Production and Enzymatic Degradation of Sup35NM_ a Prion-Like Protein from Yeast

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					                                         Abstract

ROJANATAVORN, KAWAN. Production and Enzymatic Degradation of Sup35NM, a
Prion-Like Protein from Yeast. (Under the direction of Jason C.H. Shih)


       Prion diseases, or transmissible spongiform encephalopathies (TSEs), including

human Creutzfeld-Jakob disease (CJD) and bovine spongiform encephalopathy (BSE),

are fatal neurodegenerative diseases of humans and animals that are caused by aggregates

of the inheritable, protease-resistant, self-propagating isoform of prion proteins, PrPSc.

A bacterial keratinase produced by Bacillus licheniformis strain PWD-1 was found to be

able to degrade this prion under certain conditions; however, since this disease-causing

prion is infectious and expensive to work with, a model or surrogate protein was needed

for laboratory studies. This study developed the use of a yeast prion protein system, the

prion-determining NM region of the yeast prion protein Sup35p from Saccharomyces

cerevisiae, that is structurally similar, but non-pathogenic, to study the enzymatic

degradative process. Sup35NM was expressed and purified from Escherichia coli to

form amyloid in vitro, a phenomenon similar to the amyloid formation in vivo in CJD and

BSE. Aggregation and de-aggregation of Sup35NM were examined by gel

electrophoresis, Congo red binding, fluorescence, Western blot and electron microscopy.

Proteinase K (PK) and keratinase (KE) were used to characterize the yeast prion,

Sup35NM. Protease concentrations, preheating temperatures, reaction temperatures, and

reaction times were studied to characterize its enzymatic digestion patterns.

Consequently, optimal conditions and other active enzymes can be further tested with

BSE tissues and prions. In the long run, an effective enzymatic process can be developed

to inactivate prions in food and feed to prevent the spread of TSEs.
      PRODUCTION AND ENZYMATIC DEGRADATION OF SUP35NM,

                    A PRION-LIKE PROTEIN FROM YEAST




                                          by

                            KAWAN ROJANATAVORN




A thesis submitted to the Graduate Faculty of North Carolina State University in partial

          fulfillment of the requirements for the degree of Master of Science



                                 MICROBIOLOGY



                               Raleigh, North Carolina

                                         2004



                                  APPROVED BY:




_______________________________                  _______________________________




                        _______________________________

                            Chair of Advisory Committee
DEDICATED TO MY PARENTS AND MY BROTHER




                  ii
                                     Biography

       Kawan Rojanatavorn was born on May 8, 1973. He attended primary and

secondary schools in Raleigh, North Carolina where he graduated from William G. Enloe

High School in 1990. He entered North Carolina State University in 1990 and received a

Bachelor of Science degree in Biochemistry, Biological Sciences, and Zoology and a

Bachelor of Arts degree in Chemistry in 1994. Upon graduation, he worked at Rhone-

Poulenc Ag Company in the Research Triangle Park. After developing an interest in

microbiology, Kawan returned to his alma mater to pursue and receive a Bachelor of

Science degree in Microbiology in 2000 followed by this Master of Science degree with a

minor in Biotechnology.




                                          iii
                                 Acknowledgements

       The author expresses his sincere appreciation to Dr. Jason C.H. Shih, his major

professor and committee chair, for the never-ending encouragement and guidance during

the course of his graduate work. His gratitude is also extended to the other members of

his Advisory Committee, Dr. James W. Brown and Dr. A. Clay Clark.

       Special thanks go to Dr. Ching-Ying Chen for her collaboration in this

investigation and to Dr. J.J. Wang and Mr. Brian Spencer for their continued assistance

over the years.

       The author would also like to thank his coworkers, Brian, Brad, J.J., Magi, Sandy,

Rattana, and Nasser, for their friendship and making lab and Friday lunch meetings fun.

       Finally, a special note of thanks is extended to the author’s family for all of their

love and support.




                                             iv
                         Table of Contents
                                                                 Page
List of Figures………………………………………………………………………... ...vii
List of Tables………………………………………………………………………… ..viii

Chapter I – Literature Review…………………………………………………………....1

     Prions…………………………………………………………………………... ..1
         Prion Characteristics……………………………………………………….. ..1
         Prion Diseases……………………………………………………………… ..3

     Yeast Prions……………………………………………………………………. ..5
        Yeast Prion Protein Determinant, Sup35p…………………………………. ..6
        Yeast Prion Inheritance and Maintenance…………………………………. 10

     Sup35p in Escherichia coli…………………………………………………….. 10
        Expression of Sup35p Fragments………………………………………….. 12
        Expression Constructs………………………………………………………13
        In Vitro Seeding……………………………………………………………. 15
        Analysis of NM Aggregates………………………………………………...18

     PWD-1 Keratinase……………………………………………………………... 19

     Literature Cited………………………………………………………………… 22

Chapter II – Production and Enzymatic Degradation of Sup35NM,
             a Prion-Like Protein from Yeast…………………………………………. 29

     Abstract………………………………………………………………………… 29

     Introduction…………………………………………………………………….. 31

     Materials and Methods………………………………………………………….32
        Expression Construct………………………………………………………. 32
        Expression and Production of Monomeric Sup35NM……………………... 32
        Isolation and Purification of Sup35NM……………………………………. 33
        Concentration and Storage of Purified Sup35NM…………………………. 34
        Aggregation and De-Aggregation of Sup35NM…………………………… 34
        Congo Red Binding Assay…………………………………………………. 35
        Thioflavin T Binding Assay………………………………………………...35
        Protease Resistance of Sup35NM………………………………………….. 35
        Degradation of Sup35NM………………………………………………….. 36
        Western blot analysis………………………………………………………. 36
        Keratinase and Proteinase K……………………………………………….. 37
        Electron Microscopy……………………………………………………….. 37


                                 v
Results………………………………………………………………………….. 38
   Properties of Sup35NM……………………………………………………. 38
   Enzymatic degradation……………………………………………………...42
   Pre-heating treatment………………………………………………………. 42
   Keratinase amount and digestion time……………………………………... 49

Discussion……………………………………………………………………… 54

Acknowledgements…………………………………………………………….. 58

Literature Cited………………………………………………………………… 59




                          vi
                                 List of Figures

Chapter I – Literature Review
                                                                             Page
Figure 1   The effect of [PSI+] on Sup35 and translation termination………………... ..7
Figure 2   A model for prion formation in yeast…………………………………….... 11
Figure 3   The N, M, and C regions of Sup35p……………………………………….. 14
Figure 4   pJC25 plasmid with NM gene………………………………………………16
Figure 5   DNA analysis of pJC25NMstop…………………………………………… 17
Figure 6   Comparison of enzymatic relative specific activity………………………... 21


Chapter II – Production and Enzymatic Degradation of Sup35NM,
             a Prion-Like Protein from Yeast

Figure 1   Transmission electron microscopy of Sup35NM………………………….. 39
Figure 2   Congo red binding assay during Sup35NM polymerization………………..40
Figure 3   SDS-PAGE of Sup35NM during polymerization………………………….. 41
Figure 4   Fluorescence emission of ThT bound Sup35NM………………………….. 43
Figure 5   PK and KE degradation of Sup35NM: SDS-PAGE……………………….. 44
Figure 6   PK and KE degradation of Sup35NM: Western blot…………………... .45,46
Figure 7   Various pre-heating temps, KE amounts, and reaction temps
           on Sup35NM: SDS-PAGE……………………………………………... .47,48
Figure 8   Various pre-heating temps, KE amounts, and reaction temps
           on Sup35NM: Western blot……………………………………………. .50,51
Figure 9   Various KE amounts and reaction times on Sup35NM with and
           Without pre-heating: Western blot……………………………………....52,53




                                        vii
                                List of Tables

Chapter I – Literature Review
                                                                Page
Table 1   Comparison of Yeast Prion and BSE Prion………………………………... .9




                                     viii
                                    CHAPTER 1


                                 Literature Review



                                          Prions

       Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a

unique group of fatal neurodegenerative diseases of humans and animals that are both

inheritable and infectious (Prusiner et al., 1998). They are responsible for diseases such

as kuru, Creutzfeld-Jakob disease (CJD), Gerstmann-Straussler syndrome (GSS), and

fatal familial insomnia (FFI) in humans, scrapie in sheep, and bovine spongiform

encephalopathy (BSE), widely known as mad cow disease, in cattle (Parchie and

Gambetti, 1995). All of these diseases are characterized by extensive neuronal loss

giving sectioned brain tissue a spongy appearance. In most of these diseases, deposits of

amyloid, a polymer that assembles into fibrils with a high β-sheet content, are present in

the affected tissues (Prusiner et al., 1983; Sapriel, 2001; Sunde and Blake, 1998).


