Biochimica et Biophysica Acta 1830 (2013) 5335–5341

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Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbagen

Enzyme dimension of the ribosomal protein S4 across plant and

animal kingdoms
Babu Sudhamalla a, Mahesh Kumar b, R. Sunil Kumar a, Pulikallu Sashi a, U. Mahammad Yasin a,
Dasari Ramakrishna a, P. Nageswara Rao b, Abani K. Bhuyan a,⁎
a
School of Chemistry, University of Hyderabad, Hyderabad 500 046, India
b
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India

a r t i c l e i n f o a b s t r a c t

Article history: Background: The protein S4 of the smaller ribosomal subunit is centrally important for its anchorage role in
Received 1 March 2013 ribosome assembly and rRNA binding. Eubacterial S4 also facilitates synthesis of rRNA, and restrains transla-
Received in revised form 10 May 2013 tion of ribosomal proteins of the same polycistronic mRNA. Eukaryotic S4 has no homolog in eubacterial king-
Accepted 9 June 2013 dom, nor are such extraribosomal functions of S4 known in plants and animals even as genetic evidence
Available online 19 June 2013
suggests that deficiency of S4X isoform in 46,XX human females may produce Turner syndrome (45,XO).
Methods: Recombinant human S4X and rice S4 were used to determine their enzymatic action in the cleavage
Keywords:
Ribosomal protein S4
of synthetic peptide substrates and natural proteins. We also studied autoproteolysis of the recombinant S4
Cysteine protease proteins, and examined the growth and proliferation of S4-transfected human embryonic kidney cells.
Human S4X isoform Results: Extraribosomal enzyme nature of eukaryotic S4 is described. Both human S4X and rice S4 are cysteine
Cell growth and proliferation proteases capable of hydrolyzing a wide spectrum of peptides and natural proteins of diverse origin. Whereas
rice S4 also cleaves the -XXXD↓- consensus sequence assumed to be specific for caspase-9 and granzyme B,
human S4 does not. Curiously, both human and rice S4 show multiple-site autoproteolysis leading to
self-annihilation. Overexpression of human S4 blocks the growth and proliferation of transfected embryonic
kidney cells, presumably due to the extraribosomal enzyme trait reported.
Conclusions: The S4 proteins of humans and rice, prototypes of eukaryota, are non-specific cysteine proteases
in the extraribosomal milieu.
General significance: The enzyme nature of S4 is relevant toward understanding not only the origin of ribo-
somal proteins, but also processes in cell biology and diseases.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
It is often believed that rRNAs themselves or in association with a
few RNA-stabilizing peptides are capable of synthesizing proteins, the
ribosomal proteins could just be aides to making translation efficient
and faster [1–3]. These proteins were probably independent and
self-sufficient pre-existent entities responsible for various cellular func-
tions before being integrated to ribosome structure along evolutionary
progression. The thought is supported by literature reports of a sizable
number of ribosomal proteins involved in diverse extraribosomal pro-
cesses, many of which are vital to normal functioning of the cell
[2,4–7]. A key protein component of the smaller subunit of ribosome is
S4, which generally interacts with 18S rRNA (16S in prokaryotes) and
may be involved in ribosome assembly. Besides, it appears to participate
Abbreviations: rRNA, ribosomal RNA; Z-FR↓-AMC, N-CBZ-Phe-Arg-aminometh-
ylcoumarin; Ac-LEHD↓-AFC, N-acetyl-LEHD-7-amino-4-trifluoromethylcoumarin; Mb,
horse myoglobin; IAA, iodoacetic acid; EcS4, E. coli ribosomal protein S4
⁎ Corresponding author. Tel.: +91 40 2313 4810.
E-mail address: akbsc@uohyd.ernet.in (A.K. Bhuyan).
in codon decoding, and hence could be responsible for accuracy of pro-
tein synthesis. Despite a lack of clear understanding of the role of S4
within the ribosome, its extraribosomal characters in eubacterial sys-
tems are illustrious. For example, E. coli S4 (EcS4) has been shown to
regulate self-synthesis as well as the synthesis of those ribosomal pro-
teins that are coded by the same polycistronic mRNA [8–10]. It also
serves as a general transcription antitermination factor by associating
with RNA polymerase during normal transcription. Further, EcS4 is in-
volved in rRNA operon antitermination [11]. In lower eukaryotes, such
as Dictyostelium mold, expression of the protein S4 is necessary for
phase-shift of cells from growth to differentiation [12].
The character of eubacterial S4 outside the ribosome as briefed
above cannot be assumed true for eukaryotic S4 proteins, because the
latter have no homolog in the eubacterial kingdom [1]. Further, in con-
trast with polycistronic nature of prokaryotic genes, the genes in higher
eukaryotes are generally monocistronic. In fact, no extraribosomal func-
tion of S4 has been described in plants and animals, even though genetic
evidence suggests that underexpression of the S4X isoform, which is
generally 85–90% as abundant as the S4Y, in 46,XX human females
may produce Turner syndrome [13]. Again, conclusive evidence for

