Effect of pepstatin A on structure and polymerization of intermediate filament subunit proteins in vitro

JOURNAL.

OF S’l’RUCTURAL

BIOLOGY

106,64-72

(19%)

Effect of Pepstatin A on Structure and Polymerization of Intermediate Filament Subunit Proteins in Vitro ELFFUEDE MOTHES, Max-Plnnck-htitut

fir Zellbiologie,

ROBERT

L. SHOEMAN,

AND PETER TRAUB

Rosenhof, D-6802 Ladenburg,

Fe&ml

Republic of Germany

Received November 15, 1990, and in revised form December 27, 1990

with the inhibitor was necessary to achieve maximum inhibition (Shoeman et al., 1990al. During the course of this original study, and as detailed previously (Mothes et al., 19901, we observed that pepstatin A can spontaneously polymerize into regular, higher-order structures such as helical filaments, ribbons, sheets, and cylinders. We demonstrated that the inhibitory activity of pepstatin A is largely lost as a result of self-polymerization (Mothes et al., 19901. We reasoned that pep&tin A might also be capable of interacting with IF subunit proteins and affects not only their utilization as substrates for HIV-l protease but also their polymerization properties. Data are presented here that demonstrate that this is indeed the case. After completion of this study, Wallin et al., (1990) reported that pep&&in A affects the polymerization of tubulin into microtubules in vitro, although no details were presented.

Pep&&in A, a pentapeptide aspartyl protease inhibitor, can interact with intermediate filament (IF) subunit proteins and induce their polymerization in the absence of salt into long filaments with a rough surface and a diameter of 1617 nm. This polymerization appears to be driven primarily by non-ionic interactions between pep statin A and polymerization-competent forms of IF proteins, resulting in a composite filament. Proteolytic fragments of vimentin, lacking portions of only the head domain or of both the head and tail domains, failed to copolymerize with pepstatin A into long filaments under these conditions. Rather, these peptides, as well as control proteins like bovine serum albumin, were found to decorate pep&tin A polymers (filaments, ribbons, and sheets) by sticking to their surfaces. In addition to the electron microscopy experiments, UV difference spectra, ultracentrifugation, and SDS-PAGE analysis of in vitro cleavage products of vimentin obtained with HIV-1 protease all provided independent evidence for a direct association of pepstatin A with IF subunit proteins, with subsequent alterations in the IF subunit protein conformation. These data show that non-ionic interactions can substitute for the effect of salt and effectively drive the higher-order polymerization of IF subunit proteins. 0 1991 Academic Press, Inc.

MATERIALS

Murine vimentin was prepared from Ehrlich ascites tumor cells (Nelson et al., 19821, desmin from chicken smooth muscle (Geisler et al., 1982), and GFAP from porcine spinal cord (Vorgias and Traub, 1983). T-vimentin, a thrombic peptide containing amino acid residues 71-465, was prepared from murine vimentin as described (Perides et al., 1987b). Vimentin core peptide, produced by complete digestion of vimentin with HIV-1 protease and containing amino acid residues 93-422 (Shoeman et al., 199Ob), was purified by rate velocity sucrose density gradient centrifugation (Shoeman et al., 1990b) and separated from residual HIV-1 protease by centrifugation in a Centricon 30 ultraconcentrator (Amicon, Witten, FRG), according to the manufacturer’s instructions. Bovine serum albumin (BSA) was purchased from Sigma (Deisenhofen, FRG). Pepstatin A (Fig. 1) (Sigma) was dissolved at 20 m&f in dimethyl sulfoxide (DMSG) and was added slowly to the incubation mixtures (to prevent precipitation) to the indicated fmal concentrations (see Results). IFS were formed by incubating protofilamenta (150 pg/mll at 37°C in 10 m&f Tris-acetate, pH 7.6,6 mM 2-mercaptoethanol with either 150 m&f KC1 for 60 min or with pepstatin A at concentrations ranging from 0.003 to 2 m&f for 30 min. Samples were prepared for electron microscopy and viewed in a Philips Model 400 T electron microscope, essentially as described (Perides et al., 1986a; Mothes et al., 1990). Polymerized material was separated f+om IF protofilaments or unpolymerized pepstatin A by centrifugation in a Beckman Airfuge at 130 000s for 120 min at 0°C. Assays for HIV-1 protease and in-

