No abstract

Many proteins are built from structurally and functionally distinct and domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction. Keywords molecular switch; unfolding; natively unfolded; allostery Proteins often display modular architecture that combines protein or small molecule interaction domains with catalytic domains. In such cases, the domains must be coupled, both functionally and structurally, for the protein to attain overall biological activity. For example, ligand binding or phosphorylation can induce structural changes within a regulatory domain that then trigger activity in a catalytic domain. A related type of switching mechanism is illustrated by the recent discovery of proteins that are unstructured in physiological conditions but fold upon binding to their cellular targets. 1,2 Examples include elongin C 3,4 and the GTPase-binding domain of the Wiskott-Aldrich syndrome protein. 5 In these instances, the folding/unfolding of a regulatory domain modulates function of the intact protein via propagation of structural changes. Protein folding makes a particularly effective functional switch because it is reversible and inherently cooperative. Understanding the molecular basis for this type of mechanism is important because it is widely used to regulate protein-protein interactions and in signaling pathways that control cellular behavior. The system consists of a fusion protein in which human ubiquitin (Ub) is inserted into a surface loop of the ribonuclease barnase (Bn) from Bacillus amyloliquefaciens. These proteins were chosen for the following reasons. First, Bn is extremely lethal to both prokaryotic and eukaryotic cells. It is able to be synthesized in B. amyloliquefaciens only because it is co-expressed with its intracellular inhibitor barstar (Bs). 7 This cytotoxic property allows the enzymatic activity...

Investigative genetic genealogy (IGG) is a new technique for identifying criminal suspects that has sparked controversy. The technique involves uploading a crime scene DNA profile to one or more genetic genealogy databases with the intention of identifying a criminal offender’s genetic relatives and, eventually, locating the offender within the family tree. IGG was used to identify the Golden State Killer in 2018 and it is now being used in connection with hundreds of cases in the USA. Yet, as more law enforcement agencies conduct IGG, the privacy implications of the technique have come under scrutiny. While these issues deserve careful attention, we are concerned that their discussion is, at times, based on misunderstandings related to how IGG is used in criminal investigations and how IGG departs from traditional investigative techniques. Here, we aim to clarify and sharpen the public debate by addressing four misconceptions about IGG. We begin with a detailed description of IGG as it is currently practiced: what it is and—just as important—what it is not. We then examine misunderstood or not widely known aspects of IGG that are potentially confusing efforts to have constructive discussions about its future. We conclude with recommendations intended to support the productivity of those discussions.

The mammalian JNK/p38 MAP kinase kinase kinase MEKK4 and the Saccharomyces cerevisiae Ssk2p are highly homologous. MEKK4 can replace all of the known functions of Ssk2p in yeast, including functioning in the high osmolarity glycerol (HOG) MAPK pathway and the recently described actin recovery pathway. MEKK4 and Ssk2p share a number of conserved domains and appear to be activated by a similar mechanism. Binding of an activating protein to the N-terminal region alleviates auto-inhibition and causes the kinase to auto-phosphorylate, resulting in activation. In this review we will examine the role of the MAP kinase kinase kinase isoform Ssk2p/MEKK4 in the adaptation of both yeast and mammalian systems to specific external stimuli. Recent work has provided a wealth of information about the activation, regulation, and functions of these MEKK kinases to extra-cellular signals. We will also highlight evidence supporting a role for MEKK4 in mediating actin recovery following osmotic shock in mammalian cells.

Osmotic stress induces activation of an adaptive mitogen-activated protein kinase pathway in concert with disassembly of the actin cytoskeleton by a mechanism that is not understood. We have previously shown that the conserved actin-interacting MAP kinase kinase kinase Ssk2p/MEKK4, a member of the high-osmolarity glycerol (HOG) MAPK pathway of Saccharomyces cerevisiae, mediates recovery of the actin cytoskeleton following osmotic stress. In this study, we have employed in vitro kinase assays to show that Ssk2p kinase activity is activated for the actin recovery pathway via a noncanonical, Ssk1p-independent mechanism. Our work also shows that Ssk2p requires the polarisome proteins Bud6p and Pea2p to promote efficient, polarized actin reassembly but that this requirement can be bypassed by overexpression of Ssk2p. Formin (BNI1 or BNR1) and tropomyosin functions are also required for actin recovery but, unlike for Bud6p and Pea2p, these requirements cannot be bypassed by overexpression of Ssk2p. These results suggest that Ssk2p acts downstream of Bud6p and Pea2p and upstream of tropomyosin to drive actin recovery, possibly by upregulating the actin nucleation activity of the formins.