0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bbagen.2013.06.010
5336 B. Sudhamalla et al. / Biochimica et Biophysica Acta 1830 (2013) 5335–5341
involvement of S4X in Turner syndrome has not been obtained. In re-
cent studies, we fortuitously found that wheat S4 is a cysteine protease
capable of cleaving a range of peptides and proteins [14]. An immediate
query arising from the wheat work was whether the protease nature of
S4 is a general phenomenon in eukaryotic kingdom, especially in
humans. To establish that reputed cysteine protease activity indeed is
a significant dimension of S4 in all higher eukaryotes, and to study its
extraribosomal character, we have now produced the human S4X iso-
form, referred to as human S4 hereforth, as well as the rice S4 protein
in E. coli overexpression system. We find that S4 is a cysteine protease
in both systems, albeit the human protein can be distinguished from
that of rice in respect of steady-state enzyme kinetic parameters, mod-
ulator affinities, and environmental optima for activity. Remarkably, S4
appears to annihilate itself by a process of autoproteolytic cleavage at
multiple sites. It is also found that overexpression of S4 in cultured
human cells has anti-proliferative effect. The results obtained strongly
suggest that the enzyme dimension of S4 should be common to all eu-
karyotic species because ribosomal proteins from eukaryotes have a
common set of ancestral genes [1].
2. Materials and methods
2.1. Cloning, expression, and purification of human and rice S4
Human S4 cDNA was synthesized from HeLa cells RNA by RT-PCR
using the following primers
The synthesized cDNA was cloned into pTZ57R/T by TA ligation,
and positive recombinant plasmids were isolated and sequenced.
The S4 was released from the TA vector and sub-cloned between
BamH1 and Xho1 sites of pET28 a (+) vector. Protein expression
was induced from Rosetta BL21 (DE3) cells.
For synthesis of cDNA to clone rice S4, total RNA was isolated from
~ 100 mg tissue of a-week old rice seedlings. Primers were
The S4 cloning into pEt28 a (+) was carried out as briefed above
for human S4, and the protein was expressed from BL21 (DE3) RIL
cells. Cells were grown in LB medium containing kanamycin
(50 μg mL−1), and protein expression was induced at A600 ~ 0.5
with 0.5–1 mM IPTG.
Harvested cells were washed in Tris–EDTA buffer (10 mM Tris–HCl,
1 mM EDTA, pH 8.0), suspended in Buffer A (20 mM Tris, 100 mM NaCl,
pH 8), and sonicated at 4 °C. The cell lysate was stirred with deoxycholic
acid (4 mg per gram weight of E. coli) for ~30 min, and centrifuged at
10,000 rpm for 15 min at 4 °C. The inclusion body pellet was washed
3× with ~9 volumes of Buffer B (20 mM Tris, 100 mM NaCl, 10 mM
EDTA, 0.5% Triton X-100, pH 8.0). To remove Triton X-100, the pellet
was further washed 3× with ~10 volumes of Buffer A. After a final
wash in 20 mM Tris, 1 M GdnHCl, pH 8, the inclusion bodies are stored
at −80 °C until further processing.
About 42 mg of the inclusion body aggregate was solubilized by
unfolding in 2 mL of Buffer C (50 mM Tris, 150 mM NaCl, 5 mM
EDTA, 5 mM DTT, 6.0 M GdnHCl, pH 8). The solution was vortexed
gently at room temperature for ~ 2 h, centrifuged at 12,500 rpm to re-
move any particulate material, and added in drops to 9-volumes of
Buffer D (50 mM Tris, 500 mM NaCl, 2 mM DTT, 0.5 M urea, 0.4 M ar-
ginine, 0.1% glycerol, pH 8.5) at 4 °C. The refolded protein solution,
now in ~ 0.6 M GdnHCl and 0.45 M urea, was left in cold overnight,
centrifuged, and chromatographed on a Sephadex G75 column in
20 mM Tris, 50 mM NaCl, pH 8. Collected fractions were analyzed
by SDS electrophoresis and mass spectrometry.
2.2. Assay of catalytic activity of S4 in the hydrolysis of peptide
substrate analogs
Typically, 400 μL of 1.2–1.8 μM freshly prepared and appropriately
buffered protein solution equilibrated at 37 °C in the fluorescence cell
was combined with 5–10 μL of a 1 mM solution of Ac-DEVD↓-AFC or
Z-FR↓-AMC. This provides ~ 1:10 for the ratio of molar concentrations
of S4 and substrate. Time-base emission was initiated within ~ 10 s of
mixing the two solutions. Excitation and emission wavelengths were
400 and 500 nm, respectively, for monitoring the cleavage of
Ac-DEVD↓-AFC, and 360 and 460 nm, respectively, for Z-FR↓-AMC.
Initial velocity (vo) modulated by ZnCl2 was analyzed by
V max
vo ≈ 