Prion characteristics

       The term prion was originally defined to describe the proteinaceous, infectious

particle that causes these diseases (Prusiner, 1982). In a wider sense, it refers to proteins

that have the ability to convert autocatalytically into an abnormal conformation. The

common feature of all prion diseases is the aberrant metabolism of the prion protein, PrP



                                              1
(Glatzel and Aguzzi, 2001). PrP exists in at least two conformational states with distinct

physicochemical properties. The cellular form of the prion protein, referred to as PrPC, is

a membrane-bound protein of unknown function. It has an apparent molecular mass of

33-35 kDa and is expressed at high levels in neurons and in lower levels in cells of the

immune system and in muscle (Bendheim et al., 1992). It is encoded by a single copy

mammalian gene designated PRNP in humans and prnp in animals (Oesch et al., 1985).

In humans, the PRNP gene is located on chromosome 20 (Sparkes et al., 1986). The

structure of PrPC has been resolved by nuclear magnetic resonance studies. It comprises

three α-helices and a C-terminal globular domain. The disease-associated isoform of

PrPC is termed PrPSc. In contrast to PrPC, PrPSc is insoluble and partially protease-

resistant. The structure of PrPSc has a high β-sheet content. PrPC is about 42% α-helical

and about 3% β-sheet, whereas, PrPSc is about 30% α-helical and 43% β-sheet (Pan et al.,

1993).

         In its simplest form, prions are devoid of any informational nucleic acids and

consist solely of PrPSc (Riesner et al., 1993). Aggregation is the hallmark of the change

in state associated with the conversion of PrPC to PrPSc (Lansbury and Caughey, 1995).

Interaction of PrPSc with the normal host protein, PrPC, forces PrPC to adopt the

conformation of PrPSc resulting in an amplification of the infectious agent. The key to

prion pathology is this ability of PrPSc to induce new PrPC molecules to adopt the altered

structure, producing a protein-conformation cascade that causes the disease and gives rise

to new PrPSc (Patino et al., 1996). The PrP molecule has two domains that play different

roles in the conversion of PrPC to PrPSc (Kaneko et al., 1997). First, there is the ‘stable’

or ‘ordered’ core domain that contains PrP’s two asparagine-linked oligosaccharides; two



                                              2
α-helices, helix-B and helix-C, that are stabilized by a disulfide bridge; a

phosphatidylinositol glycolipid (GPI) which anchors PrPC to the plasma membrane; and

protein binding sites believed to lower the energy barrier for conversion of PrPC to PrPSc

(Telling et al., 1995). Second, there is the ‘variable’ or ‘disodered’ domain that contains

the portion of PrPC that interacts with PrPSc and changes its conformation from primarily

unstructured to β-sheet (Prusiner et al., 1998).

          PrPC is completely degraded when limited proteinase K (PK) digestion is carried

out on healthy animal brain and visceral tissue extracts. In contrast, PrPSc is only

partially cleaved in infected brain extracts subjected to the same treatment. This

differential resistance to proteolysis, being able to hydrolyze PrPC and leaving the PrPSc

core intact, PK is commonly used in the diagnosis of TSE and the detection of PrPSc

(Bousset and Melki, 2002). Depending on host species and TSE strain, PK removes 55 to

70 residues from the N-terminal domain of PrPSc, yielding a distinct product, PrPres,

which consists mainly of three glycoforms: aglycosyl, monoglycosyl, and diglycosyl

fractions (Thuring et al., 2004). The relative concentrations of these three glycoforms

(glycosylation profile) are used as biochemical targets for discriminating infecting TSE

agents.


Prion diseases

          Prion diseases have unusual properties in that they have extremely long

incubation periods from a few months to several years, there is no inflammation and no

disease-specific immune response, and they have three different manifestations that are

unlikely related; infectious, inherited and sporadic disorders (Prusiner, 1998). Based on

their neuroanatomical features and the properties of the pathogenic PrPSc protein in the


                                              3
brain, prion diseases in animals and humans can be divided into three broad categories

(DeArmond and Bouzamondo, 2002). The first category includes the vast majority of

prion diseases including scrapie in sheep and rodents; BSE; kuru; sporadic, familial and

iatrogenic CJD (sCJD, fCJD, and iCJD); and sporadic and familial fatal insomnia (SFI

and FFI respectively). This category is characterized by vacuolar (spongiform)

degeneration of gray matter, accumulation of protease resistant PrPSc in the gray matter

neuropil and little or no PrP amyloid plaque formation.

       The only diseases in the second category are the seven dominantly inherited

syndromes designated GSS. The defining neuropathological characteristic is the

deposition of numerous PrP immunopositive amyloid plaques in multiple cortical and

subcortical brain regions that are composed of highly truncated PrP peptides spanning

residues 90-160 (Ghetti et al., 1996). These peptides are highly amyloidogenic and,

when released into the extra-cellular space, polymerize into the large numbers of amyloid

plaques. The majority of mutations linked to GSS neuropathological changes occur in

this domain. Unlike the protease-resistant, full-length mutated PrP that accumulates in

the gray matter in familial CJD (designated ∆PrP) is protease-sensitive (Hedge et al.,

1999; Hsiao et al., 1994). Similarly, ∆PrP in the gray matter of transgenic mouse models

of GSS is protease sensitive.

       The third category of human prion disease is represented by a new variant of CJD,

designated vCJD, that was transmitted to humans by ingestion of BSE-contaminated food

products in Great Britain and to a lesser extent in the rest of Europe (Scott et al., 1999).

The first cases of BSE in the United Kingdom were reported in 1986. Beginning in 1994,

the first cases of vCJD were discovered in young men and women with a mean age of 28



                                              4
(Wells et al., 1987). Like GSS, there is abundant PrP amyloid deposition and likeCJD

and scrapie, there is intense vacuolation of the gray matter and accumulation of protease-

resistant PrPSc in the neuropil.



                                      Yeast Prions


       Prion proteins are also linked with stable, heritable traits in two fungi,

Saccharomyces cerevisiae and Podospora anserina. Although none of the four known

fungal prions can be considered ‘disease-causing,’ some are actually beneficial to the

host (Uptain and Lindquist, 2002). In yeast, [PSI +] is a genetic element of S. cerevisiae

for which a heritable change in phenotype is caused by a heritable change in the

conformational state of the Sup35 protein, Sup35p (Wickner, 1994). [PSI +] yeast cells

are metastable, occurring spontaneously in normal cells at low frequency, but have an

inheritable, non-Mendelian pattern of transmission (Sapriel, 2001). A [PSI +] and [psi -]

cross of haploid strains results in diploid yeast with the [PSI +] phenotype. Meiotic

division of these diploids produces four progeny, all with the [PSI +] phenotype (Cox,

1965). [PSI +] transmissibility from one cell to another by cytoplasmic fusion, although

unlinked to a nucleic acid, behaves like a dominant, cytoplasmically inherited genetic

element because of its prion-like ability to self-perpetuate in an alternate protein

conformation (Conde and Fink, 1976). Its unusual relation to the nuclear-encoded

protein Sup35p is reminiscent of the relation between mammalian prions and nuclear-

encoded PrPC (Prusiner et al., 1998; Wickner et al., 1999).