INTRODUCTION

Intermediate filaments (IFS) are major constituents of the cytoskeleton of eukaryotic cells. IFS are thought to play a structural role (Lazarides, 1980), although a growing body of evidence suggests they may have additional roles in regulating expression of genetic information (Traub et al., 1987; Chan et al., 1989). We have recently demonstrated that the IF subunit proteins vimentin, desmin, and glial fibrillary acidic protein (GFAP) can be efficiently cleaved in vitro by human immunodeficiency virus type 1 (HIV-11 protease (Shoeman et al., 1990a). The aspartyl protease inhibitor pepstatin A (Richards et al., 19881 was found to prevent this cleavage of IF proteins, although preincubation of HIV-l protease 64 1047~3477/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All righta of reprodwtion in any form reserved.

AND METHODS

PEPSTATIN CH3

A AND IF SUBUNIT

65

PROTEINS

CH3

CH3

CH3-CH

CH3 CH-CH3 CH3 I I CH-CH3 CH-CH3 CH2 CH2 CH3 I I I I CO-NH-CH-CO-NH-CH-CO-NH-CH-CH-CH-CH2-CO-~H-CH-CO-~H-CH-CH-CH2-COOH

I

OH

CH-CH3 I CH2

I

OH

FIG. 1. Structure of pepstatin A ~N-isovaleryl-~-valyl-3-hydroxy-6-methyl-~-aminoheptanoyl-~-alanyl-3-hydroxy-6-methyl-~aminoheptanoic acid; molecular weight = 686). hibitory activity of pepstatin A and its polymers were performed as described (Mothes et al., 1990). IF subunit proteins and their cleavage products were analyzed by electrophoresis on 9-1596 polyacrylamide gradient SDS gels (SDS-PAGE) and stained with Coomassie blue (Egberts et al., 1976). Quantitative estimates of the distributions of IF subunit proteins were made by excising bands of stained proteins and scanning at 590 nm with a Gilford Model 2600 ~pectrophotometer system. UV difference spectra of IF proteins, pep&&in A, and their complexes were also determined with this system and results of both analyses were plotted with a HewletkPackard Model 7225A graphics plotter. For UV difference spectra determinations, 2-mercaptoethanol, due to ita variable absorption upon oxidation, was omitted from the incubation mixtures and removed from the IF protein stock solutions by dialysis. RESULTS

We have previously observed that pepstatin A inhibits the cleavage by HIV-l protease of IF subunit proteins at low ionic strength (where IF subunit proteins are in the form of tetramers) and that IFS require 10 times the HIV-l protease activity to achieve equivalent extents of digestion (Shoeman et al., 1990a). This raised the seemingly unlikely possibility that pepstatin A prevented the cleavage of IF subunit protein tetramers by inducing their polymerization into filamentous (and hence HIV-l protease-resistant) forms. We therefore investigated the influence of pepstatin A on the polymerization state of several IF subunit proteins at low ionic strength (Fig. 2). Vimentin (M, = 54 OOO), desmin (M, = 53 000), and GFAP (M, = 51000) were employed at a concentration of 150 cl,g/ml, which is approximately 0.003 n&f. At low ionic strength, vimentin protofilaments were visualized as short rods (Fig. 2a). When pepstatin A was added to final concentrations of 0.003 to 0.01 mM, numerous short rods or filaments were observed (Fig. 2b). As the pepstatin A concentration was raised to 0.1 (Fig. 2c) and 1 mM (Fig. 2d for vimentin, Fig. 2e for desmin, and Fig. 2f for GFAP), thin pepstatin A filaments formed (arrowheads in Fig. 2d), and increasingly long, regular, filamentous structures containing the IF subunit proteins were observed. Relative to vimentin IFS (Fig. 2g) (formed in the presence of 150 mM KC1 and equivalent concentrations of the pepstatin A solvent DMSO as a control), which possess the characteristic smooth surface and diameter of approximately 10 nm, the filaments formed in the presence of pepstatin A (Figs. 2c-2f1 have a rough