Km ½Zn2þ 
1þ ½S  1 þ K app
i
where Kapp
i (≈Kdiss) is the inhibition or dissociation constant of Zn2+.
Dependence of vo on MgCl2 was fitted to a Michaelis–Menten equation
h i
V max Mg2þ
vo ≈c þ  
K diss þ Mg2þ
by introducing an offset, c, to accommodate the residual activity.
The buffer for pH dependence studies contained 7 mM each of
PIPES, HEPES, Tris, and CAPS. The dependence of vo on pH was fitted
allowing for two ionizations involving multiple protons, n.
c
vo ≈ :
1 þ 10n1 ðpH−pK a;1 Þ þ 10n2 ðpK a;2 −pHÞ
2.3. Detection of proteolytic activity of S4
The mixture of substrate protein and recombinant S4 in 2:1 molar
ratio buffered in 20 mM Tris, 50 mM NaCl was incubated at 37 °C. Sam-
ple aliquots withdrawn at different times after initiating the reaction
were boiled immediately after withdrawal. Proteolysis was detected
qualitatively by silver-staining the protein bands separated electropho-
retically in 15% polyacrylamide gels. The extent of proteolytic cleavage
was estimated by quantifying the stained band intensities.
3. Results and discussion
3.1. E. coli expression and primary structures of human and rice S4
Aligned sequences of human and rice S4 expressed and purified in
this study (Fig. 1a) show 71% sequence homology with little variation
in hydrophobicity and net charge content. Accordingly, the same op-
timized protocol for expression and purification was used for both
proteins. In brief, BL21 (DE3) E. coli cells harboring the S4 gene-
containing plasmid were allowed to grow at 37 °C for 3 h after
 B. Sudhamalla et al. / Biochimica et Biophysica Acta 1830 (2013) 5335–5341 5337

a) Homo sapiens MARGPKKHLK RVAAPKHWML DKLTGVFAPR PSTGPHKLRE CLPLIIFLRN 50

Oryza sativa MARGLKKHLK RLNAPKHWML DKLGGAFAPK PSSGPHKSRE CLPLILIIRN 50

Homo sapiens RLKYALTGDE VKKICMQRFI KIDGKVRTDI TYPAGFMDVI SIDKTGENFR 100

Oryza sativa RLKYALTYRE VISILMQRHV LVDGKVRTDK TYPAGFMDVI SIPKTGENYR 100

Homo sapiens LIYDTKGRFA VHRITPEEAK YKLCKVRKIF VGTKGIPHLV THDARTIRYP 150

Oryza sativa LLYDTKGRFR LQSVKDEDAK FKLCKVRSVQ FGQKGIPYLN TYDGRTIRYP 150

Homo sapiens DPLIKVNDTI QIDLETGKIT DFIKFDTGNL CMVTGGANLG RIGVITNRER 200

Oryza sativa DPIIKANDTI KIDLETNKIV DFIKFDVGNV VMVTGGRNTG RVGVIKNREK 200

Homo sapiens HPGSFDVVHV KDANGNSFAT RLSNIFVIGK GNKPWISLPR GKGIRLTIAE 250

Oryza sativa HKGSFETIHV EDALGHQFAT RLGNVFTIGK GNKPWVSLPK GKGIKLSIIE 250

Homo sapiens ERDKRLAAKQ SSG 265

Oryza sativa EQRKRDAAAQ AAANA 265

b) c) d)
1.0
M 1 2 3 4 5 6 7 8 9 10
kDa

OD600 nm
C
97.4 0.5 C
66 N N
43
29
0.0
14.3 0 4 8 112
Time (h)

