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Background: The protein S4 of the smaller ribosomal subunit is centrally important for its anchorage role in ribosome assembly and rRNA binding. Eubacterial S4 also facilitates synthesis of rRNA, and restrains translation of ribosomal proteins of the same polycistronic mRNA. Eukaryotic S4 has no homolog in eubacterial kingdom, nor are such extraribosomal functions of S4 known in plants and animals even as genetic evidence 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 of synthetic peptide substrates and natural proteins. We also studied autoproteolysis of the recombinant S4 proteins, and examined the growth and proliferation of S4-transfected human embryonic kidney cells., Results: Extraribosomal enzyme nature of eukaryotic S4 is described. Both human S4X and rice S4 are cysteine 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 ribosomal proteins, but also processes in cell biology and diseases., (Copyright © 2013 Elsevier B.V. All rights reserved.)

S4 is an integral protein of the smaller subunit of cytosolic ribosome. In prokaryotes, it regulates the synthesis of ribosomal proteins by feedback inhibition of the α-operon gene expression, and it facilitates ribosomal RNA synthesis by direct binding to RNA polymerase. However, functional roles of S4 in eukaryotes are poorly understood, although its deficiency in humans is thought to produce Turner syndrome. We report here that wheat S4 is a cysteine protease capable of abrogating total protein synthesis in an actively translating cell-free system of rabbit reticulocytes. The translation-blocked medium, imaged by atomic force microscopy, scanning electron microscopy, and transmission electron microscopy, shows dispersed polysomes, and the disbanded polyribosome elements aggregate to form larger bodies. We also show that human embryonic kidney cells transfected with recombinant wheat S4 are unable to grow and proliferate. The mutant S4 protein, where the putative active site residue Cys 41 is replaced by a phenylalanine, can neither suppress protein synthesis nor arrest cell proliferation, suggesting that the observed phenomenon arises from the cysteine protease attribute of S4. The results also inspire many questions concerning in vivo significance of extraribosomal roles of eukaryotic S4 performed through its protease activity.

Background: Ribosomal proteins often carry out extraribosomal functions. The protein S4 from the smaller subunit of Escherichia coli, for instance, regulates self synthesis and acts as a transcription factor. In humans, S4 might be involved in Turner syndrome. Recent studies also associate many ribosomal proteins with malignancy, and cell death and survival. The list of extraribosomal functions of ribosomal proteins thus continues to grow., Methods: Enzymatic action of recombinant wheat S4 on fluorogenic peptide substrates Ac-XEXD↓-AFC (N-acetyl-residue-Glu-residue-Asp-7-amino-4-trifluoromethylcoumarin) and Z-FR↓-AMC (N-CBZ-Phe-Arg-aminomethylcoumarin) as well as proteins has been examined under a variety of solution conditions., Results: Eukaryotic ribosomal protein S4 is an endoprotease exhibiting all characteristics of cysteine proteases. The K(m) value for the cleavage of Z-FR↓-AMC by a cysteine mutant (C41F) is about 70-fold higher relative to that for the wild-type protein under identical conditions, implying that S4 is indeed a cysteine protease. Interestingly, activity responses of the S4 protein and caspases toward environmental parameters, including pH, temperature, ionic strength, and Mg(2+) and Zn(2+) concentrations, are quite similar. Respective kinetic constants for their cleavage action on Ac-LEHD↓-AFC are also similar. However, S4 cannot be a caspase, because unlike the latter it also hydrolyzes the cathepsin substrate Z-FR↓-AMC., General Significance: The eukaryotic S4 is a generic cysteine protease capable of hydrolyzing a broad spectrum of synthetic substrates and proteins. The enzyme attribute of eukaryotic ribosomal protein S4 is a new phenomenon. Its possible involvement in cell growth and proliferations are presented in the light of known extraribosomal roles of ribosomal proteins., (Copyright © 2012 Elsevier B.V. All rights reserved.)

S4 is a paradigm of ribosomal proteins involved in multifarious activities both within and outside the ribosome. For a detailed biochemical and structural investigations of eukaryotic S4, the wheat S4 gene has been cloned and expressed in Escherichia coli, and the protein purified to a high degree of homogeneity. The 285-residue recombinant protein containing an N-terminal His(6) tag along with fourteen additional residues derived from the cloning vector is characterized by a molecular mass of 31981.24 Da. The actual sequence of 265 amino acids having a molecular mass of 29931 Da completely defines the primary structure of wheat S4. Homology modeling shows a bi-lobed protein topology arising from folding of the polypeptide into two domains, consistent with the fold topology of prokaryotic S4. The purified protein is stable and folded since it can be reversibly unfolded in guanidinium hydrochloride, and is capable of hydrolyzing cysteine protease-specific peptide-based fluorescence substrates, including Ac-DEVD-AFC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin) and Z-FR-AMC (N-CBZ-Phe-Arg-aminomethylcoumarin)., (Copyright © 2011 Elsevier Inc. All rights reserved.)