Protein splicing and autoproteolysis mechanisms

292

Protein splicing and autoproteolysis Francine B Perler*t, It has generally

been assumed

inactive

precursors

protein

is mediated numerous

by specific examples

processing

been discovered, of hedgehog enzyme

reactions,

which

functions

ranging

feature

groups.

rearrangements

Addresses *New England

However, rearrangements

of

autoprocessing

in diverse

splicing

biological

to protein

and the generation

their coupling steps

are

to diverse

drives the protein

to completion.

Biolabs

Inc, 32 Tozer Road, Beverly,

Opinion

in Chemical

Biology

1997,

MA 01915, Street,

USA

Boston,

1:292-299

http://biomednet.com/elecref/1367593100100292 0 Current

Biology

Ltd ISSN

1367-5931

Abbreviations C-extein

carboxy-terminal

Hh

hedgehog protein Hh carboxyl domain Hh amino domain Hh amino domain esterified

Hh-C Hh-N Hh-N, HissHh-C

extein

with cholesterol

Hh with all but nine residues

of autoproteolysis. The protein splicing pathway consists of an acyl rearrangement, transesterification, cyclization of an asparagine and a second acyl rearrangement. As of April 1, 1997, 40 putative intein sequences have been deposited in public databases. These sequences derive from 32 genes in 17 different organisms distributed across all three domains of life-eukarya, bacteria, and archaea [l”]. In many cases, inteins are bifunctional molecules which also act as homing endonucleases that can initiate intein gene mobility [l**].

targeting,

of enzyme-bound

such acyl rearrangements

irreversible

and

of a peptide

te-mail: [email protected] *Boston Biomedical Research Institute, 20 Staniford MA 02114, USA; e-mail: [email protected] Current

splicing

The initial formation

unfavorable,

of self-catalyzed

of all

proteins

protein

of all of these

Although

Xu”, and Henry Paulus*

glycosylasparaginases

themselves

from protein

thermodynamically types

including proteins,

precursors.

manifest

activation,

prosthetic

protein

by the acyl rearrangement

bond is a common

proenzyme

active

enzymes.

of self-catalyzed

have recently

an ester bond

that the conversion

to biologically

autoproteolysis and pyruvoyl

Ming-&n

mechanisms

of Hh-N replaced

by six

Autoprocessing of glycosylasparaginases, pyruvoyl enzyme precursors, and hedgehog proteins involves the cleavage of a single peptide bond. Glycosylasparaginase is a member of the amino-terminal nucleophile hydrolase family, which includes glutamine S-ribosyl-1-pyrophosphate (PRPP) amidotransferase, penicillin acylase, and the proteasome. Cleavage of the glycosylasparaginase precursor generates the amino terminus of the P-subunit, which is required for enzyme activity. The self-catalyzed cleavage of pyruvoyldependent enzymes also generates an amino-terminal moiety that is essential for enzymatic activity, except that the amino-terminal amino acid is further modified in the course of the cleavage reaction. The hedgehog proteins are signaling proteins that function in developmental patterning of all multicellular animals, from nematodes to mammals. The processing of hedgehog proteins results in removal of the carboxy-terminal processing domain and the addition of a cholesterol moiety at the carboxyl terminus of the hedgehog protein amino-terminal signaling domain. This cholesterol moiety mediates the association of the signaling domain with cell surfaces.

histidines

MBP N-extein

maltose-binding protein amino-terminal extein

Introduction Until recently, post-translational including processes such as

processing proteolytic

of proteins, activation of

modification of amino acid sidechains by proenzymes, phosphorylation, alkylation, or acylation, and conjugation with prosthetic groups, was thought to be catalyzed by other proteins. Indeed, many enzymes that modify proteins have been characterized. Within the last decade, however, four types of self-catalyzed protein modifications that do not require the intervention of other enzymes have been identified (Figure 1). The most complex of these modifications is protein splicing, which requires the co-ordinated cleavage of two peptide bonds that flank the protein splicing element (the intein) and the ligation of the external protein domains (the exteins) to form a functional protein. The formation of a native peptide bond between the exteins differentiates protein splicing from other forms