Fig. 1. Expression of human S4X isoform and rice S4 in E. coli. (a) Sequence alignment showing differences at 77 amino acid positions accounting for 71% sequence homology of
human and rice S4. (b) The purification protocol described in the text is identically applicable to the two proteins. Here, a coomassie-stained 12% SDS-polyacrylamide gel is
presented showing step-wise purification of overexpressed rice S4. Lane labels are: M, marker proteins; 1, uninduced cell lysate; 2, IPTG-induced whole cell lysate; 3, supernatant
from the cell lysate; 4, precipitated material from the cell lysate; 5, Triton X-100 washed inclusion bodies; 6, supernatant after washing the inclusion bodies in 1 M GdnHCl; 7,
pelleted inclusion bodies washed in 1 M GdnHCl; 8, inclusion bodies solubilized in 6 M GdnHCl (buffer A); 9, soluble proteins in the refolded protein solution; 10, Sephadex
G75-purified rice S4. (c) Stalled growth of E. coli cells induced to express human S4 (●). Control cells not harboring the S4 gene continue to grow normally after IPTG induction
(□). (d) Energy-minimized structural model of human and rice S4 (left and right, respectively) generated from S. cerevisiae (3IZB) and T. acidophilum (3KBG) templates.

induction. Because S4 was largely localized in the cytoplasmic inclu-
sion body fraction at all temperatures tested for cell growth, the
harvested cells were processed to isolate the inclusion body aggre-
gates. Solubilization of inclusion bodies in 6 M GdnHCl unfolds the
S4 protein, which is then refolded at 4 °C under optimized conditions,
and purified to a high degree of homogeneity by size exclusion chro-
matography (Fig. 1b). Localization of most of the overexpressed S4 in
the cytoplasmic inclusion body fraction was somewhat unexpected,
because both human and rice S4 are highly charged under the intra-
cellular pH condition of E. coli. Considering the net charge of + 25 at
pH 7.5, calculated from the sequences of human and rice S4
(Fig. 1a), one expects the proteins to be soluble. Perhaps other deter-
minants of protein solubility, including turn-forming residue fraction
and folding rate [15], contribute adversely to S4 solubility.
E. coli cells transformed with human or rice S4 gene-containing plas-
mids show stalled growth within 3 h of IPTG-induced protein expression
(Fig. 1c), similar to an earlier observation of inhibited cell growth when
intrinsic EcS4 is overproduced under conditions of stimulated synthesis
[8]. However, the reasons and mechanisms of inhibition of cell growth
due to overexpression of eukaryotic S4 and the non-homologous eubac-
terial EcS4 must be different. In prokaryotes like E. coli, stimulated pro-
duction of EcS4 feedback-inhibits self synthesis along with the synthesis
of ribosomal proteins EcS13 and EcS11 both in vivo and in vitro, because
these proteins are coded by the same polycistronic mRNA of the
α-operon [8,9]. The repressive regulation of EcS4 on the production of
EcS13 and EcS11 leads to defects in ribosome assembly, and hence is
harmful to cell growth [8]. This mechanism is not applicable in eukaryotes
like human and rice where mRNAs are monocistronic. We believe that, it
is the enzymatic disposition of eukaryotic S4 that is responsible for
stalling cell growth. Presumably, the accumulation of a large pool of
overexpressed S4 in its free and active state results in nonspecific prote-
olysis of soluble proteins of the E. coli cytoplasm.
3.2. Both human and rice S4 proteins are cysteine proteases
The central theme of this work is to demonstrate the cysteine protease
character of S4 across plant and animal kingdoms. The energy-minimized
structural models of human and rice S4 based on the templates of Saccha-
romyces cerevisiae (PDB: 3IZB) and Thermoplasma acidophilum (PDB:
3KBG), respectively, show that both chains fold into two globular do-
mains (Fig. 1d), a fold feature common to the family of cysteine proteases
[16,17]. Experimentally, enzyme activities of human and rice S4 were
determined using Z-FR↓-AMC (N-CBZ-Phe-Arg-aminomethylcoumarin),
a standard fluorogenic substrate specific for cathepsins B and L, and
papain-like cysteine proteases (Supporting Information). Enzymatic
cleavage of Z-FR↓-AMC is inhibited by iodoacetic acid (IAA), a cysteine
thiol blocker, as well as by CA-074, a selective irreversible inhibitor
(Supplementary Information, Fig. S1), suggesting that the catalytic
group of S4 is a cysteine. To determine the catalytic residue, mutants
were produced by systematically replacing the cysteines with alanine.
For both human and rice S4, the C124A mutant was found enzymatically
inactive, implicating this cysteine in enzyme activity.
3.3. Effect of ions and common environmental variables on the activity of
human and rice S4
To study the modulation of S4 activity so as to optimize conditions
for enzyme reactions, the initial velocity of Z-FR↓-AMC cleavage, vo,
was measured as a function of ZnCl2, MgCl2, pH, and temperature
(Fig. 2). Extracted dissociation constants, Kdiss, for S4 and divalent
metal ion interactions, and pH and temperature optima for catalysis
are listed in Table 1. Zinc readily inhibits catalytic activity of both
human and rice S4 (Fig. 2a, b), consistent with the proposal that
they are cysteine proteases, and the apparent binding affinity of the
latter for Zn2+ is slightly larger. Presented values of Kdiss (Table 1)
5338 B. Sudhamalla et al. / Biochimica et Biophysica Acta 1830 (2013) 5335–5341