surface and a diameter of 15-17 nm, suggesting that pepstatin A is incorporated into these modified filaments. The long, thin pepstatin A filaments seen in these and later figures are indistinguishable from those obtained by self-polymerization in the absence of IF subunit proteins (Fig. 2k) (Mothes et al., 1990). As a control, vimentin-pep&&in A copolymers were first applied to a grid, followed by normal vimentin IFS formed in the presence of 150 m&f KCl. Selected fields (Fig. 21) contained both types of structures seen individually in Figs. 2d and 2g. IF subunit proteins polymerize into filaments in a time- and salt concentration-dependent fashion. Vimentin in the presence of 50 n&f KC1 formed only short, irregular rods (Fig. 2h) under our standard incubation conditions, as previously reported (Ip et al., 1985). In identical reaction mixtures supplemented with 1 mM pepstatin A, the predominant polymers observed, besides the expected pepstatin A filaments (Fig. 2i, arrowhead), were long, regular filaments (Fig. 2i, open arrow), reminiscent of normal lo-run IFS (compare Figs. 2g and 2i), and many unraveled vimentin IF polymers (Fig. 2i, arrows> (Aebi et al., 1983,1988). Polymerization of vimentin in the presence of both 1 mM pepstatin A and 150 mM KC1 resulted in the formation of filaments (Fig. 2j, open arrow) with a morphology and diameter like those observed with KC1 alone, as well as pepstatin A filaments (Fig. 2j, arrowhead). As previously noted, pepstatin A alone can polymerize into various higher-order structures, including filaments (Fig. 2k), ribbons, sheets, and cylinders (Mothes et al., 1990). These polymers are found with the highest frequency at the higher pepstatin A concentrations employed, for example, 2 mM, although they are infrequently found at concentrations down to 0.1 mM. At low ionic strength in reaction mixtures containing IF subunit proteins and pepstatin A, large-diameter ribbons of pepstatin A decorated with protofilaments of GFAP, vimentin, and desmin were sometimes observed (data not shown). Previous studies have demonstrated the importance of the N-terminal, non-o helical head domain of vimentin for polymerization of IFS (Geisler et al., 1982, Kaufmann et al., 1985; Nelson and Traub, 1983; Shoeman et al., 199Ob; Traub and Vorgias, 1983,1984). T-vimentin, a thrombic peptide lacking

66

MOTHES, SHOEMAN,

AND TRAUB

PEPSTATIN

A AND IF SUBUNIT

the first 70 amino acids of the intact protein, is not capable of forming filaments in physiological salt solutions. Likewise, the large secondary cleavage product produced by a limit digest of vimentin by HIV-l protease, which encompasses amino acids 93421 and will hereafter be referred to as vimentin core peptide, is not only incapable of polymerizing into filaments under normal conditions, but also is released from preformed filaments (Shoeman et al., 199Ob). Typical structures found in polymerization reactions containing 150 mM KC1 with T-vimentin and vimentin core peptide were short rods (data not shown), grossly similar to the smallest vimentin polymers seen at low ionic strength (presumably tetramers) in Fig. 2a. When various concentrations of pepstatin A were substituted for the KC1 in these reactions, essentially identical results were obtained with both peptides: pepstatin A filaments and ribbons were covered with short rods of the respective peptides. No long IF-type filaments were observed to be formed by these peptides in the presence of pepstatin A, in contrast to the long filamentous structures obtained with intact IF proteins (data not shown). In addition, no long copolymers or filaments were found in reactions containing pepstatin A and non-filament forming proteins, such as BSA; however, BSA was observed to decorate the surface of preformed pepstatin A polymers in the same manner as T-vimentin or vimentin core peptide (data not shown). Since the analysis of extent of polymerization by electron microscopy is not quantitative and one might argue that the association of the vimentin proteolytic fragments with the pepstatin A polymers was simply fortuitous, we chose to isolate polymeric forms of vimentin (IFS), vimentin peptides, and pepstatin A by centrifugation. The distribution of the proteins was analyzed by SDS-PAGE and densitometry (Table I). In the presence of 150 n&f KCl, vimentin assembled quantitatively into pelletable filaments. Under the same conditions, only 14% of Tvimentin was found in the pellet. In the absence of