Although at first they were considered unrelated, these four examples of protein processing are all initiated by a self-catalyzed N-O or N-S acyl rearrangement. In this review we suggest that such acyl rearrangements may represent a common method of activation of autoproteolysis in diverse biological processes. The ester linkage formed after the acyl rearrangement can provide a reactive site for additions or modifications, thus defining a new method of generation of enzyme-bound prosthetic groups that, in the case of the hedgehog protein, participate in protein targeting. The protein splicing mechanism also involves an uncommon type of asparagine peptide bond cleavage rather

cyclization, which than deamidation.

leads

to

N-S or N-O acyl rearrangements Peptide bonds involving serine, threonine, and cysteine are usually considered to be quite stable. Nevertheless, the sidechains of these amino acids can attack the carbonyl carbon of the preceding amino acid to generate a hydroxyoxazolidine or hydroxythiazolidine intermediate (Figure 2) [2]. This cyclic intermediate can follow one of

Protein

splicing

and autoproteolysis

mechanisms

Perler, Xu and Paulus

293

Figure 1

[a) Protein Splicing

(c) Glycosylasparaginase

Tc C-extein

lntein

N-extein

A

8

HNS

DE

C

Protein C

H

1

a

a

: Excised

exteins

I_ -DE

F

B151T152

T’

HN

K295

-+-

H

FG

p subunit

with

catalytic

site

a subunit

intein

(d) Pyruvoyl enzyme formation

autoprocessing Signaling

domain

K295

Autocleavage

splicing

-+?i

(b) Hedgehog

~15~152 --If

FG

x

Ligated

autocleavage

Autoprocessing domain (Hh-C)

(Hh-N)

-

A + Cholesterol

B471

H 329

C258

-85

i

j

B Autoprocessing

a -85 -Cholesterol

G 257

Signaling protein (Hh-Np)

Autoprocessing of protein precursors. precursors proceed via self-catalyzed

~258 +

H329

B471

7 Non functional cleavage

s’ t

S30 +

F32

Y31

Pyruvoyl-_ a subunit

p subunit

product

Current Owxon

Protein splicing and processing of hedgehog proteins, glycosylasparaginases pathways initiated by an acyl rearrangement at a conserved serine, threonine

on Chemd

Bioloa

and pyruvoyl enzyme or cysteine residue, often

assisted by a conserved histidine. For each process only the initial precursor and final product are depicted. Protein splicing nucleophilic displacements to cleave the peptide bonds flanking the intein and to ligate the exteins (see Figure 3). Hedgehog

requires four proteins undergo

an autoproteolysis reaction that results in addition of cholesterol to the hedgehog signaling domain. This cholesterol anchor is essential for hedgehog protein compartmentalization. Autoproteolysis of glycosylasparaginases generates the amino-terminal threonine required for enzyme activity, whereas autoproteolysis of pyruvoyl enzyme precursors results in the generation of the pyruvoyl moiety. (a) A typical intein. (b) Drosophila hedgehog protein. (c) Flavobacterium meningosepticum glycosylasparaginase. (d) Lacrobacillus 30a prohistidine decarboxylase. Boxes within the intein and hedgehog protein represent conserved motifs; their alphabetic designations and the most important conserved residues (one-letter amino acid code) are indicated above the box.

two fates, depending on whether cyclization is followed by N- or 0-protonation [3]. The latter process yields a cyclic product which, upon dehydration and dehydrogenation, gives rise to a stable oxazole or thiazole ring. This type of reaction is seen in the formation of certain bacterial antibiotics such as microcin B [3], but is not a focus of this review. Rather, we will concentrate on the consequence of N-protonation, which leads to an ester intermediate. Because the equilibrium between amides and esters favors amides at neutral pH [Z], one does not expect to observe significant amounts of the ester under ordinary conditions. Nature, however, has taken advantage of the reactive potential of esters by using them as intermediates in the first step in a broad spectrum of self-catalyzed protein rearrangements. In each of these, an intramolecular acyl transfer sets the stage for a second, irreversible step that drives the protein rearrangement to completion. As outlined in Figure 2, these reactions are quite diverse and involve unrelated catalytic elements.

are indicated

in italics below

the box,

In the case of thioesters, which are highly reactive towards nitrogen nucleophiles such as hydroxylamine and can be displaced by an excess of a thiol, the ester intermediate can be trapped [Z]. This has been the basis for establishing the occurrence autoproteolysis

of ester intermediates in several of these pathways [4”,5*,6**-8**,9,10]. Likewise,

the selective hydrolysis of oxygen esters with alkali [Z] was critical in helping to determine the structure of the protein splicing branched intermediate [ 1 l] and the covalent ester linkage between cholesterol amino domain [ lP].