a c e
ZnCl2 (mM)

Fluorescence460 nm(au)
0.01 1.0
0.8 1.6

Relative vo
Relative vo
0.02
0.7
0.07 1.2 0.5
0.6 0.10
0.40
0.5 0.8 0.0
0 5 10 10-3 10-2 10-1 100 101 7.0 7.5 8.0 8.5
Time (min) MgCl2 (mM) pH

b d f
1.0 1.0 1.0

Relative vo

Relative vo
Relative vo

0.5 0.5 0.5

0.0 0.0
0.0
-3 -2 -1 0
10 10 10 10 20 30 40 50 60 70 5 6 7 8 9 10 11
ZnCl2 (mM) Temperature (OC) pH

Fig. 2. Effect of ions and environmental parameters on enzyme activity. (a) Representative time-base fluorescence due to cleavage of Z-FR↓-AMC by rice S4 in the presence of in-
dicated concentrations of ZnCl2. (b) Dependence of initial velocity on ZnCl2 for human (●) and rice (○) S4 provides apparent inhibition constant, Kapp
i (≈Kdiss) of 0.03(±0.005) and
0.02(±0.005) mM, respectively. (c) Dependence of vo on MgCl2 yields Kdiss values of 0.035(± 0.01) and 0.45(±0.05) for human (●) and rice (○) S4, respectively. (d) The optimum
temperature, Topt, for catalysis is ~46 °C for human S4 (●) and ~50 °C for rice S4 (○). (e) The vo vs pH profile shows an optimum value of ~8 for catalytic activity of human S4
involving two pKa values, 7.7 and 8.3. (f) For rice S4, the activity is maximum in the pH range of 8–9.5 involving pKa values of 6.7 and 10.

represent apparent affinity only, since zinc reacts with cysteine thiols Ionization of active-site residues at higher or lower pH reduces en-
irrespective of cysteine protease character of a protein. The rate of zyme activity.
substrate hydrolysis by both S4 increases in the presence of the acti-
vator magnesium (Fig. 2c), but the affinity of the human protein for 3.4. Affinity between S4 and Z-FR↓-AMC, and the catalytic efficiency
Mg2+ is at least an order of magnitude higher than that of the rice
protein (Table 1). These differences possibly originate from both Assuming that S4-catalyzed substrate hydrolysis obeys the
structures and adaptability of S4 proteins to intracellular concentra- Michaelis–Menten model, kinetic parameters for Z-FR↓-AMC cleavage
tions of Zn2+ and Mg2+ in plant and animal cells. were determined at pH 7.1, 37 °C (Fig. 3a, b). The functional depen-
Regarding temperature dependence of activity, the optima for dence of initial velocity on substrate concentration is described by a
human and rice S4 are found at 46 and 50 °C, respectively (Fig. 2d). hyperbola, providing values for Michaelis–Menten constant, Km, and
Both S4 proteins are enzymatically active in the 6–9.5 range of pH maximal velocity, Vmax. More accurate values of Km and Vmax were
(Fig. 2e, f), the rice protein being more active between pH 8 and 9.5 extracted by graphical analysis [18] of data (Fig. 3a, b, inset, and
(Fig. 2f). While the bell-shaped pH profiles suggest a single active Table 1). Interestingly, Km values for human and rice S4 are fairly
form of S4, the basic optimal pH for activity offers a reducing environ- comparable, 20 and 18 μM, respectively, indicating very similar affin-
ment where the active site cysteines are less susceptible to oxidation. ities of the two proteins for Z-FR↓-AMC under these experimental
conditions. However, the substrate turnover reflected by Vmax and
kcat is five-fold lower for human S4 (Table 1). Similar values of Km
Table 1 but significantly different catalytic efficiencies (kcat / Km) of the two
Binding parameters for Zn2+ and Mg2+, temperature and pH optima for enzymatic hy- proteins (Table 1) imply that the rate-limiting energy barrier for
drolysis of Z-FR↓-AMC in 20 mM HEPES, pH 7.1, 37 °C, and basic kinetic parameters for breakdown of the enzyme–substrate complex to product is relatively
the cleavage of Z-FR↓-AMC by human and rice S4, and for Ac-LEHD↓-AFC by rice S4a at
higher for human S4.
37 °C, 20 mM Tris, 50 mM NaCl, pH 7.4.