PROTEINS

67

KCl, both vimentin and T-vimentin remained largely (>96%) in the supernatant fraction. When pepstatin A was added to reaction mixtures lacking KC& detectable amounts of T-vimentin and large amounts of vimentin were found in the pellets. The relatively low amounts of T-vimentin pelleted are probably a function of the limiting number of pepstatin A polymers in the reaction mixtures: at higher pepstatin A concentrations, where a greater proportion of polymers are found, more T-vimentin was found in the pellets. At 2 mM pepstatin A, the amount of vimentin recovered in a polymeric form was only half that recovered at 1 mM pepstatin A (Table I); this presumably is due to the reduction in free pepstatin A available for copolymerization as a result of increased self-polymerization. Prior to centrifugation, aliquots of the incubation mixtures were analyzed by electron microscopy. The predominant polymers observed are listed in Table I and are in good agreement with the data presented in Fig. 2 (and data not shown). In short, pepstatin A can largely substitute for or relieve the requirement for salt (KCl) in the polymerization of intact vimentin. T-vimentin is essentially incapable of polymerizing into long filaments, even in the presence of pepstatin A; however, it can decorate pepstatin polymers in a stable fashion. Ifpepstatin A indeed can directly interact with IF proteins and induce their polymerization, it is conceivable that differences in optical properties of the proteins might be detectable. Pepstatin has been shown to bind to pepsin and alter its optical activity between 279 and 293 run (Aoyagi and Umezawa, 1975). Therefore, the UV absorption spectra for IF proteins, pepstatin A, and mixtures of the two were measured and difference spectra were calculated over the range of 240-300 nm for vimentin (Fig. 3a) and T-vimentin (Fig. 3b). Large increases in absorption of IF protein-pepstatin A complexes, with a peak at 274 nm, were measured with increasing concentrations of pepstatin A. At the highest concentration of pepstatin A tested (2 miW, these peak ab-

FIG. 2. Copolymerization of IF subunit proteins and pep&tin A. Structures formed after incubation of vimentin (a-d), desmin (e) and GFAP (f) in 10 mM Tris-HCl, pH 7.4, alone (a) or containing pepstatin A at 0.01 (b), 0.1 (c), and 1 mM (d-f). In (d), the arrowheads point to typical pepstatin A helical filaments, the filled arrow points to a thin composite filament and the open arrow points to a thick, 15 to 17-nm composite vimentin-pepstatin A filament. Vimentin filaments (g) formed in the presence of 150 mM KC1 and 5% DMSO (the solvent for pep&tin A present in the other incubations) have a normal appearance. Salt and pepstatin A can act synergistically, as evidenced by polymers formed in 10 miU Tris-HCl, pH 7.4, in the presence of(h) 50 m&4 KCl, (i) 50 n&f KC1 and 1 m&f pepstatin A, and (i) 150 miW KC1 and 1 mM pep&tin A. In (i and j), the arrowheads point to a typical pepstatin A filament, the closed arrows point to unraveled vimentin IF polymers and the open arrow points to a vimentin-pepstatin A composite fllament in (i) and to a fllament very similar to typical vimentin IFS in (i). Note the elongation of long filaments in the presence of pepstatin A (compare h and i) and the strong resemblance of filaments formed in the presence of both 150 mM KC1 and pepstatin A to those formed only in 150 mM KC1 (compare g and j). Typical pepstatin A filaments (k) are readily distinguished from vimentin-pepstatin A copolymers (1, open arrow) and vimentin IFS formed in the presence of 150 mM KC1 (1, double arrow). As a control, the vimentin-pepstatin A copolymers and vimentin IFS were formed in separate reactions and applied sequentially to the same carbon film for the image presented in (1). The differences seen between these structures are indistinguishable from those seen between d and g. For (a-j), the magnification was x 92 000, and the bar represents 100 mn. For (k) and (l), the magnification was x 300 000, and the bar represents 50 nm.