and

the

hedgehog

protein

Protein splicing The mechanism of protein splicing has been defined using a combination of approaches. Eight conserved intein motifs (Blocks A-H) have been identified (Figure 1) [1’*,13]. Comparisons of intein sequences implicated four conserved splice-junction residues. All inteins begin with serine or cysteine and end in asparagine, and all but three

294

Mechanisms

Figure 2

MICROCIN

B 0

N-C-bH432 R4’

’

$H

\,/

(X = S or 0; aa-XH = Cysteine, Serine,

(X = S or 0)

or Threonine residue) OXAZOLE or THIAZOLE

AMIDE

HYDROXYTHIAZOLIDINE HYDROXYOXAZOLIDINE

or

INTERMEDIATE

(X =o; Threonine instead of serine)

N-S or N-O ACYL Rearrangement RI-!-XH +

0

k&NH-R, I CH,

Current Opinion in Chemfcal Biology

(X = 0)

The role of acyl rearrangements in the initiation of autoprocessing pathways. Although originally thought to be unrelated, the four autoprocessing pathways depicted, and the synthesis of the bacterial antibiotic microcin B, are all initiated by a self-catalyzed acyl rearrangement (see text). The sidechain hydroxyl or thiol group of a conserved serine, threonine or cysteine attacks the carbonyl group of the preceding residue to form a cyclic intermediate. 0-protonation of the cyclic intermediate leads to formation of oxazoles or thiazoles found in bacterial antibiotics whereas N-protonation of the in this addition

prosthetic

have a asparagine. Little,

cyclic intermediate Nature groups,

has protein

preceding an); sequence

in ester four different targeting.

reactive

This ester of irreversibly oxygen

the is obsemed

the exteins, for the first residue the C-extein threonine or and a towards hydrophobic preceding some Intein Block contains a histidine which usually
a veq process, the obsemed only The protein splicing 3) development of in vitro on the Pal-1 intein of extreme thermophile

sulfur atoms

is common the ester

all of

in ester

spp. GB-D revealed a of a intermediate to of the N-extein linked an ester linkage [ 111. This intermediate of serine

autoprocessing to effect are represented

pathways activation, an ‘X’.

Incubation of precursor in migrating species the kinetic intermediate. The migrating two amino corresponding and the [18], with alkali-labile i.e. through structure is analogous to the acyl proteases.

Rlutagenesis data for the Psp Pol-1 intein indicate that only Serl and none of the carboxy-terminal splice junction residues (His536, Asn.537 or Ser538) are required for amino-terminal splice-junction cleavage [-I”]. hlutation of Ser538 blocked branch formation, but not cleavage at either splice junction, suggesting that Ser538 is the branch point. hlutation of Asn537 blocked branch resolution and carboxy-terminal cleavage, whereas mutation

Protein splicing and autoproteolysis

carboxy-terminal

Figure 3

mechanisms Perler, Xu and Paulus

cleavage

with

the assistance

295

of His536.

The presence of partial reactions, manifested by cleavage at either splice junction, indicates that cleavage at each splice junction is independent of splicing or cleavage at the opposite splice junction [4**,7”,11,14-171.

The mechanism N-X acyl rearrangement

ester intermediate, site-directed mutagenesis was used to replace Serl of the Psp Pol-1 intein with cysteine, to generate a thioester that would be subject to nucleophilic displacement by hydroxylamine or thiols [4”,5’]. Indeed, treatment of the Serl+Cys intein linked to the carboxyl terminus of the maltose-binding protein (MBP) with hydroxylamine leads to rapid cleavage at the intein amino terminus and the production of the carboxy-terminal hydroxamate derivative of MBP [4**,5*]. Hydroxylamine cleavage of the Serl+Cys mutant is inhibited by the thiol blocking reagent iodoacetamide (M-Q Xu, unpublished data). Cleavage can also be effected by high concentrations of cysteine or thiols [4”].