                                              5
Yeast Prion Protein Determinant, Sup35p

        The altered conformation of Sup35p, the yeast homolog of the eukaryotic release

factor eRF3, is the protein determinant of [PSI +] (Didichenko et al., 1995). In its native

state, Sup35p forms a functional translation termination complex with Sup45p, the yeast

homolog for eRF1, that directs the faithful termination of translation at stop codons in

[psi -] cells (Stansfield et al., 1995). In [PSI +] cells, the function of Sup35p is

sequestered from translation and unfaithful termination leads to read-through of nonsense

codons and the expression of ORFs that are usually interrupted (Fig. 1). This epigenetic

loss of Sup35p activity results from a unique type of Sup35p aggregation, an insoluble

aggregate composed of amyloid fibrils of the Sup35p protein that has only been isolated

from [PSI +] strains (Patino et al., 1996). It is this prion conformation of Sup35p that is

protease-resistant, especially in vitro (King et al., 1997).

        Based on its amino acid composition and homology to other translation factors,

Sup35p is a multidomain protein composed of three regions: N, the amino-terminal

region, M, the middle region, and C, the carboxy-terminal region (Kushnirov et al.,

1988). The N region, amino acids 1-123, is the prion-determining domain of Sup35p and

is necessary for the propagation of [PSI +] (Doel et al., 1994; Ter-Avanesyan et al.,

1994). Transient overexpresssion of this region alone is enough to induce new [PSI +]

elements in all [psi -] strains expressing full-length Sup35p (Derkatch et al., 1996;

Chernoff et al., 1992; Chernoff et al., 1993). N has a high propensity for self-association

(Patino et al., 1996). It is always aggregated when expressed as an isolated domain in

yeast and has been shown to be highly amyloidogenic in vitro (Serio et al., 1999). It is

insoluble in physiological buffers and forms amyloid even in the presence of denaturant




                                               6
Figure 1. The effect of [PSI+] on Sup35 and translation termination. (A) In [psi-]
cells, a complex of Sup35 (see legend at right) and Sup45 (not shown) binds
ribosomes at stop codons to mediate translational termination. (B) In [PSI+]
cells, prion-forming domains of the majority of Sup35 adopt the prion
conformation and self-assemble into an aggregated structure. This
conformational change impairs the ability of Sup35 to participate in translational
termination; consequently, stop codons are read through occasionally.
(Adapted from Chernoff et al., 2002).




                                        7
(Glover et al., 1997). 77% of the N region is comprised of the amino acid residues

glycine (G, 17%), tyrosine (Y, 16%), asparagines (N, 16%), and glutamine (Q, 28%)

(Serio and Lindquist, 2001a). Its primary structure includes five imperfect repeats of the

nonapeptide PQGGYQQYN, which bears a striking resemblance to the octapeptide

repeat of the mammalian PrP responsible for BSE (Sapriel, 2001). The glutamine-rich

repeats play a central role in the self-assembly of N in vivo and in vitro (Liu and

Lindquist, 1999). Deletion of N results in the irreversible loss of the [PSI +] phenotype.

A comparison of yeast Sup35p and BSE PrPSc is summarized in Table 1.

       The M region of Sup35p, amino acids 124-253, is not essential for the induction

of [PSI +]; however, it does enhance the solubility of the prion-determining N region and

profoundly alters the N region’s behavior both in vivo and in vitro (Derkatch et al., 1997).

The highly-charged residues of the M region is strongly biased to two amino acids,

glutamic acid (18%) and lysine (19%) (Masison, 2000; Tuite, 2000). In conjunction with

N, NM can exist in multiple states in vivo modeling the differences in Sup35p solubility

that are characteristic of [PSI +] and [psi -] strains (Paushkin et al., 1996). NM assembly

accelerated by the addition of preformed NM amyloid or lysates from [PSI +], but not

[psi-] cells, link properties of amyloid formation in vitro to the propagation of [PSI +] in

vivo (Glover et al., 1997). This is the basis of protein-conformation self-perpetuation and

the protein-only mode of inheritance for [PSI +] (Serio et al, 1999).

       The C region of Sup35p, amino acids 254-686, is responsible for the protein’s

translation termination activity (Serio and Lindquist, 2001b). It is evolutionarily

conserved and is structurally similar to the yeast translational elongation factor EF-1α.

The C region forms a complex with Sup45p and has a polypeptide chain release function




                                              8
                             Yeast Prion (Sup35pPSI+)          BSE Prion (PrPSc)

Solubility                   Insoluble (aggregate)          Insoluble (aggregate)

Protease (common conditions) Resistant                      Resistant core

Structure                    Rich in β-sheet                Rich in β-sheet

Polymerization               Yes                            Yes

Isoform (normal cellular form) Monomer, Sup35p              Monomer, PrPC

Characteristic Sequence      5 imperfect repeats (PQGGYQQYN) 5 copies (PHGGGWGQ)

Infectivity                  Infectious, non-pathogenic     Infectious, pathogenic




Table 1. Comparison of Yeast Prion and BSE Prion.




                                          9
(Paushkin et al., 1997). Unlike the N region, it is essential for viability but dispensable

for both the maintenance and induction of [PSI +].


Yeast Prion Inheritance and Maintenance

       The inheritance and maintenance of [PSI +] and the physical state of Sup35p in

vivo depend on the protein chaperone Hsp104, a heat shock protein (Fig. 2). Hsp104, the

most crucial thermotolerance-related protein found in Saccharomyces cerevisiae, is a

stress tolerance factor that promotes the reactivation of heat-damaged proteins in yeast

(Sanchez and Lindquist, 1990). When present at its normally low constitutive level,

Hsp104 partially unfolds Sup35p or disassembles Sup35p/Sup45p heterodimers, thereby

overcoming conformational or kinetic barriers to the formation of ordered Sup35p

aggregates (Patino et al., 1996). Alternatively, Hsp104 can also dissociate large,

preformed Sup35p aggregates into smaller, aggregation-prone particles that seed the

aggregation of newly synthesized Sup35p more efficiently (Glover and Lindquist, 1998).

Either too much or too little Hsp104 can cure cells of [PSI +]. Hsp104, however, is not

essential for the actual Sup35p prion-like conversion. Although Hsp104 is required for

the inheritance of the [PSI +] prion state in vivo, it is not required for the polymerization

of Sup35p into fibrils in vitro (Kushnirov and Ter-Avanesyan, 1998).




                              Sup35p in Escherichia coli


       The fact that mammalian prions and Sup35p, which are both able to undergo self-

propagating conformation changes, are also able to adopt filamentous structures suggests

that filament growth by seeding can be a common mechanism for prion propagation



                                              10
Figure 2. A model for prion formation in yeast. 1: Newly synthesized Sup35p
(white shapes at left) interacts with the chaperone Hsp104 (black ovals at
center). 2: Hsp 104 helps Sup35p achieve a protein-folding transition state that
is required for prion formation but is inherently unstable. 3: In the absence of
[PSI +], Sup35p reverts to its normal functional state. 4: Preexisting [PSI +]
elements capture and stabilize transition-state conformers; Sup35p is
sequestered from translation and unfaithful termination leads to nonsense
suppression. 5: Transient overexpression of Sup35p nucleates prions de novo
because the high concentration of transition-state conformers increases the
likelihood of stabilizing intermolecular interactions. These interactions may be
facilitated by the simultaneous binding of several Sup35p proteins to an Hsp104
hexamer or by rapid sequential binding and release of individual conformers in its
immediate vicinity. 6: In the absence of Hsp104, the transition state is difficult to
attain and prions cannot be perpetuated. 7: Overexpression of Hsp104 might
disturb the equilibrium in several ways: Hsp104 might bind prion-state
conformers and disaggregate them; rebind monomers, reducing their ability to be
captured by [PSI +] elements; or reduce the local concentration of transition-state
conformers because they are dispersed in association with larger numbers of
Hsp104. (Adapted from Patino et al., 1996).




                                         11
(King et al., 1997). Recent studies have linked the process of amyloid formation in vitro

to the propagation of [PSI+] in vivo (Serio et al., 1999). Fragments of Sup35p capable of

inducing [PSI+] in vivo form amyloid in vitro. Lysates from [PSI+] but not [psi-] strains

accelerate the formation of amyloid in vitro, as do preformed fibrils (Glover et al., 1997).