MOTHES, SHOEMAN,

68

AND TBAUB

TABLE I

Effect of Pep&din A or Salt on the Polymerization of Vimentin and T-Vimentin Addition

KCL (mM) Protein Vimentin: Form T-vimentin: Form

polymer (%)

polymer (%I

0 4 Protofilaments 1 Protofilaments

50 78 Short filaments 1

Pepstatin A (mM) 150 99 Filaments 14 Aggregates

0.1 9 F&we filaments 3

1

2

63

31 Abundant filaments

4 6 Pepstatin filaments, surface decorated with T-vimentin

The proteins were incubated in 10 mM Tris-acetate, pH 7.6,6 m&f 2-mercaptoethanol with the indicated additions as described under Materials and Methods. The prevailing shape of polymers formed was determined by electron microscopy prior to ultracentrifugation. The distribution of protein in supernatant and pellet fractions was analyzed by SDS-PAGE (data not shown). The numbers tabulated here were obtained from densitometry of these gels.

sorbance values of the IF protein-pepstatin A complexes were lower than those measured at 1 mM pepstatin A, presumably due to the reduction in free pepstatin A as a result of increased self-polymerization as also seen in the centrifugation experiments (Table I). These data provide a clear indication that the interaction of pepstatin A with IF proteins affects the local environment of the tyrosine and tryptophan residues, which are primarily responsible for the absorption at this wavelength. Changes in the UV difference spectra at lower wavelengths (200-230 nm) were also noted (data not shown). However, since absorption in this range is largely a property of the peptide bond, it was not possible to differentiate between changes in the IF proteins, pepstatin A, and the various complexes. At higher pepstatin A concentrations, there is a competition between the association of pepstatin A with these proteins and self-polymerization. We have shown previously (Mothes et al., 1990) that pepstatin A polymers are ineffective at inhibiting HIV-l protease. It was therefore of interest to examine whether IF protein-pep&tin A complexes serve as good substrates for HIV-l protease. SDS-PAGE analysis (Fig. 4) of incubation mixtures showed, as previously described, that vimentin (Fig. 4, lane 2) was efficiently cleaved by HIV-l protease (Fig. 4, lane 3) and that this cleavage could be largely inhibited by preincubation of the HIV-l protease with pepstatin A (Fig. 4, lane 4). Unpolymerized pepstatin A was more effective (Fig. 4, lane 5) than pelleted pep&tin A polymers (Fig. 4, lane 6) in inhibiting cleavage of vimentin by HIV-l protease (Mothes et al., 1990). Dissolving the pepstatin A polymer pellets in DMSO largely restores the inhibitory activity (Fig. 4, lane 7; compare to Fig. 4, lane 61, suggesting that the polymerization state of pep-

statin A is critical for its ability to inhibit HIV-l protease. When vimentin and pepstatin A were coincubated at low ionic strength, most of the vimentin formed filaments and was pelleted; the small amount of vimentin that remained in the supernatant was protected from cleavage by HIV-l protease by the unpolymerized pepstatin A (Fig. 4, lane 8). Pelleted vimentin-pepstatin A complexes were only partially cleaved by HIV-l protease (Fig. 4, lane 9) and no effect of resuspension in DMSO (Fig. 4, lane 10) was observed. The details of cleavage of the vimentin-pepstatin A complexes are different from those observed for protofilaments (compare Fig. 4, lanes 9 and 10 to lanes 5-7) or intact vimentin IFS (Shoeman et al., 1990a,b) in that relatively little of the primary cleavage product (M, = 50 700) is observed. Instead, intact vimentin, the smallest vimentin core peptide (M, = 40 100) and another intermediate cleavage product (M, = 46 400) are observed. This intermediate cleavage product has reproducibly been observed to occur in reactions containing less than fully inhibitory concentrations of pepstatin A (Fig. 4) and a monoclonal antibody specific for the C-terminal tail domain (Shoeman et al., 1990b) reacts with it, whereas several monoclonal antibodies that recognize epitopes in the N-terminal head domain do not (data not shown). Based on the &f, of the cleavage products, this M, = 46 400 intermediate cleavage product probably arises from cleavage of vimentin in the N-terminal head domain between amino acid residues 62 and 63 (i.e., secondary cleavage site 2 in Shoeman et al., 1990a,b). These data suggest that the interaction of pepstatin A with vimentin causes conformational changes such that both the C-terminal primary cleavage site and the N-terminal secondary site between amino acid residues 92 and 93 are no longer cleaved (ac-