Linear ester intermediate

Asparagine

Transesterlflcation

0

Branched

Asparagine

lntein

X-N acyl rearrangement

wth C-terminal or isoasparaglne

splicing

acyl rearrangement The ester bond

The second step in protein splicing involves attack of the ester at the amino-terminal splice junction by the sidechain of the serine, threonine, or cysteine residue at the downstream splice junction to yield the branched ester

Ligated &ems

Current Op~monI” Chemical Biology

The protein

Similar experimental results were obtained with the VMA intein of Saccharomycescemxsiae (See), which is naturally flanked by cysteines [7*-l. These results, supported by the genetic studies described above, constitute strong evidence that the first step in protein splicing involves the rearrangement of the peptide bond involving the serine or cysteine residue at the upstream splice junction to form an ester intermediate. An earlier suggestion that the formation of the ester intermediate involves participation of the asparagine residue at the carboxy-terminal splice junction [19] is ruled out by the observation that replacement of this asparagine with aspartate or glutamate does not block the early steps in protein splicing [4”].

cyclization

Hydrolysis

asparqne

mechanism.

Protein

splicing

of the intein amino-terminal

in the linear ester intermediate

is initiated

by an

serine or cysteine. is cleaved

through

transesterification with the serine, cysteine or threonine at the amino terminus of the downstream extein, resulting in the formation of the branched

ester intermediate.

resolved

by cyclization

excised

intein contains

The branched

ester intermediate

of the intein carboxy-terminal a carboxy-terminal

succinimide

is

asparagine. ring which

The can

be hydrolyzed to form asparagine or isoasparagine. Finally, a peptide bond between the exteins is formed by a second, spontaneous acyl rearrangement, which can occur in the absence of the intein. The oxygen

or sulfur atom of the reactive

represented

of protein splicing

Protein splicing involves four successive nucleophilic displacements (Figure 3). In order to demonstrate a linear

serine, threonine

or cysteine

is

by an ‘XL

of His536 resulted in the absence of carboxy-terminal cleavage and accumulation of the branched intermediate, suggesting that Asn537 mediates branch resolution and

intermediate described above (Figure 3). The equilibrium constant for this transesterification reaction is about 1 if the esters involved are of the same type, i.e. thioester or oxygen ester, or about 50 if it involves the conversion of a thioester to an oxygen ester [ll]. The metastable branched intermediate then undergoes an essentially irreversible transformation, which involves the cyclization of the asparagine residue preceding the downstream splice junction (Figures 3 and 4). Asparagine cyclization can result in deamidation or peptide bond cleavage [20,21]. Spontaneous asparagine rearrangements at neutral pH most commonly involve the attack of the peptide-bond nitrogen on the asparagine sidechain carbonyl carbon atom, resulting in release of ammonia and formation of a five membered succinimide ring (Figure 4). The succinimide ring then undergoes rapid hydrolysis at either the a- or P-carbonyl group to generate isoaspartate or

296

Mechanisms

Figure 4

/ c, CH,

R’-NH -

NHz

CH

’ \

4

R’-NH -

NH-R”

CH

\

/NH-R”

C’

II 0

0 Cyclization

and

Cyclization

cleavage

and

deamidation

0

II

CHLC

\

CHAC

\

NH + R”-NH,

N-R” + NH,

R’-NH -CH

R’-NH -CH /

\

Carboxyterminal

C II 0 Internal

aminosuccinimide

Hydrolysis

,A’

yo_

I R

-CH

-6

‘NH, CHz 2

Or

N’-‘z

\ C’

II

NH -CH

Lc

lsoasparagine

Ho-

II

R

0

II c, /

0 Asparagine

I R

II c, /

O-

NH-R’

(342

01

I

NH

1

0

0

0

CHz NH

aminosuccinimide

Hydrolysis

0

,!

/

\

C II 0

-CH

NH-R”

\ C’

il

NH

I R

0 Aspartate

-CH \

,OC

II

0 lsoaspartate

Current Optmon an Chemical Biology

Asparagine cyclization pathways. Asparagine can cyclize to form a succinimide ring when one of its carbonyl groups is attacked by one of its nitrogens. Attack by the peptide bond nitrogen on the sidechain carbonyl results in deamidation and formation of aspartate or isoaspartate upon aminosuccinimide hydrolysis. Attack by the sidechain nitrogen on the mainchain carbonyl results in peptide bond cleavage and formation of asparagine

or isoasparagine

upon aminosuccinimide

hydrolysis.