Deletions within the prion-determining N region, as well as specific point mutations,

slow the process of assembly into amyloid as well as block the induction of new [PSI+]

elements (DePace et al., 1998). Simlarly, the expansion of repeated sequences in the N

region accelerates amyloid formation in vitro and increases the efficiency of [PSI +]

induction in vivo (Liu and Lindquist, 1999).


Expression of Sup35p Fragments

       The abilities of N, M, NM and NMC, have been assessed, expressed and purified

from E. coli to form amyloid in vitro (Glover et al., 1997; King et al., 1997). E. coli is

the most studied bacterium and the most easiest-to-use recombinant tool. It provides

several options with regard to plasmids and strains available for cloning. Although all

Sup35p fragments containing the N region are capable of the ordered assembly of

amyloid, NM and NMC fragments most accurately reflect [PSI +] metabolism in vivo

(Derkatch et al., 1997).

       Fragments containing the N region alone are insoluble in physiological buffers

and assemble into fibers rich in β-sheet structure too rapidly in 2M urea (Glover et al,

1997). In contrast, fragments encompassing both the N and the highly charged M domain

(NM) are soluble in non-denaturing buffers and assemble into fibers only after a

considerable lag. These fibers more closely share characteristics with protein amyloids

implicated in several human diseases (Serpell et al., 1997). For example, Sup35p fibers


                                             12
(NMC) are protease resistant and bind to the dye Congo red, producing the characteristic

spectral shift and apple-green birefringence under polarized light (King et al., 1997; Serio

and Lindquist, 1999). Most convincingly, by X-ray diffraction, NM fibers exhibit the

dominant reflections characteristic of the cross-pleated β-sheets of other amyloid

proteins.

       The M region and the C region alone do not form fibers in vitro, but the whole

protein, NMC, does (Fig. 3). However, NMC does not form fibers as reproducibly as

NM, perhaps because the large C region is not in its native state after purification and is

prone to other types of aggregation (Serio and Lindquist, 1999). By electron microscopy,

N and NM fibers are smooth, with diameters of 7 and 10 nm, respectively. NMC

fibers exhibit shaggy protuberances from a central fiber core of 10 nm (Glover et al.,

1997). Thus, just as the N region is necessary and sufficient for the induction of [PSI+]

in vivo, it seems to be the primary component directing fiber formation in vitro, with the

M and C domains entering the fiber only because they are attached to it. Furthermore,

NM seems to serve as the better model in vitro by aggregating as well as N fragments,

but without sacrificing the characteristics of the full protein, NMC.


Expression Constructs

       The expression of all cloned fragments of Sup35p were driven by T7 polymerase.

Fragments were cloned into either pJC45 encoding an amino-terminal 10 residue

histidine tag (His10) or pJC25, lacking a tag (Serio et al., 1999). These plasmids are high

copy number, containing the pUC origin of replication, and have a consensus T7

promoter and the lacI operator at the 5’ end of the multiple cloning site. Sup35p




                                             13
Figure 3. The N, M, and C regions of Sup35p and results of the expression of
their various combinations. (Adapted from Serio and Lindquist, 2001).




                                      14
fragments were inserted between the NdeI and the BamHI sites, allowing in-frame fusion

as in the case of pJC25NMstop (Fig. 4,5). The plasmids impart ampicillin resistance.

       Each construct was expressed in BL21 [DE3] pAP lacI q. This strain contains a

[DE3] lysogen for the high-level expression of T7 polymerase following induction with

isopropyl-β-D-thiogalactopyranoside (IPTG). The strain also expresses a low level of the

lacI product to repress leaky expression of the polymerase and, therefore, the target

protein. The strain is also kanamycin resistant.


In Vitro Seeding

       The delay in NM assembly into amyloid fibers in vitro provides a window in

which the effects of known modifiers of [PSI+] propagation can be assessed. The prion

hypothesis and the behavior of NM-GFP fusions in vivo predicted that conversion to the

[PSI+] state would be accelerated by protein already in that state. Indeed, in

physiological buffers, small quantities of pre-formed fibers greatly accelerate the rate at

which NM forms fibers (Glover et al., 1997). Preformed fibers also accelerate the rate at

which N forms fibers in 40% acetonitrile (King et al., 1997). Second, when NM

fragments contain a deletion in the N region of Sup35p, which inhibits [PSI+] formation

in vivo ( BstEII), fibers form only after a very prolonged lag phase, even with the

addition of preformed fibers (Glover et al., 1997). Third, point mutations in the N region,

which interfere with [PSI+] propagation in vivo, reduce the rate of Sup35p self-assembly

in vitro (DePace et al., 1998). Finally, a mutant that enhances the rate at which [PSI+]

elements form in vivo, enhances the rate at which NM forms fibers in vitro (Liu and

Lindquist, 1999).




                                             15
Figure 4. pJC25 plasmid with NM gene insertion between NdeI and BamHI.




                                    16
                                   3087 bp – pJC25plasmid with NM gene

                                   2333 bp – pJC25 plasmid without NM gene




                                   754 bp – Sup35p NM gene fragment




Figure 5. DNA analysis of Expression Plasmid pJC25 with & without NM gene.




                                    17
Analysis of NM Aggregates

       Several methods for analyzing NM amyloid formation have been established over

the past ten years. Similar to PrPSc, differential resistance to PK digestion is also useful

in detecting NM aggregates. NM fibers, or polymers, are more resistant to proteinase K

digestion than NM monomers (Uptain and Lindquist, 2002). By digesting samples of

NM with dilute concentrations of PK and running the samples on SDS-PAGE, NM

aggregates will appear on the gel while NM monomers will not.

       Like many other amyloidogenic proteins, NM fibers also bind to the diagnostic

dye, Congo red (Serio et al., 1999). Monitoring Congo red binding over an assembly

time course is a sensitive probe for fiber formation. NM fibers exhibit a spectral shift in

absorbance, with a new peak at 540nm, in comparison to unpolymerized protein or

Congo red alone.

       Binding to thioflavin T (ThT) is another way to detect NM aggregates in vitro

(Chernoff et al., 2002). ThT is a yellow dye that fluoresces faintly at 438nm when

excited at 350nm in the absence of amyloid. In the preence of amyloids, including NM

fibers, ThT fluoresces brightly at 481nm when excited at 450nm. There are several

advantages of using ThT instead of Congo red. Unlike Congo red, ThT does not inhibit

NM fiber formation; thus, fiber formation may be performed in the presence of the dye.

Second, ThT is much more sensitive and has a greater dynamic range. Third,

fluorescence of ThT is more specific for amyloid than the spectra shift of Congo red

(LeVine, 1999). Finally binding of ThT is very rapid so a prolonged incubation period is

not required.




                                             18
       Assembly of NM into amyloid can also be monitored by the degree of

solubilization by 2% (w/v) SDS (Serio et al., 1999). Unpolymerized protein remains

soluble in 2% (w/v) SDS. In contrast, once amyloid has formed, these structures are

largely insoluble in 2% (w/v) SDS at room temperature. The same amount of

unpolymerized NM enters the gel whether or not samples have been boiled. Conversely,

NM fibers only enter the gel in boiled samples. The predicted molecular weight of NM is

28.5 kDa; however, due to the presence of the highly charged M region, NM can migrate

aberrantly by SDS-PAGE up to 45 kDa. This difference in SDS solubility, combined

with SDS-PAGE, is utilized as a qualitative assay to monitor fiber formation.

       The most important method to date for identifying the presence of amyloid is by

microscopy. Transmission electron microscopy (TEM), scanning transmission electron

microscopy (STEM), and atomic force microscopy (AFM) have been utilized to monitor

the assembly ofNM into amyloid (Chernoff et al., 2002). Although each of these

techniques provides distinct information about the structure and size of complexes

formed by NM, EM is most often used due to the general accessibility of this technique.

NM fibers are routinely negatively stained for EM analysis.