PEPSTATIN

A AND IF SUBUNIT

69

PROTEINS 12

34

5

6

7

8

91011

0.6 - a IAE

AE

0 , 250

I

I 270

I

I 290

nm

FIG. 3. UV absorption difference spectra of (a) vimentinpepstatin A copolymers and (b) T-vimentin-pepstatin A copolymers. Absorption spectra of IF protein-pepstatin A complexes, IF proteins (alone), and pepstatin A (alone) were recorded and the difference spectra were determined by subtracting the absorption of the pep&tin A and buffer components from the absorption of the complexes. Thus, these curves represent the absorbance due to vimentin in the presence of varying amounts of pepstatin A. The pepstatin A concentrations employed are indicated on the curves and ranged from 0.1 to 2 mM.

cessible) and the normally secondary site at amino acid residues 62-63 becomes, under these special conditions, the primary cleavage site. DISCUSSION

Three independent experimental procedures, electron microscopy, optical absorption, and ultracentrifugation, demonstrate that pepstatin A can influence the polymerization state of IF subunit proteins. In this respect, the effect of pepstatin A can substitute for the effect of moderate salt concentrations (i.e., 150 n&f KC11 normally employed for in vitro filament formation, although it is quite clear that completely different features of filament structure and assembly are influenced by these two treatments. Pepstatin A itself only allows polymerization competent forms to polymerize; it does not drive the polymerization of truncated forms of vimentin lack-

FIG. 4. SDS-PAGE analysis of the influence of pepstatin A and vimentin polymerization state on the ability of HIV-l protease to cleave vimentin in vitro. Aliquots of M, standards (lane 1, 11) or incubations containing the following components were applied to the gel: Lane 2, vimentin alone; lane 3, vimentin incubated with HIV-l protease; and lane 4, vimentin incubated with HIV-l protease, pretreated with pepstatin A. For lanes 5-7, pepstatin A polymerization reactions were separated into supematant and pellet fractions by ultracentrifugation. Vimentin and HIV-l protease were then added and incubated before applying to the gel. The pep&tin A fractions were: lane 5, supematant (unpolymerized) fraction; lane 6, pellet (polymerized) fraction, resuspended in HIV-l protease incubation mixture; and lane 7, pellet (polymerized) fraction, dissolved in DMSO and then added to the HIV-l protease incubation mixture. Lanes 6-10 are the same as lanes 5-7, respectively, except that pep&tin A and vimentin were allowed to copolymerize before ultracentrifugation and addition of the HIV-l protease to the individual fractions. Note that the dissolution of pepstatin A polymers in DMSO restores significant inhibitor activity (compare lane 7 to lane 61, whereas the addition of DMSO to vimentin-pepstatin A copolymers has no effect (compare lane 10 to lane 9). The M, values of the standards (Pharmacia-LKB) are, from top to bottom, 94 000,67 000,43 000, 30 000,20 000, and 14 000.

ing the head domain (known to be essential for polymerization) nor does it cause “normal” (i.e., nonfilament forming) proteins, such as BSA, to polymerize. IF subunit proteins all contain a central rod domain, with both a high propensity to form an a-helix and a highly conserved amino acid sequence, as well as head and tail domains of variable length and sequence, which are largely responsible for the unique properties of the individual molecules (reviewed by Weber and Geisler, 1984; Aebi et al., 1988; Steinert and Roop, 1988). The central rod domain contains many repeats of the sequence u-&c-&e-~-g, where positions a and d are usually apolar, and charged residues commonly occur at positions e and g (basic residues being favored at position e and acidic residues at position g). The rod domains of two molecules twist about each other in axial register in a coiled-coil arrangement, such that the apolar residues of the parallel chains are brought together on

