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The idea of establishing the amyloid-like fibrillation tendency of pro- and antisurvival proteins of human apoptotic pathways is relevant for delineating the conditions that lead to aberrant differentiation, development, and tissue homeostasis. As the first step in this direction, we report here that the caspase recruitment domain (CARD) of recombinant human apoptotic protease activating factor-1 (Apaf-1) can be induced to undergo amyloid-like fibrillation. The study was initiated with a set of biophysical investigations into the possibility and in vitro conditions for fibril growth. By scanning the pH-induced conformational transitions, protein stability, and stopped-flow folding-unfolding kinetics, we detected a molten globule (MG) transition of the CARD at pH <4. In a bid to reduce the surface-accessible hydrophobic patches in the MG state, the CARD monomer undergoes self-association to produce soluble oligomers that serve as precursor aggregates for protofibril formation. The monomer-to-oligomer self-association process is akin to the well-known homophilic CARD-CARD interaction by which CARDs of the same or different apoptotic proteins associate to transduce and regulate the apoptotic signal. The fibrillation reaction of the Apaf-1 CARD was conducted at pH 2.1 and 60 degrees C, because reduction of exposed hydrophobic surfaces in the MG state is more favored under the moderated solution condition. The Gaussian distributions of diameters of the fibril population suggest values of 2.1 and 2.7 nm for the mean diameter of precursor aggregates and protofibrils or elongated fibrils, respectively.

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.