 2001.[
]
References
{{Viral proteins
Apoptosis
Viral nonstructural proteins