70

MOTHES, SHOEMAN,

the inner surface of the structure and the charged residues are distributed across its surface. Thus hydrophobic and non-ionic interactions are primarily responsible for stabilizing the coiled-coil dimer structure and the association of dimers into higher order structures is thought to be, to a large extent, influenced by the charged residues exposed on the surface of the dimers. This latter feature of models (Conway and Parry, 1988) of IF formation is a consequence of calculations that demonstrate that the specific distribution of acidic and basic residues (each with a slightly different repeat frequency) within subdomains of the central rod domain results in ionic interactions that lead to stable, higher-order polymers. Non-ionic interactions are thought to play a more minor, but nonetheless significant, role in determining or stabilizing these interactions of dimers. Experimental confirmation of this feature of the model has previously been lacking. This background into the generally accepted mechanism of IF polymerization provides a ready explanation for our results. At low ionic strength, pep&tin A interacts with IF subunit proteins probably through both hydrophobic interactions and hydrogen bonding (e.g., from both the peptide bonds as observed in a-helices and p-sheets and from the two unusual y-amino acid statine moieties in pepstatin A (Fig. 1) (Aoyagi and Umezawa, 1975)) with moieties not only in the rod domain, but also in the head domain. Ionic interactions are probably of little significance in this interaction since the pentapeptide pepstatin A has only one ionizable group, the weakly acidic, terminal carboxyl group (the Nterminus is acylated with an isovaleryl group), which would be insufficient to neutralize all the charges on the surface of the rod domains, as can be done by salts, such as KCl. Of course, an ionic interaction between the weakly acidic pepstatin A and the strongly basic head domain of vimentin may also contribute to this association, although it must be emphasized that this can play only a minor role as there would still be a large number of unbalanced charges throughout the vimentin molecule. It is also possible that the strongly basic, N-terminal head domain of vimentin associates with pepstatin A through non-ionic interactions. Precedence for this notion has been established by the strong interaction of N-terminal head domain peptides with hydrophobic matrices: related peptides differing by a few amino acids can be separated from each other by chromatography on phenyl-Sepharose 4B and bind very tightly to octyl-Sepharose 4B (Traub et al., 1986). In addition, the N-terminal domain of vimentin can be specifically photoafhnity-labeled by lazidopyrene or a phosphatidylcholine derivative in vitro, as a result of its association with lipid vesicles (Perides et al., 1987a).

AND TRAUB

We thus envision that the composite structures formed from copolymerization of pepstatin A and polymerization competent forms of IF proteins differ from IFS formed in the presence of salt in that there probably are parallel, small pepstatin A polymers lying in the grooves between adjacent tetramers or subfilaments (Aebi et al., 1983, 1988), where they serve an “adhesive” function as a result of non-ionic interactions with at least two, otherwise unassociated IF subunit protein tetramers or subfilaments. This would be reminiscent of the observations that detergent may facilitate the ordered packing of proteins by binding to their hydrophobic surfaces in a micellar manner (Both et al., 1990 and references therein). In our previous study (Mothes et al., 19901, we have observed small, flexible pepstatin A filaments with diameters (2-3 run) and lengths (>lOO nm) compatible with such a role and, additionally, the lateral association of individual pepstatin A polymers (presumably through non-ionic interactions) is a hallmark of pepstatin A self-polymerization. Noteworthy is the appearance of polymers formed from vimentin in the presence of both KC1 and pepstatin A (Fig. 1); they seem indistinguishable from normal IFS polymerized in the presence of salt only. This suggests both that pepstatin A is excluded from these polymers and that they represent the energetically favored form. Although not exhaustively studied, we have also observed that the IF subunit proteins, as well as other proteins such as BSA, can interact with pepstatin A polymers. This is usually manifested as a coating of the surface of the pepstatin A filament or ribbon with the soluble (nonpolymeric) form of the proteins. In this reaction, no appreciable differences were noted between polymerizable and nonpolymerizable proteins, including BSA. The specific, large increase in absorbance at wavelengths characteristic of the aromatic amino acids tyrosine and tryptophan suggests that significant changes in the structure of IF subunit proteins take place as a result of their interactions with pepstatin A. Whereas pepstatin A contains no aromatic amino acids and has no significant absorption at these wavelengths, murine vimentin (Wood et al., 1989; Hennekes et al., 1989) has five tyrosine residues in the N-terminal, non-a-helical head domain and eight tyrosine and one tryptophan residues in the central, a-helical rod domain. It is plausible, although unproven, that there are direct interactions between pepstatin A and the aromatic amino acids themselves. This change in structure of IF subunit proteins is further evidenced by a decrease in susceptibility to cleavage by HIV-l protease, although this must be viewed cautiously since pepstatin A is also an inhibitor of the HIV-l protease. More convincing evidence is provided by the shift in utiliza-