Parameter Human Rice 3.5. Rice S4 also cleaves the substrate analog of caspase-9, but human S4
Kdiss ≈ Ki (mM) does not
Zn2+ binding 0.030 0.02
Mg2+ binding 0.035 0.45 Another interesting note of difference between the two proteins
Temperature optimum (°C) ~46 ~50
pH optimum ~8 8–9.5
regards the ability of rice S4 to cleave Ac-LEHD↓-AFC, a fluorogenic
(pKa,1 ~ 7.7, pKa,2 ~ 8.3) (pKa,1 ~ 6.7, pKa,2 ~ 10) peptide substrate assumed to be specific for caspase-9 [19]. Human
Z-FR↓-AMC cleavage S4 does not hydrolyze this substrate. Since the Ac-XEXD↓-AFC class
Km (μM) 19.8(±0.2) 17.3(±0.2) of peptide substrates is also hydrolyzed by wheat S4 [14,20], it ap-
Vmax (μM min−1) 0.015(±0.002) 0.1(±0.01)
pears that plant ribosomal protein S4 generally recognizes the
kcat (min−1) 9.2 × 10−3 62.5 × 10−3
Ac-LEHD↓-AFC cleavage caspase recognition motif as well. Here, we simply quantify the kinet-
Km (μM) 32.3(±0.3) ic parameters for rice S4-catalyzed cleavage of Ac-LEHD↓-AFC (Fig. 3c
Vmax (μM min−1) 0.04(±0.01) and Table 1) without commenting on the significance of this func-
kcat (min−1) 24.0 × 10−3 tional difference between animal and plant S4. To note, however,
a
Ac-LEHD↓-AFC is not cleaved by human S4. the serine protease granzyme B is the only non-caspase enzyme
 B. Sudhamalla et al. / Biochimica et Biophysica Acta 1830 (2013) 5335–5341
a)
0.010
Initial velocity, vo
µM min-1)
0.02
0.005
(mM
0.01
40 20 0 20 40
0.000
0 10 20 30 40 50
Z-FR-AMC (µµM)
M)
Fig. 3. Determination of kinetic constants for the cleavage of Z-FR↓-AMC by (a) human S4, and (b) rice S4, and of Ac-LEHD↓-AFC by (c) rice S4. In each panel, the lower inset shows
graphical analysis of Km. The upper inset in (c) presents time-base fluorescence due to AFC released by catalytic cleavage of Ac-LEHD↓-AFC by rice S4. Values of extracted kinetic
constants are listed in Table 1.
hitherto known to recognize the -XXXD↓- consensus sequence [19].
The present study along with the earlier report of cleavage of
Ac-XEXD↓-AFC by wheat S4 [20] describes the ribosomal protein S4
of plants as the third group of enzymes capable of hydrolyzing the
-XXXD↓- motif.
3.6. Cleavage of protein substrates by human and rice S4
To examine proteolysis, native protein substrates were presented
to S4 for several hours in 20 mM Tris, pH 7.4, 37 °C, and the reaction
mixture analyzed by 15% SDS-PAGE and gel band quantification. All
protein substrates tested, including recombinant Bcl–xL, and com-
mercial casein, lysozyme, and myoglobin, were cleaved. For example,
in a mixture of 2 μM human S4 and 4 μM hen lysozyme, the latter is
completely cleaved within 2 h of incubation (lanes 2–6, Fig. 4a). Pro-
teolysis of at least 50% lysozyme population was observed within
15 min of mixing with S4 (Supplementray Information, Fig. S2).
Therefore, proteolysis of lysozyme in the rice S4–lysozyme mixture
(1.6 and 4 μM, respectively) was followed up to 2 h at intervals of
5 min initially followed by longer time increments (Fig. 4b). Virtually
complete absence of full-length lysozyme within 2 h of incubation
indicates complete cleavage. Proteolysis of horse myoglobin (Mb)
by human S4 appears relatively slow, because detectable fraction of
uncleaved myoglobin persists at the end of 2 h (Fig. 4a). Further,
the band intensity of full-length Mb appears to increase somewhat
after 2 h up to 12 h of incubation. This observation has been made
in several repeats of the experiment. We suspect that, this effect is
produced by time-dependent increase in the fraction of full-length
myoglobin aggregates, which are less susceptible to proteolytic attack
by S4. If so, the time-dependent increase in the population of
uncleaved aggregates in the reaction mixture would show up as
uncleaved monomers of Mb in SDS electrophoresis (Fig. 4a, lower
right panel). The PAGE analysis does not reveal cleaved fragments of
Mb and lysozyme, suggesting that S4 cleaves the protein substrates
at multiple sites to produce small peptides that run out of the gel.
