Quantification of cell cycle-arresting proteins.

Cellular senescence, which can be defined as a stress response preventing the propagation of cells that have accumulated potentially oncogenic alterations, is invariably associated with a permanent cell cycle arrest. Such an irreversible blockage is mainly mediated by the persistent upregulation of one or more cyclin-dependent kinase inhibitors (CKIs), including (though not limited to) p16( INK4A ) and p21( CIP1 ) and p27( KIP1 ). CKIs operate by binding to cyclin-dependent kinases (CDKs), de facto inhibiting their enzymatic activity. Here, we provide an immunoblotting-based method for the detection and quantification of CKIs in vitro and ex vivo, together with a set of guidelines for the interpretation of results.

Defining structural domains of an intrinsically disordered protein: Sic1, the cyclin-dependent kinase inhibitor of Saccharomyces cerevisiae.

The cyclin-dependent kinase inhibitor Sic1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation in the yeast Saccharomyces cerevisiae. Notwithstanding many studies on its biological function, structural characterization has been attempted only recently, fostering the development of production and purification protocols suitable to yield large amounts of this weakly expressed protein. In this study, we describe the identification of protein domains by the heterologous expression, purification, and characterization of Sic1-derived fragment. Four C-terminal fragments (Sic1(C-ter)) were produced based on functional studies and limited-proteolysis results. The N-terminal fragment (Sic1(1-186)) was complementary to the most stable C-terminal fragments (Sic1(Δ186)). Both Sic1(1-186) and Sic1(C-ter) fragments were, in general, less susceptible to spontaneous proteolysis than the full-length protein. The boundaries of the C-terminal fragments turned out to be crucial for integrity of the recombinant proteins and required two rounds of design and production. Sic1 fragments were purified by a simple procedure, based on their resistance to heat treatment, at the amount and purity required for structural characterization. Circular dichroism (CD) measurements and nuclear magnetic resonance (NMR) spectra of N- and C-terminal fragments confirm their disordered nature but reveal minor structural differences that may reflect their distinct functional roles.