aspartate. .4 less common reaction involves the attack of the asparagine sidechain amide nitrogen on the following peptide bond carbonyl, resulting in peptide bond cleavage and formation of a carboxy-terminal aminosuccinimide. The latter, in turn, is hydrolyzed during protein splicing to form isoasparagine or asparagine (Figure 4). The Psp PO]-1 and See MIA intein carboxy-terminal aminosuccinimides were identified by mass spectrometry and comparison with synthetic model peptides [7”,11,22]. hlutagenesis data indicate that the adjacent histidine assists in asparagine cyclization. The intein aminosuccinimide

is hydrolyzed to asparagine [ll]. In model peptide studies, hydrolyzed with a half-time of 7.4, as determined with model the succinimide ring j71 viuo is

or isoasparagine in aitro the aminosuccinimide is about 80 hr at 25’C and pH peptides [Xl. The fate of not known.

After release of the intein, the exteins are linked by an ester bond, which rapidly undergoes an acyi rearrangement to the thermodynamically more stable. normal peptide bond, with a half-time of less than 1 min at neutral pH in model peptides (Y Shao, H Paulus, unpublished data). This rearrangement, which is presumably uncatalyzed

Protein splicing and autoproteolysis

because

it occurs

after the intein

makes

protein

splicing

essentially

Given protein

the relative autonomy splicing (Figure 3),

has already

been

excised,

irreversible.

of the steps involved in it appears that the intein

must have painstakingly evolved in its splicing context to assure that the relative rates of these steps are perfectly balanced so as to prevent abortive side reactions. It is interesting that the splicing of chimeric proteins is usually accompanied by significant cleavage at both splice suggesting that transposition of an junctions [7”,18], intein into a different environment tends to disturb the balance between the various splicing steps. hloreover, each intein has evolved a specific architecture and/or chemical environment to optimize the nucleophilic attack by the native splice junction residues. Replacement of the serine, threonine, or cysteine residues at the splice junctions with other hydroxyl or thiol containing amino acids does not always result in protein splicing or cleavage [4**,7**,14-171. In general, cysteine can substitute for seine or threonine, albeit poorly, but the reverse situation is not observed. A possible explanation for this lack of reciprocity may be that hydroxyl groups have a higher pK, than thiols. As a result, serine or threonine needs assistance from surrounding residues for deprotonation of their sidechain hydroxyls, whereas cysteine does not because some cysteinyl sulfhydryl groups will be deprotonated at neutral

pH.

Autoproteolysis precursors

of glycosylasparaginase

Glycosylasparaginase hydrolyses AspNHGlcNAc and corresponding glycans, and its deficiency in humans leads to aspartylglycosaminuria, a common genetic disorder [23]. The human and Flavobactetium meningosepticum glycosylasparaginases have a high degree of amino acid sequence and structural similarity, since both enzymes are (@)z tetramers [24]. The C( and p subunits are synthesized as a single polypeptide chain, which is converted to the a and p chains by post-translational cleavage adjacent to a threonine residue [23,24] (Figure lc). An interesting aspect of this reaction is that the new amino terminus is essential for enzyme activity. In this respect, glycosylasparaginase resembles a group of other amidohydrolases termed amino-terminal nucleophile hydrolases, which have similar three-dimensional structures and whose activity depends on an amino-terminal serine, threonine, or cysteine residue that is generated by the cleavage of a precursor protein, probably through self-catalysis [ZS]. Autoproteolysis of bacterial glycosylasparaginase occurs adjacent to Thrl52, which resides in a highly conserved region (Figure lc). Replacement of Thr152 with amino acids other than serine or cysteine prevents autoproteolysis, whereas replacement with cysteine or serine reduces its rate [6**]. The use of ThrlSZ+Ser and ThrlSZ+Cys mutant proteins fused to the carboxyl terminus of ~L~BP, in conjunction with the discovery

that

glycine

and

mechanisms Perler, Xu and Paulus

small

I,-amino

acids

severely

297

inhibit

autocleavage of these mutants in viva, made possible the purification of glycosylasparaginase precursors fused to MBP and the study of the autocleavage mechanism [6”]. The with

cleavage respect

of the purified fusion protein is first order to protein concentration under conditions

where the precursor is monomeric, suggesting cleavage involves a self-catalyzed intramolecular

that the reaction.