                                 PWD-1 Keratinase


       A feather-degrading bacterium, Bacillus licheniformis strain PWD-1, was

discovered and isolated from a thermophilic waste digester (Williams and Shih, 1989;

Williams et al., 1990; Shih, 1993). Subsequently, PWD-1 keratinase (Lin et al., 1992)

and the gene encoding the enzyme (Lin et al., 1995) were isolated and sequenced.

Characterization of the PWD-1 keratinase confirmed that it is a serine protease capable of


                                           19
the hydrolysis of all kinds of protein tested. Fermentation production of this enzyme was

scaled up to a 150-liter fermentor (Wang and Shih, 1999). In feed applications, PWD-1

keratinase is able to convert feathers into digestible feed protein (Williams et al., 1991)

and potentially improve digestibility of common feed and the growth of animals

(Odetallah et al., 2003). Like Sup35p aggregates and PrPSc, feather keratin has a β-sheet

rich protein structure, and is resistant to most proteases.

       Purified PWD-1 keratinase was compared with other proteases, including

elastase, collagenase, proteinase K (PK) and trypsin in reacting with various kinds of

protein substrates. Relative specific activities of the proteases against different substrates

were compared (Fig. 6). It demonstrated that PWD-1 keratinase has a wide range of

substrates and is more active than all other proteases tested including PK. From a series

of in vitro tests, it has been concluded that, after a pre-cooking step, BSE and scrapie

PrPSc can be degraded by PWD-1 keratinase digestion (Langeveld et al., 2003). Brain

stem tissues of BSE and sheep scrapie were homogenized and cooked at 115 oC for 40

min. They were then digested with PWD-1 keratinase at 50 oC for 30 min. The detection

of PrPSc was analyzed by the standard Prionics method, including gel electrophoresis,

Western blot and immunochemical reactions with specific monoclonal antibodies.




                                              20
Figure 6. Comparison of relative specific activity of PWD-1 keratinase, elastase,
collagenase, proteinase K and trypsin with four different substrates.




                                       21
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                                            28
                                   CHAPTER 2


      Production and Enzymatic Degradation of Sup35NM,

                      a Prion-Like Protein from Yeast



                                        Abstract

       Transmissible spongiform encphalopathies (TSEs) such as mad cow disease

(Bovine spongiform encephalopathy, BSE) and variant Creutzfeld-Jakob disease (vCJD)

have become a worldwide problem over the last two decades affecting everything from

the cattle industry to human health. These neurodegenerative disorders are believed to be

caused by an unconventional infectious agent, the prion protein. The infectious isoform,

PrPSc, is able to aggregate and form amyloid fibrils, very stable and resistant to most dis-

infecting processes and common proteases. Under specific conditions, PrPSc in BSE

brain tissue was found degradable by a bacterial keratinase and some other proteases.

Since this disease-causing prion is infectious and dangerous to work with, a model or

surrogate protein was needed for the degradation study. A non-pathogenic yeast prion-

like protein, Sup35NM, was produced and characterized for this purpose. The gene

fragment was cloned and over-expressed in E. coli and the Sup35NM protein was

purified. Aggregation and de-aggregation of Sup35NM were examined by gel

electrophoresis, Congo red binding, fluorescence, Western blot and electron microscopy.



                                             29
The degradability of Sup35NM aggregates and the conditions of degradation by

keratinase and proteinase K were studied and compared. These results will be of value in

understanding the mechanism and optimization of the degradation process. In the long

run, an effective enzymatic process for cleaning and disinfecting equipment used to

handle contaminated materials may be developed.




                                           30
                                     Introduction


       Prion diseases, or transmissible spongiform encephalopathies (TSEs), are a

unique group of fatal neurodegenerative disorders found in humans and animals (Prusiner

et al., 1998). They include diseases such as kuru, Creutzfeld-Jakob disease (CJD) in

humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE), widely known

as mad cow disease, in cattle (Parchie and Gambetti, 1995). All of these diseases are

characterized by extensive neuronal loss giving brain tissue a spongy appearance.

       The pathological isoform of prion protein (PrPSc) aggregates in diseased tissues

and, in common conditions, is resistant to digestion by proteases (Caughey et al., 1991;

Pan et al., 1993). Recently, it was found that PrPSc in diseased tissues can be degraded

by a feather-degrading keratinase and other microbial proteases when the tissue

homogenate was pre-heated at 115 oC (Langeveld et al., 2003). This result has indicated

a potential method of enzymatic inactivation of prion protein.

       Because PrPSc is pathogenic and dangerous to handle, a safe prion surrogate

protein is needed to study the degradation process in a standard laboratory. Sup35p from

Saccharomyces cerevisiae is a translation termination factor that can also convert into an

insoluble aggregate form, a property very similar to prion protein (Paushkin et al., 1996;

Masison and Wickner, 1995). Sup35p can be divided into three regions, namely, N, M,

and C, based on their positions and different functions. Individual regions and

combinations were assessed, expressed and purified from E. coli to form aggregates, or

amyloid, in vitro (Glover et al., 1997; King et al., 1997). Sup35N, capable of

aggregation, was found to be the prion-forming domain, but Sup35NM and Sup35NMC

more accurately reflected [PSI+] metabolism in vivo (Derkatch et al., 1997). The


                                            31
aggregates of Sup35N and Sup35NM have also been found to be proteinase K resistant

(Kushnirov et al., 2000).

       For these reasons, Sup35NM was selected for this study to be produced, purified

and characterized for its properties as a prion-like protein. Its degradation by keratinase

and proteinase K under various conditions were studied and compared. Being non-

pathogenic, Sup35NM is believed to be a candidate for a safe prion surrogate protein.



                               Materials and Methods

Expression Construct

       The expression plasmid pJC25NMstop containing a DNA fragment encoding the

NM region of Sup35p was cloned into the plasmid pJC25 (Serio et al., 1999). The

plasmid was gifted from Dr. Susan L. Lindquist, the Whitehead Institute at MIT. The

plasmid was stored in E. coli DH5α strain A6798 as a shuttle vector. In order to induce

the expression of Sup35NM with isopropyl-thio-β-D-galactopyranoside (IPTG, Fisher),

the plasmid was transformed into an E. coli expression strain.


Expression and Production of Monomeric Sup35NM

       Competent cells of E. coli strain Rosetta (DE3) pLacI were used for

transformation of the plasmid (Serio et al., 1999). Transformed colonies were inoculated

into a 50 ml LB-Ampicillin (50 ug/ml) flask and incubated for 5 hours at 37 oC with

300rpm agitation. Twenty ml of the growing culture were inoculated into a fresh 500ml

LB-Amp (50 ug/ml) medium. After shaking incubation for 3-4 hours, OD600 ~0.6, the

flasks were induced with 1.0 mM IPTG. After 3 hours, E. coli cells were harvested by



                                             32
centrifugation. The intracellular expression of Sup35NM was confirmed by SDS-PAGE

analysis of total cellular proteins. Cell pellets were collected by centrifugation and stored

at –80 oC.


Isolation and Purification of Sup35NM

       Cell pellets from 1-L culture were lysed in 50 ml lysis buffer [10 mM Tris-HCl,

pH 7.2, 1 mM DTT, 1mM PMSF, 8 M urea] and the resuspended pellet was incubated for

30 min at 25 oC with occasional agitation (Chernoff et al., 2002). The lysate was then

cleared by centrifugation at 30,000g for 20 min at 10 oC. The cleared supernatant from

cell lysis was applied to a 20-ml Q Sepharose Fast Flow column (Pharmacia) pre-

equilibrated with lysis buffer at a flow rate of 3 ml/min. The column was washed with 5x

bed volumes of Q wash buffer I [10mM Tris-HCl, pH 7.2, 1 mM DTT, 1mM PMSF, 8 M

urea, 85 mM NaCl], and 5x volumes of Q wash buffer II [10mM Tris-HCl, pH 7.2, 8 M

urea, 150 mM NaCl]. The protein was eluted in 3 volumes of Q elution buffer [10mM

Tris-HCl, pH 7.2, 8 M urea, 200 mM NaCl]. The eluate from the Q Sepharose was then

loaded directly onto a 25-ml Macro Prep Ceramic Hydroxyapatite Type I 40-um column

(Bio-Rad) pre-equilibrated with Q elution buffer. The column was washed with 2x

volumes of HA wash buffer I [1 mM potassium phosphate, pH 6.8, 8 M urea, 1 M NaCl]

and then with 2x volumes of HA wash buffer II [25 mM potassium phosphate, pH 6.8, 8

M urea]. The protein was eluted using a step gradient of 75 mM and 125 mM potassium

phosphate, pH 6.8 in 8M urea. Fractions (5 ml) were analyzed by 15 % SDS-PAGE

(loading 10 ul per lane) followed by staining with Coomassie Brilliant Blue R-250.