PEPSTATIN

A AND

tion of the secondary cleavage sites of vimentin seen under conditions where the HIV-l protease is only partially inhibited. The fact that secondary site 2 (Shoeman et al., 1990a,b) now becomes the primary site could be easily explained if the conformation of the head domain of vimentin changes as a result of interaction with pepstatin A such that cleavage at this site is favored. We have previously observed a similar phenomenon as a result of changing the pH of the filament assembly buffer (Shoeman et al., 1990b), which is known to influence the assembly of IFS presumably as a result of changes in subunit protein conformation (Aebi et al., 1988). It seems likely that other reagents, such as membrane lipids, might also exert an effect similar to that of pepstatin A in altering IF subunit protein structure and/or inducing or supporting polymerization of IFS through primarily non-ionic interactions. This speculation is raised to call attention to a current dilemma in IF research: IFS have been observed to traverse the cell, from cell periphery to nuclear envelope, and have been proposed to form a continuum with the nuclear lamina (French et al., 1989). Yet, to date, no universally accepted theory has been proposed as to how the cytoplasmic IFS might penetrate the nuclear membrane. Our present results, that IF subunit proteins can form filamentous structures stabilized through primarily non-ionic interactions, implies that IFS may indeed be capable of traversing the nuclear membrane in an atypical, yet stable form. This hypothesis, of course, awaits experimental confirmation, but is nonetheless in accordance with some current ideas and previous observations concerning IF interactions with lipids (Asch et al., 1990; Perides et al., 1986a,b). Lastly, these results have more general implications for the interpretation of experiments employing pepstatin A and other similar compounds. Not only can pepstatin A self-associate to form higherorder structures (with reduced ability to associate with proteases), but pepstatin A can also interact with other proteins in an orderly fashion primarily through non-ionic interactions, as has been shown for pepsin (Aoyagi et al., 1972; Kunimoto et al., 1974). We have demonstrated in this study that pepstatin A additionally induces conformational changes in vimentin and propose that this may also occur with other proteins. After this study was completed, Wallin et al. (1990) noted that pepstatin A induced the formation of aberrant forms of microtubules in in vitro assembly assays for the cleavage of microtubule-associated proteins by retroviral proteases (including HIV-l protease). These results, mentioned only as data not shown, fit well with our prediction that pepstatin A may interact with many different proteins and thereby change their aggregation properties.

IF SUBUNIT

71

PROTEINS

We thank Swapan Roy, Emil Russoman, and Seth Monkarsch (HoEmar-LaRoche, Inc., Nutley, NJ) for pure HIV-l protease, Mrs. Margot Bialdiga and Mrs. Ulrike Traub for tissue culture cells, Ms. Annemarie Scherbarth for assistance in protein purification, Ms. Cornelia Kesselmeier for assistance in gel electrophoresis, Mrs. Christel Fabricius for the artwork, Mrs. Erika Schindler for the photographs, and Mrs. Heidi Klempp for secretarial work. REFERENCES AEBI, U., FOWLER, W. E., REW, Biol. 97, 1131-1143.

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