Autocleavage occurs most rapidly at pH 6.0-7.5, and is prevented by the replacement of His150 with serine, consistent with a catalytic role of the histidine sidechain. hlutation of the corresponding histidine residue (His204) in the human glycosylasparaginase also interferes with autoproteolysis [26]. Evidence for the involvement of an ester intermediate comes from the observation that the cleavage of the ThrlX?+Cys mutant protein is promoted by hydroxylamine and inhibited by the thiol-blocking reagent iodoacetamide [6”]. Cleavage occurs by the hydrolysis of the ester intermediate, but little is known about the catalytic groups other than His150 that are required for this process [6”].

Autoproteolysis formation

leading to pyruvoyl enzyme

Although autoproteolysis of pyruvoyl enzymes was the first example of autoprocessing involving an acyl rearrangement at a serine residue, this reaction will not be reviewed fully as our focus is on recent publications. It was the pioneering work of Snell and co-workers (summarized in [lo]) that established that the processing of prohistidine decarboxylase involves cleavage of the peptide bond between Ser81+Ser82 after a self-catalyzed acyl rearrangement. The ester intermediate was identified by hydroxylamine cleavage experiments using a Ser82-+Cys mutant and by labeling experiments with 180. The autoproteolysis reaction involves p-elimination at the serine residue to form an amino-terminal pyruvate residue, rather than ester hydrolysis (Figures Id, 2).

Autoprocessing

of hedgehog

proteins

The hedgehog protein (Hh) from Drosophda is a secreted protein that undergoes polypeptide cleavage to yield a cell surface-associated 20 kDa amino domain, the carboxyl terminus of which is esterified with cholesterol (Hh-N,), and a 25 kDa carboxyl domain, which has cysteine at its amino terminus (Hh-C) (Figure lb) [8**,9,12**,27]. The Hh-N, autoprocessing product is responsible for developmental signaling, the Hh-C domain for autoprocessing [8”,9,12**,27]. The biological advantage of generating an extracellular signaling molecule by a self-catalyzed reaction is that such a reaction is concentration-independent and does not require additional protein cofactors. The mechanism of hedgehog autoprocessing was studied using bacterially expressed modified hedgehog proteins in which the Hh-N domain was replaced by other proteins, or by a hexahistidine sequence that replaced

298

Mechanisms

all but In the cysteine,

residues of the Hh-N domain (HisbHh-C). presence of thiols, such as dithiothreitol and or hydroxylamine, the purified HisbHh-C pre-

nine

cursor undergoes time-dependent cleavage at the normal processing site, leading to the expected adducts at the new carboxyl terminus of the amino domain [8”,9]. This reactive thioester bond in HisbHh-C can also be cleaved by peptides containing cysteine at their amino terminus, resulting in ligation of the peptide to the new carboxyl terminus of the processed N-domain [8”]. Autoproteolysis is blocked by thiol reagents such as it’-ethylmaleimide or by the replacement of CysZ58 with alanine [8”]. However, replacement of Cys258 with serine allows cleavage to occur at a reduced rate, consistent with the lower reactivity of oxygen esters to nucleophilic displacement by thiols. Replacement of His329 with tyrosine blocks the autoprocessing reaction [9]. The inability of tagged mutant proteins lacking the conserved histidine to undergo cleavage when mixed with wild-type hedgehog protein [27], and the observation that autoprocessing is concentration-independent [9], support the idea that autoproteolysis involves intramolecular catalysis. The second step in the hedgehog protein autoprocessing pathway involves an intermolecular transfer of the acyl moiety of the thioester intermediate to the 3p hydroxyl group of cholesterol [ 12**]. This reaction occurs with purified His(,Hh-C containing only nine residues of Hh-N, and it appears that the catalysis of this transesterification reaction is effected entirely by the Hh-C domain. Aucoproteolysis of hedgehog proteins occurs adjacent to a highly conserved cysteine residue. Sequence similarities have been found between the amino-terminal motifs and histidine-containing motifs of inteins and Hh-C, suggesting mechanistic similarities between protein splicing and hedgehog autoprocessing [1”,9,13,28]. The discovery of six new genes in Caenorhabditis efegarrs with a high degree of similarity to the hedgehog Hh-C protein suggests that proteins other than hedgehog may undergo a similar type of autoproteolysis