Protein concentrations were determined with the calculated extinction coefficient of 0.90

for a 1 mg/ml NM solution at 280 nm absorbance (Gill and von Hippel, 1989).



                                             33
Concentration and Storage of Purified Sup35NM

       For long-term storage, NM was methanol precipitated to remove urea and the

precipitate stored at –80 oC (Serio et al., 1999). Anhydrous methanol (100%) was added

to eluates containing NM on ice at a ratio of 5:1. The mixture was incubated on ice for

30 min, and the precipitate was collected by centrifugation at 14,000g for 30 min at 4 oC.

The pellet was then washed with 100% methanol (1/2 volume of supernatant) and

collected by centrifugation again. The supernatant was removed and the pellet was stored

in 70% (v/v) methanol (1/2 volume of supernatant) at –80 oC.


Aggregation and De-Aggregation of Sup35NM

       Methanol precipitated NM was collected by centrifugation at 14,000g for 30 min

at 4 oC (Chernoff et al, 2002). The methanol was removed and the pellet was damp-dried

under vacuum without heat for 5 min. The protein precipitate was then resuspended in

Congo red binding buffer [CRBB: 5mM potassium phosphate, pH 7.4, 150 mM NaCl] or

in 25 mM potassium phosphate pH 8.0. The protein concentration was confirmed by

diluting samples in 8M urea and measuring at 280nm. Aggregation of Sup35NM began

to occur naturally at room temperature, 25 oC, when separated from the denaturant and

re-suspended in buffer. To help produce aggregates in a shorter time, 2% preformed

fibers were added to freshly re-suspended Sup35NM or re-suspended Sup35NM

underwent slow rotation at 5 rpm. For de-aggregation, aggregated Sup35NM was boiled

at 100 oC for 10 min.




                                            34
Congo Red Binding Assay

       Congo red binding is a sensitive probe for fibril formation (Klunk et al., 1989).

Sup35NM protein was diluted to 2 mM with 10 mM Congo red in CRBB. Sup35NM

fibrils bound to Congo red caused a spectral shift in the absorbance peak from 477nm to

540 nm. To calculate moles of Congo red bound/L of solution, the following equation

was used: mol CRbound/L = (A540/25295) - (A477/46306).


Thioflavin T Binding Assay

       Thioflavin T (ThT) is a yellow dye that binds amyloid and fluoresces. A stock

solution of 1mM ThT in water was prepared fresh for each experiment (Chernoff et al.,

2002). Sup35NM (10 uM) was incubated in CRBB and at indicated times, protein was

diluted to 0.2 uM in the presence of 20 uM ThT in CRBB. ThT fluorescence was

monitored using a LS50B fluorometer (Perkin-Elmer Life Sciences), with excitation at

450 nm and emission at 481 nm.


Protease Resistance of Sup35NM

       This method was modified from a Sup35pN proteinase K resistance assay (King

et al., 1997). About 50 ug of Sup35NM samples (both fibers and freshly diluted

Sup35NM) were suspended in 25ul potassium phosphate buffer (pH 8.0). Two ul of

solution containing enzyme was added and the mixture was incubated for various periods

of time at the regular assay temperature (37 oC) or at the keratinase enzyme optimum

temperature (50 oC) (Lin et al., 1992). Reactions were terminated by adding PMSF

(phenylmethylsulfonyl fluoride) to a final concentration of 5 mM. SDS sample buffer




                                            35
was immediately added at 1x and boiled for 10 min. Samples were run on a 15% PAGE

gel and intact or degraded Sup35NM was detected with Coomassie Brilliant Blue R-250.


Degradation of Sup35NM

       Protease concentrations, preheating temperatures, reaction temperatures, and reaction

times were studied to characterize the enzymatic digestion patterns of Sup35NM. Proteinase

K (PK) and keratinase (KE) were used to characterize the yeast prion, Sup35NM. After

protease digestion, samples were assayed by SDS gel electrophoresis and Western blot

analysis. In each reaction, 50µg samples of Sup35NM underwent various treatments:

digestion by PK or KE with different enzyme units (1.5 U or 7.3U), preheat treatments at

different temperatures (115 oC, 100 oC, 80 oC, and 0 oC), reaction with KE at various

temperatures (37 oC and 50 oC), and varying lengths of incubation time with PK or KE (15

min, 30min, and 60min).


Western blot analysis

       For minute amounts of Sup35NM, Western blot analysis, which is more sensitive,

was performed using a modified ECL Western blotting analysis system (Amersham

Pharmacia). Two synthetic peptides, Ac-QGGYQQYNPDAGYQ-amide (Liu et al.,

2002) and Ac-CAPKPKKTLKLVSSSG-amide (Serio, personal communication), were

used to generate anti-Sup35NM polyclonal anti-peptides against amino acids 55-68 (anti-

N) and 135-148 (anti-M), respectively. These two peptides were synthesized and used

for production of polyclonal antibodies in rabbits by Biosource, Inc (Camarillo, CA).

The proteins were detected using ECL detection reagents. Briefly, Sup35NM proteins

were analyzed by 15% SDS-PAGE and transferred to PVDF membranes (Bio-Rad). The




                                            36
membranes were incubated with primary antibody (1:2000) in PBS buffer with 0.1%

Tween 20 (PBST) and 5% skim milk for 1hour, washed with PBST, and incubated with

secondary anti-rabbit antibody (1:5000) conjugated to horseradish peroxidase (Bio-Rad)

for 45 min. The proteins were then visualized with enhanced chemiluminescence reagent

(Pierce).


Keratinase and Proteinase K

       The keratinase was produced and purified as previously described in this laboratory

(Lin et al., 1992; Lin et al., 1997). Proteinase K was purchased from Sigma Chemical Co.

(St. Louis, MO). Keratianse activity and general protease activity was measured by the

hydrolysis of azo-keratin (Lin et al., 1992) and azo-casein (Sarath et al., 1989), respectively.

Azo-keratin was purchased from Sigma Chemical Co. (St. Louis, MO).


Electron Microscopy

       Sup35NM fibers were negatively stained according to Chernoff et al. (2002).

Five ul of a 5 uM protein solution was applied to a glow-discharged 400 mesh carbon-

coated copper grid for 30 sec followed by staining with several drops of 2% (w/v)

aqueous uranyl acetate. Excess liquid was removed with a filter paper wick and the grid

was allowed to air dry. Samples were then observed in a JEOL – 100S Transmission

Electron Microscope (Peabody, MA) at an accelerating voltage of 120kV in low-dose

mode at a magnification of 50,000X. The images were recorded on Kodak SO163 film.

This work was carried out at the NCSU Center of Electron Microscopy.




                                             37
                                         Results

Properties of Sup35NM

       Sup35NM was expressed, isolated and purified from E. coli as previously

published (Serio et al., 1999). When allowed to aggregate, Sup35NM formed polymer-

like fibrils as examined by transmission electron microscopy. They appeared to be

smooth and rigid (Fig. 1). Congo red binding was used to measure the aggregation under

two different conditions. One was slow 5 rpm rotation at room temperature without

seeding and the other, with 2% seeding of preformed aggregates but without rotation.

With the 2% seed, peak values of Congo red binding were reached within 5 hours, while

the unseeded, rotated sample took almost 24 hours to fully aggregate (Fig. 2).