3.7. Autoproteolysis of S4
Curiously, the electrophoretic analyses show a time-dependent de-
crease in the band intensity of S4 itself (Fig. 4a, b). At the end of an
8 hour-incubation of the mixture of human S4 and lysozyme or myoglo-
bin at 37 °C, for example, 60% of the full-length S4 population is lost
(Fig. 4a). Similarly, the intensity of the full-length rice S4 noticeably de-
creases in the reaction mixture containing the lysozyme substrate
(Fig. 4b). In fact, when incubated alone in the absence of any protein sub-
strate, the full-length human S4 disappears completely at the end of 15 h
of incubation (Fig. 4c). This observation is made consistently in all reac-
tions of S4 analyzed, and probably originates from self-degrading
5339
b) c) 2
Fl (500 nm)
0.10 0.015
1
0 2 4 6
Time (min)
0.2 0.010
0.05
0.1 0.02
0.005 0.01
40 20 20 50 25 0 25 50
0
0.00 0.000
0 20 40 60 0 20 40
Z-FR-AMC (µµM)
M) µM)
Ac-LEHD-AFC ((mM)
multiple-site autoproteolysis, the chemistry of which remains largely
unknown at present. The alleged autoproteolysis appears to produce
small fragments of S4 not retained in the 15% polyacryalamide gel
(Fig. 4). Many cysteine proteases are known to be activated by limited
autoproteolytic mechanisms, but multiple-site autoproteolysis of an al-
ready active protease has not been described. The proposal of multiple-
site autoproteolysis of S4 seems consistent with an earlier report of de-
tection of smaller fragments of human S4 in electrophoretically resolved
40S ribosomal proteins [13].
3.8. Inhibited cell viability due to overexpressed S4
As already discussed, the growth of E. coli cells overexpressing
human S4 stalls soon after IPTG induction (Fig. 1c). To test if such is
the case for eukaryotic cells as well, HEK 293T cells were transfected
with pcDNA 3.1-mycHis-human S4, and cell proliferation was moni-
tored with time. Compared to normal proliferation of untransfected
cells or those infected with the vector alone (Fig. 5a), the wild-type
S4-expressing kidney cells exhibit lack of confluence and surface ad-
herence (Fig. 5b). Such a block on cell viability is also observed in
wheat S4-transfected HEK 293T cells [14], suggesting a general
anti-proliferative activity of overly expressed eukaryotic S4. Further,
cells transfected with the C124A mutant of human S4 appear to pro-
liferate normally, since C124 is the putative catalytic cysteine.
3.9. Significance of cysteine protease character of S4
A pertinent question arising from our enzyme work of S4 regards its
physiological importance. Unfortunately, conclusive data are not avail-
able at this stage to associate the phenomenon with a particular regula-
tory process or metabolic pathway. Available results establish a wide
spectrum of protein substrates across species that are proteolyzed by
S4 in vitro, tending to suggest that it simply is an enzyme with no spe-
cific target protein. In this perspective, S4 could be regarded as a
pre-existent cysteine protease functioning independently, and was
later recruited to the ribosome assembly due to its ability to bind
rRNA — a thought in the line of Wool hypothesis [1,2]. The proteolytic
disposition is silent while it is held tightly in the 40S ribosomal subunit,
but can function as an active enzyme if the ribosomal proteins are
disassembled and released into the cytoplasm during starvation or
ribophagy, for example, when the cell cannot afford to spend energy
for ribosome maintenance. Such an event could be considered inconse-
quential, because random proteolytic action on a wide spectrum of sub-
strates does not establish a regulatory role in cellular function. The
extraribosomal role could therefore be manifested when S4 is
overproduced, and is free prior to ribosome assembly. Overexpression
of genes of several human ribosomal proteins, including S3, is thought
to be associated with colorectal tumors and polyps [21].
5340 B. Sudhamalla et al. / Biochimica et Biophysica Acta 1830 (2013) 5335–5341