The chemical reactions highlighted in this review, namely acyl rearrangement and asparagine cyclization, can potentially be applied to protein engineering. Porter et al. [ lZ**] have already demonstrated that amino-terminal cysteine containing peptides can be added to the newly generated carboxyl terminus of proteins by attacking self-generated thioesters in the hedgehog system. Similar addition of amino-terminal cysteine-containing peptides to N-exteins has also been demonstrated with mutated inteins (hl-Q Xu, unpublished data). hlutation can convert inteins into autoproteolytic reagents that only cleave at single splice junctions [4”,7”,14-16,291. Such modified inteins can be used for protein purification when combined with affinity tags [29] or to generate reactive thioesters at the carboxyl terminus of target proteins preceding inteins that would allow nucleophilic addition of a labeling reagent, a lipid or a peptide. The independently developed technique for the synthesis of proteins by native chemical ligation [30] proceeds by a mechanism analogous to protein splicing. Peptide ligation requires the presence of a thioester on one peptide which is then attacked by the thiol group of an amino-terminal cysteine thiol group from a second peptide. One can imagine combining in vitro synthetic techniques with in vivo derived thioester-tagged proteins to build all sorts

recently discovered examples of prothat lead to the conversion of inactive to biologically active forms. A common

mechanisms

the context of the biological function of the autoprocessing reaction. A second common theme in these reactions is a conserved histidine which is required for autoproteolysis. Protein splicing is the most complex of these processing events. reauirinp three co-ordinated nucleoohilic diselacements after the initial acyl rearrangement. Protein splicing also involves an unusual asparagine rearrangement that 0

terminus, addition of a prosthetic group, and addition of lipids directing protein compartmentalization within the cell. Each of these pathways has changed our view of how functional proteins can be synthesized, activated or targeted.

of chimeric proteins. As we become more familiar with the chemical mechanisms that nature employs to build and breakdown proteins, we will be able to apply these

feature of these self-catalyzed protein rearrangements is an N-O or N-S acyl rearrangement at a specific serine, threonine, or cysteine residue, yielding an unstable ester intermediate. Further processing of this intermediate to the stable end products can occur by four different routes, which appear to have evolved independently in

I>

the more remarkable aspects of these four autoprocessing pathways is that, although they are all initiated by an acyl rearrangement, they generate diverse and fundamentally important end results: peptide excision and ligation, peptide cleavage to generate a new catalytic amino

[8”].

Conclusions We have described tein autoprocessing precursor proteins

results in peptide-bond cleavage instead of deamidation. It is likely that more examples of autoprocessing initiated by an acyl rearrangement will soon be discovered. One of

I

References

to our advantage.

and recommended

Papers of particular interest, published have been highlighted as: . l

0

reading

within the annual period of review,

of special interest of outstanding interest

1.

Perler FB, Olsen GJ, Adam E: Compilation and analysis of intein sequences. Nucleic Acids Res 1997, 25:1087-l 093. ;kis catalog lists published intein sequences, as well as the phylogenetic relationships among inteins. A new intein motif, Block H, is described and the intein motifs first defined in 1131 are listed for all inteins. An updated intein database is maintained on the New England Biolabs Web site (http:l/www.neb.coml).

I

2.

Elliott DF: A search for specific chemical methods for fission of peptide bonds. 1. The N-acyl to 0-acyl transformation in the degradation of silk fibroin. Biochem / 1952, 50:542-550.

Protein

3.

Li YM, Milne JC, Madison LL, Kolter R, Walsh CT: From peptide precursors to oxazole and thiazole-containing peptide antibiotics: microcin Bi 7 synthase. Science 1996, 274:11881193.

4. Xu M-Q, Perler FB: The mechanism of protein splicing and its .. modulation by mutation. EMBO J 1996, 15:5146-5153. Mutation of conserved splice junction residues of the Psp ~01-1 intein defines the roles of each residue. A cold-sensitive mutation allows splicing at temperatures of 37-C or above, but not below 37-C. The rate of hydrolysis at the amino-terminal splice junction in MBP-intein fusions is 1 O-fold higher if a serine is also present at the carboxy-terminal splice junction. The presence of the linear ester intermediate in a precursor containing a Ser-1 +Cys mutation is confirmed by cleavage with hydroxylamine, free cysteine or dithiothreitol. Shao Y, Xu M-Q, Paulus H: Protein splicing: evidence for an N-O acyl rearrangement as the initial step in the splicing process. Biochemistry 1996, 35:381 O-381 5. Provides evidence for a linear ester intermediate by demonstrating cleavage by hydroxylamine and by identifying the N-extein carboxy-terminal hydroxamate using mass spectrometry and a calorimetric assay for hydroxamic acids.