       SDS-PAGE and western blotting analyses showed that unseeded samples of

purified Sup35NM did not fully polymerize until nearly 24 hours. Based on the

insolubility of fully formed fibers, SDS-PAGE and Western blots showed a gradual

decrease in soluble, monomeric Sup35NM over 44 hours (Fig. 3A). At 44 hours, soluble

protein was almost gone; however, heating of the same 44 hour sample at 100 oC for 10

minutes produced a band at the same mobility and intensity as the one at 0-time. This

indicated that de-aggregation to monomer form occurs as a result of heating. Western

blot revealed the same result by detecting polymerized Sup35NM as insoluble protein

trapped in the wells (Fig. 3B). Over the course of 44 hours, a gradual increase in the

amount of insoluble polymer in the wells was observed in correlation with the gradual

decrease of monomer in the resolving gel.




                                            38
      A




      B




Figure 1. Transmission electron microscopy of negatively stained Sup35NM
amyloid fibers (A) and Sup35NM after heated at 100 ℃ for 10 min.




                                     39
   A
                                    3.5

                                    3.0
             µM Congo Red bound
                                    2.5

                                    2.0                                     CRBB
                                                                            2% seed
                                    1.5                                     5 rpm

                                    1.0

                                    0.5

                                    0.0
                                          0   1    2          3    4    5
                                                   Time (hours)




   B
                                    3.5

                                    3.0
               µM Congo red bound




                                    2.5

                                    2.0                                     CRBB
                                                                            2% seed
                                    1.5                                     5rpm

                                    1.0

                                    0.5

                                    0.0
                                          0   10   20        30   40   50
                                                   Time (hours)




Figure 2. Congo red binding assay during Sup35NM polymerization, first 5
hours (A) and entire 48 hours (B).




                                                        40
      A
              Lane 1 2 3 4 5 6 7 8 9 10 11 12
              hrs  0 0.5 1 1.5 2 2.5 3 4 5 22 44




               NM




      B
              Lane 1 2 3 4 5 6 7 8 9 10 11 12
              hrs 0 0.5 1 1.5 2 2.5 3 4 5 22 44
           wells




             NM




Figure 3. SDS-PAGE of soluble, monomeric Sup35NM during polymerization at
25 ℃ over a 44 hour time course stained with Coomassie blue (A) and a western
blot of an identical gel with stacking gel intact (B). Stacking gel contains
insoluble, polymerized Sup35NM. Both gels, Lane 1-11: sample at respective
time point; 12: 44 hr sample heated at 100 ℃ for 10 min.




                                     41
       Fluorescence emission of ThT binding clearly showed that unseeded, rotated

Sup35NM took 24 hours to completely polymerize (Fig. 4A). Fully polymerized

Sup35NM was used as a control at each time point to distinguish the extent of

polymerization. SDS-PAGE samples also taken at each time point to accompany the ThT

binding samples supported the fluorescence data (Fig. 4B).


Enzymatic degradation

       Soluble Sup35NM monomer was as readily degradable by keratinase and

proteinase K as PrPC. Sup35NM aggregates were found to be resistant to these two

enzymes at low level, but partially degradable when the enzymes were at high level. The

degradability of Sup35NM aggregates by keratinase and proteinase K were compared at

different levels of enzyme units under the same conditions (Fig. 5 and 6). SDS-PAGE

analysis (Fig 5A, B) indicated degradation at the highest enzyme level (7.3 U). Western

blot analyses with anti-M (Fig. 6 A, C) and anti-N (Fig. 6B, D) clearly showed that the

high enzyme level displayed partial degradation and keratinase had higher activity than

proteinase K (A vs. C; B vs.D). Coomassie blue staining of SDS-PAGE (Fig. 5) also

revealed that digestion from the two different enzymes produced different patterns of

fragments (A vs. B). These enzymatic digestions were conducted without a pre-heating

treatment.


Pre-heating treatment

       Pre-heating treatment of polymeric Sup35NM prior to keratinase digestion at two

different temperatures, 37 and 50 oC (Fig. 7A, B), was tested. Three different pre-heating

temperatures, 15 min each, were tested: 80, 100, and 115 oC (Fig. 7). It was clear that




                                            42
          A
                                        1400

                                        1200




                  Fluorescence 481 nm
                                        1000




                       (arb. units)
                                        800                                                   5 rpm
                                                                                              Polymer
                                        600

                                        400

                                        200

                                          0
                                               0       10       20       30     40       50
                                                                Time (hours)



          B
                                        Lane       1        2    3      4      5     6
                                        hrs        0        5   10     24      48




                                         NM




Figure 4. Fluorescence emission of thioflavin T (ThT) binding during Sup35NM
polymerization (A). SDS-PAGE of same samples at respective time points (B).
Lane 1-5: sample at respective time point; 6: 48 hour sample heated at 100 ℃ for
10 min.




                                                                43
      A                                   B
      PK amt   0    L    M    H           KE amt   0     L   M    H




Figure 5. Proteinase K (PK) degradation of Sup35NM polymer (A) and
keratinase (KE) degradation of Sup35NM polymer (B) at 37 ℃ for 15 min
compared at the same enzyme amounts: 0, Low (L): 0.3 U, Medium (M): 1.5 U,
and High (H): 7.3 U.




                                     44
Figure 6. PK and KE degradation of Sup35NM polymer at 37 ℃ for 15 min

compared at the same enzyme amounts: 0, L: 0.3 U, M: 1.5 U, and H: 7.3 U. PK

degradation using anti-M for detection (A) and anti-N (B). KE degradation using

anti-M for detection (C) and anti-N (D).




                                           45
A                                  C
    PK amt   0   L   M   H   ---        KE amt 0     L   M   H   ---




                                   NM                                  NM




                                   anti – M




B                                  D
    PK amt   0   L M     H   ---        KE amt   0   L   M   H   ---




                                   NM                                  NM



                                   anti – N




                                   46
Figure 7. The effect of various preheating temperatures at two levels of

keratinase (KE) from low (L, 1.5 U) to high (H, 7.3 U) at two different digestion

temperatures, 37 ℃ (A) and 50 ℃ (B) for 30 min on Sup35NM polymer. Also,

Sup35NM monomer treated with M and H amounts of KE and Sup35NM polymer

preheated at 115 ℃ for 15 min followed by no KE.




                                         47
A   37℃ for 30 min

          Mono               Poly

Preheat    ----    115℃ 100℃ 80 ℃   ---- 115℃
KE                                       0



                                                NM




B   50℃ for 30 min

          Mono               Poly

Preheat     ----   115℃ 100℃ 80 ℃ ---- 115℃
KE                                       0



                                                NM




                        48
heating the Sup35NM polymer at 115 oC alone without enzyme had little effect on

degradation. At the lower enzyme level (1.5 U), pre-heating at 115 and 100 oC made the

polymer, like the monomer, degradable. Preheating at 80 oC, like polymer without

preheat, was partially degradable. However, at the high enzyme level (7.3 U), they were

all degradable, the same result as in Fig. 5. Keratinase was less active at 37 oC than at 50
o
    C (7A vs. 7B), the optimum temperature of the enzyme.

          The temperature effects on the pre-heating of Sup35NM polymer and the

digestion by keratinase were confirmed by Western blot analyses with anti-M and anti-N

(Fig. 8). Pre-heating at 115 and 100 oC rendered the polymer degradable as the monomer.

The effect was clearly demonstrated at the lower enzyme level (1.5 U) and the enzyme

optimum temperature, 50 oC.


Keratinase amount and digestion time

          As the pre-heating was set at 115 oC, the digestion conditions for keratinase were

further determined for the enzyme amount and digestion time. At a reaction temperature

of 50 oC with and without preheating at 115 oC, Sup35NM monomers and polymers were

treated with two levels of keratinase, 1.5 U (Fig. 9A, B) and 7.3 U (9C, D) and three

digestion times, 15, 30 and 60 min. The results were analyzed by Western blot (Fig. 9).

Both the monomers and pre-heated polymers underwent complete degradation by 30

minutes for both enzyme levels. Sup35NM polymer without preheat was still detectable

after digestion with the lesser 1.5 U of keratinase.