The enzyme nature, nevertheless, emphasizes on strict regulation

of S4 synthesis and constrained kinetics of 40S ribosome core forma-
tion in the post-translational time bin. Regarding regulated produc-
tion of S4, oversynthesis inhibits cell growth and viability, as shown
in this study for both E. coli and human cells, but less synthesis of
the S4X isoform in 46,XX cells may produce Turner syndrome as per
available genetic evidence [13,22,23]. Evidences in favor of a definite
involvement of S4X in Turner syndrome is still lacking, nor do the
available data presented here allow drawing a relationship between
the proteolytic function of S4 and the syndrome. The enzyme charac-
ter of S4 should limit the time of its incorporation following transla-
tion into the 40S ribosome core assembly, because a large pool of
free and uninhibited S4 could lethally cleave cytosolic proteins by
proteolysis and annihilate itself by autoproteolysis. To this end, a

Fig. 4. Proteolysis by S4 at 37 °C. (a) Action of human S4 (lane 1) on hen lysozyme

(lane 2) at 2, 4, 6, and 8 h (lanes 3 to 6, respectively) and on horse myoglobin (lane
7) at 2, 4, 6, 8, and 12 h (lanes 8 to 12 in order) after preparing the enzyme–substrate
mixture. A gradual decrease in the intensity of S4 is also clearly noticed. The graphs
below depict the time-dependence of intensities of gel bands corresponding to lyso-
zyme (cyan colored circles) and human S4 (black circles), and myoglobin (red colored
diamonds) and human S4 (black circles), respectively. (b) Proteolysis by rice S4 (lane
1) of lysozyme (lane 2) at 5, 10, 15, 20, 25, 30, 45, 60, 90, and 120 min (lanes 3 to 13,
respectively) after mixing the two proteins. Here again, the intensity of the S4 band di-
minishes with time. Time-dependence of normalized intensities of lysozyme (cyan col- Fig. 5. Anti-proliferative activity of human S4 in transfected HEK 293T cells after 24 h
ored circles) and rice S4 (unfilled circles) are also produced below the gel image. The of transfection. (a) Control cells transfected with the empty vector. (b) Cells overly ex-
×-haired squares are outliers. (c) Population of full-length human S4 at 0, 0.25, 0.5, pressing wild-type S4 show drastic reduction in cell growth and cell-shape transforma-
0.75, 1, 1.5, 2, 3, 5, 7, 9, 12, and 15 h (lanes 1 to 13, respectively) of incubation at tion. (c) Cells transfected with the vector carrying the gene for C124A mutant appear to
37 °C without a substrate. Quantified gel band intensities are produced below. proliferate normally.
 B. Sudhamalla et al. / Biochimica et Biophysica Acta 1830 (2013) 5335–5341
detailed understanding of the extraribosomal enzymatic function of
S4 will broadly impact the field of cell biology and disease.
Author contributions
A.K.B. conceived the ideas; B.S., M.K., P.N.R., and A.K.B. designed
the research; B.S., M.K., R.S.K., P.S., U.M.Y., D.R., P.N.R., and A.K.B.
performed the research; B.S., P.N.R., and A.K.B. analyzed the data;
and A.K.B. wrote the paper.
Funding sources
This research was supported by grants from CSIR (38(1037)/02/
EMR-II), and Departments of Biotechnology (BRB/10/622/2008) and
Science & Technology (4/1/2003-SF) to A.K.B.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.bbagen.2013.06.010.
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