splicing

Guan C, Cui T, Rao V, Liao W, Benner J, Lin CL, Comb D: Activation of glycosylasparaginase. Formation of active Nterminal threonine by intramolecular autoproteolysis. J i3iol Chem 1996, 271 :1732-l 737. Describes the isolation of the bacterial glycosylasparaginase precursor and its processing in viva. 7. ..

Chong S, Shao Y, Paulus H, Benner J, Perler FB, Xu Ma: Protein splicing involving the Saccharomyces cerevisiae VMA intein: the steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system. J Biol Chem 1996, 271:22159-22168. The mesophilic Saccharomyces cerevisiae VMA intein, bounded by cysteine residues, splices by the same pathway described for the thermostable Psp pot-1 intein bounded by serine residues. Several lines of evidence demonstrate the occurrence of a thioester intermediate. Labeling of the carboxyl terminus of the upstream extein by radioactive cysteine is described, as are mutations that allow controllable cleavage at the intein amino terminus by thiols.

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16.

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1 7.

Hodges RA, Perler FB, Noren CJ, Jack WE: Protein splicing removes intervening sequences in an archaea DNA polymerase. Nucleic Acids Res 1992, 20:6153-6157.

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22.

Shao Y, Xu M-Q, Paulus H: Protein splicing: characterization of the aminosuccinimide residue at the carboxyl terminus of the excised intervening sequence. Biochemistry 1995, 34:1084410850.

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Tikkanen R, Riikonen A, Oinonen C, Rouvinen J, Peltonen L: Functional analyses of the active site residues of human lysosomal aspartylglucosaminidase: implications for catalytic mechanism and autocatalytic activation. EMBO J 1996, 15:2954-2960.

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Tarentino AL, Quinones G, Hauer CR, Changchien LM, Plummer TH: Molecular cloning and sequence analysis of Flavobacterium meningosepticom glycosylasparaginase: a single gene encodes the cx and p subunits. Arch Biochem Biophys 1995, 316:399-406.

25.

Brannigan JA, Dodson G, Duggleby HJ, Moody PCE, Smith JL, Tomchick DR, Murzin AG: A protein catalytic framework with an N-terminal nucleophile is capable of self-activation. Nature 1995, 278:416-419.

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Annu

Porter JA, Young KE, Beachy PA: Cholesterol modification of hedgehog signaling proteins in animal development. Science 1996, 2741255-259. This paper identifies the lipid adduct on the hedgehog protein amino domain (Hh-N) as cholesterol and positions it at the carboxyl terminus of the Hh signaling domain. The absence of cholesterol inactivates the Hh protein signaling domain and prevents its accumulation at the cell surface. Cholesterol is covalently attached to Hh-N by an alkali labile linkage, suggesting that the cholesterol moiety is linked to Hh-N by an ester bond. Cholesterol is found on amino domains of hedgehog proteins in the cells of many species, from monkey to mouse.

Perter, Xu and Paulus

Pietrokovski S: Conserved sequence features of inteins (protein introns) and their use in identifying new inteins and related proteins. Protein Sci 1994, 3:2340-2350.

8. ..

Porter JA, Ekker SC, Park WJ, von Kessler DP, Young KE, Chen CH, Ma Y, Woods AS, Cotter RJ, Koonin EV, Beachy PA: Hedgehog patterning activity: role of lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 1996, 86:21-34. This paper reports groundbreaking studies that demonstrate that the hedgehog protein (Hh) signaling domain is modified by addition of a lipid group during autoprocessing. The absence of lipid inactivates the hedgehog protein signaling domain by preventing its accumulation in the outer membrane. The Hh carboxyl domain (Hh-C) contains sufficient information for lipid addition. The authors identify several proteins with similarity to Hh-C and cysteinedependent cleavage in one of these proteins is demonstrated. The authors suggest that Drosophila Hh residues Cys258, His329 and Asp282 are conserved in Hh-C-related proteins and constitute the autoproteolytic catalytic triad.

mechanisms

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5. .

6. ..

and autoproteolysis

family