                                              49
Figure 8. The effect of various preheating temperatures at two levels of

keratinase (KE) from low (L, 1.5 U) to high (H, 7.3 U) at two different digestion

temperatures, 37 ℃ for 30 min on Sup35NM polymer using anti-M for detection

(A) and anti-N (B) and 50 ℃ for 30 min using anti-M for detection (C) and anti-N

(D). Also, Sup35NM monomer treated with M and H amounts of KE and

Sup35NM polymer preheated at 115 ℃ for 15 min followed by no KE. Std is

0.75ug of untreated, purified Sup35NM.




                                         50
A                37℃ for 30 min                 C            50℃ for 30 min


          Mono        Poly            Std           Mono           Poly            Std

Preheat   ---- 115℃ 100℃ 80 ℃ ----                  ---- 115℃100℃ 80 ℃ ---- 115℃

 KE                                                                            0




                                           anti – M



B                37℃ for 30 min                 D            50℃ for 30 min

          Mono        Poly                Std       Mono           Poly            Std

Preheat   ---- 115℃ 100℃ 80 ℃ ---- 115℃               --- 115℃ 100℃ 80 ℃ ---- 115℃

 KE                                   0                                        0




                                           anti – N




                                     51
Figure 9. The effect of different KE amounts, 1.5 U (A, B) and 7.3 U (C, D) at 50

℃ and reaction times, 15 min, 30 min, and 60 min, on Sup35NM monomer,

polymer preheated at 115 ℃, and polymer without preheating. (A) and (C) used

anti-M for detection. (B) and (D) used anti-N for detection. Control, C, is

Sup35NM polymer preheated at 115 ℃ for 15 min followed by no KE and Std is

0.75ug of untreated, purified Sup35NM.




                                         52
A               50℃ with 1.5U KE               C             50℃ with 7.3U KE

            Mono    115℃     Poly      C Std          Mono     115℃     Poly      Std

Rx time   15 30 60 15 30 60 15 30 60                 15 30 60 15 30 60 15 30 60
(mins)




                                          anti – M




B               50℃ with 1.5U KE               D             50℃ with 7.3U KE

            Mono     115℃     Poly     C Std           Mono      115℃     Poly    Std

Rx time   15 30 60 15 30 60 15 30 60                 15 30 60 15 30 60 15 30 60
(mins)




                                          anti – N




                                     53
                                       Discussion

       The prion-like protein from yeast, Sup35NM, was produced and purified. Its

property of aggregating into polymer-like fibrils was confirmed by electron microscopy

(Fig. 1) and many other analyses including Congo red binding (Fig. 2), SDS-PAGE (Fig.

3, 4B) and fluorescence with ThT dye (Fig. 4A). Sup35NM is similar to its parent

protein, Sup35p, except that its molecular size is smaller and its morphology is more rigid

and smooth under the electron microscope (Serio et al., 1999). Being nonpathogenic and

fairly easy to prepare, Sup35NM is a good candidate as a prion surrogate protein for

many research and technological applications. It could also serve as a safe prion marker

for food processing, disinfection processes, metabolic studies, and fate in different

environments. In this study, it was evaluated specifically for its use in the study of

enzymatic degradation.

       In previous tests, PWD-1 keratinase and some other proteases were found to be

able to degrade PrPSc in brain stem tissues from infected cattle and sheep (Langeveld et

al., 2003). However, a specific condition, pre-heating treatment of the tissue homogenate,

is needed for complete digestion of PrPSc by these enzymes. It was believed that the pre-

heating at 115 oC caused a change in the structure of the PrPSc subjected to enzymatic

degradation. In this study with Sup35NM aggregates or polymer, the same scenario was

observed. Sup35NM polymer was found relatively resistant to both keratinase and

proteinase K except at a very high level of the enzymes tested (Figs. 5,6). Pre-heating at

115 oC alone without enzyme had no effect (Figs. 7,8). The pre-heating temperature for

maximal degradation turned out to be the same as that for PrPSc in tissues, 115 oC

(Langeveld et al., 2003); however, unlike the tissue homogenates, Sup35NM polymers


                                             54
were also digestible at 100 oC pre-heat. This may be in part due to the state of the prion

protein being tested. In this study, we tested conditions for digestibility of purified

Sup35NM prion protein aggregates that were not in tissue. The environment of the tissue

may help contribute to the stability of PrPSc. Regardless, based on proteinase K

resistance tests, Sup35NM polymer is still more degradable than purified scrapie PrPSc

(Bolton et al., 1984). Therefore, Sup35NM is close to, but still not exactly the same as,

the prion that causes TSE.

        Nevertheless, Sup35NM can be developed as a model system to investigate

several combinations of conditions to optimize the enzyme’s cleaving ability and also to

determine its mechanism of enzyme action. The tests for optimal pre-heating

temperatures, pre-heating time, relative amounts of enzyme, digestion temperature and

time, and pilot scale test for industry, are all important but difficult, if not impossible, to

carry out with the pathogenic isoform, PrPSc. Furthermore, Sup35NM offers an

opportunity to study the mechanism of enzymatic degradation. For example, results from

this study (Figs. 7-9) indicated that the mechanism of pre-heating may be due to the de-

aggregation of the polymer because Sup35NM polymer was converted to monomer after

being heated for 10 min at 100 oC (Fig.3, 4B). De-aggregated, monomeric Sup35NM is

more readily degradable by the enzyme. The relationship between the structure and

degradability of prion molecules has yet to be determined.

        The keratinase is more active than proteinase K in degrading polymeric

Sup35NM when they were compared at the same level of enzyme units, based on the azo-

casein assay, and at the same digestion temperature, 37 oC (Fig. 6). Their patterns of

degradation products or fragments are different (Fig. 5 and Fig. 6A, C). This indicated




                                               55
that the cleavage sites of keratinase and proteinase K are different. It will be of interest to

find out these enzyme-specific cleavage sites. The keratinase acts optimally at 50 oC in

degrading feather keratin (Lin et al., 1992). This remained to be the case with the

digestion of Sup35NM polymer. More complete digestion occurred at 50 oC than at 37 oC

under identical conditions.

       Analysis of Sup35NM polymerization using established techniques in detecting

amyloid revealed key differences in how amyloid was detected. On a time course from 0

to 44 hours, SDS samples of polymerizing Sup35NM were prepared and run on a gel. At

the same time points, Congo red and ThT samples were also prepared and analyzed.

Congo red binding assay showed a gradual increase in Congo red binding from 0 to 5

hours before leveling off. ThT binding also showed a gradual increase in binding;

however, ThT binding occurred over a course of 24 hours before total saturation. SDS-

PAGE supported the ThT fluorescence data. The difference between these detection

assays is that Congo red binding detected changes in protein folding, specifically, the

formation of β-sheets, as opposed to the actual aggregates of Sup35NM that ThT detects.

Since ThT binding and fluorescence turns out to be the better method by directly

detecting polymer formation, in future studies, it should also be used to detect polymer

degradation.

       In conclusion, various conditions to optimize partial and complete degradation of

Sup35NM amyloid fibrils were studied. Ultimately, the most effective means can be

verified with infected brain tissue and PrPSc and determined by infectivity tests in

animals. Although Sup35NM polymers are less resistant to enzymatic degradation than

PrPSc, Sup35NM still fulfills the larger role of a safer, simpler and more cost-effective




                                              56
surrogate protein that serves as an important model for screening and optimizing

experimental conditions. Further studies will focus on the protein intermediates produced

from partial degradation and the weak points in the Sup35NM polymer structure. To do

so, further modifications of degradation conditions from this study need to be made in

order to produce intermediate state structures and determine the cleavage sites that follow

suit. With this knowledge, the applications of enzymatic degradation of Sup35NM can

then be applied to PrPSc prion and other disease-causing amyloids.




                                            57
                                Acknowledgements

       This research is the result of the collaboration of Dr. Ching-Ying Chen and the

author. The gift of the plasmid from Dr. S.L. Lindquist and consultation from Drs. T.R.

Serio and J.J. Wang are greatly appreciated. The technical support of Ms. Valerie

Knowlton of the NCSU Center of Electron Microscopy and the grant support from US

FDA (FD-U-002258) made this study possible.




                                           58
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                                             60