Codominance and toxins: A path to drugs of nearly unlimited selectivity

The effectiveness of drugs is often limited by their insufficient selectivity. I propose designs of therapeutic agents that address this problem. The key feature of these reagents, termed comtoxins (codominance-mediated toxins), is their ability to utilize codominance, a property characteristic of many signals in proteins, including degradation signals (degrons) and nuclear localization signals. A comtoxin designed to kill cells that express intracellular proteins P1 and P2 but to spare cells that lack P1 and/or P2 is a multidomain fusion containing a cytotoxic domain and two degrons placed within or near two domains P1* and P2* that bind, respectively, to pi and P2. In a cell containing both P1 and P2, these proteins would bind to the P1* and P2* domains of the comtoxin and sterically mask the nearby (appropriately positioned) degrons, resulting in a long-lived and therefore toxic drug. By contrast, in a cell lacking P1 and/or P2, at least one of the comtoxin's degrons would be active (unobstructed), yielding a short-lived and therefore nontoxic drug. A comtoxin containing both a degron and a nuclear localization signal can be designed to kill exclusively cells that contain P1 but lack P2. Analogous strategies yield comtoxins sensitive to the presence (or absence) of more than two proteins in a cell. Also considered is a class of comtoxins in which a toxic domain is split by a flexible insert containing binding sites for the target proteins. The potentially unlimited, combinatorial selectivity of comtoxins may help solve the problem of side effects that bedevils present-day therapies, for even nonselective delivery of a comtoxin would not affect cells whose protein "signatures" differ from the targeted one

Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: A hypothesis

Despite extensive understanding of sleep regulation, the molecular-level cause and function of sleep are unknown. I suggest that they originate in individual neurons and stem from increased production of protein fragments during wakefulness. These fragments are transient parts of protein complexes in which the fragments were generated. Neuronal Ca^(2+) fluxes are higher during wakefulness than during sleep. Subunits of transmembrane channels and other proteins are cleaved by Ca^(2+)-activated calpains and by other nonprocessive proteases, including caspases and secretases. In the proposed concept, termed the fragment generation (FG) hypothesis, sleep is a state during which the production of fragments is decreased (owing to lower Ca^(2+) transients) while fragment-destroying pathways are upregulated. These changes facilitate the elimination of fragments and the remodeling of protein complexes in which the fragments resided. The FG hypothesis posits that a proteolytic cleavage, which produces two fragments, can have both deleterious effects and fitness-increasing functions. This (previously not considered) dichotomy can explain both the conservation of cleavage sites in proteins and the evolutionary persistence of sleep, because sleep would counteract deleterious aspects of protein fragments. The FG hypothesis leads to new explanations of sleep phenomena, including a longer sleep after sleep deprivation. Studies in the 1970s showed that ethanol-induced sleep in mice can be strikingly prolonged by intracerebroventricular injections of either Ca^(2+) alone or Ca^(2+) and its ionophore. These results, which were never interpreted in connection to protein fragments or the function of sleep, may be accounted for by the FG hypothesis about molecular causation of sleep

RGS4 is arginylated and degraded by the N-end rule pathway in vitro

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. We used an expression-cloning screen to search for mouse proteins that are degraded by the ubiquitin/proteasome-dependent N-end rule pathway in a reticulocyte lysate. One substrate thus identified was RGS4, a member of the RGS family of GTPase-activating proteins that down-regulate specific G proteins. A determinant of the RGS4 degradation signal (degron) was located at the N terminus of RGS4, because converting cysteine 2 to either glycine, alanine, or valine completely stabilized RGS4. Radiochemical sequencing indicated that the N-terminal methionine of the lysate-produced RGS4 was replaced with arginine. Since N-terminal arginine is a destabilizing residue not encoded by RGS4 mRNA, we conclude that the degron of RGS4 is generated through the removal of N-terminal methionine and enzymatic arginylation of the resulting N-terminal cysteine. RGS16, another member of the RGS family, was also found to be an N-end rule substrate. RGS4 that was transiently expressed in mouse L cells was short-lived in these cells. However, the targeting of RGS4 for degradation in this in vivo setting involved primarily another degron, because N-terminal variants of RGS4 that were stable in reticulocyte lysate remained unstable in L cells

Loss of unstably amplified dihydrofolate reductase genes from mouse cells is greatly accelerated by hydroxyurea

Previous work has shown that mammalian cells that carry unstably amplified genes for dihydrofolate reductase (DHFR) gradually lose the amplified DHFR genes when grown in the absence of the DHFR inhibitor methotrexate (MTX). Unstably amplified genes occur on small acentric chromosomes called double minutes (DMs) or even smaller chromatin fragments, in contrast to stably amplified genes, which reside in centromere-containing chromosomes. We have found that the rate of loss of the unstably amplified DHFR genes can be greatly increased by growing the cells in the presence of a nonlethal concentration of hydroxyurea. For example, in one MTX-resistant subline studied, approximate to 90% of the original DHFR gene dosage is lost in 25-30 cell doublings in the absence of MTX. The same degree of loss is achieved, however, in <4 doublings if cells are grown in the presence of 50 μM hydroxyurea. This new effect of hydroxyurea does not appear to be due to changes in plating efficiency or selective cytotoxicity. In particular, no increase in cell death occurs at 50 μM hydroxyurea, and cells continue to multiply, albeit 1/2 to 2/3 as fast as in the absence of hydroxyurea. The ability to selectively accelerate the loss of amplified genes from mammalian cells as shown in the present work may have important implications both for the problem of drug resistance in cancer chemotherapy and for curing mammalian cells of extrachromosomally maintained DNA genomes of pathogenic viruses

N-degron and C-degron pathways of protein degradation

This perspective is partly review and partly proposal. N-degrons and C-degrons are degradation signals whose main determinants are, respectively, the N-terminal and C-terminal residues of cellular proteins. N-degrons and C-degrons include, to varying extents, adjoining sequence motifs, and also internal lysine residues that function as polyubiquitylation sites. Discovered in 1986, N-degrons were the first degradation signals in short-lived proteins. A particularly large set of C-degrons was discovered in 2018. We describe multifunctional proteolytic systems that target N-degrons and C-degrons. We also propose to denote these systems as “N-degron pathways” and “C-degron pathways.” The former notation replaces the earlier name “N-end rule pathways.” The term “N-end rule” was introduced 33 years ago, when only some N-terminal residues were thought to be destabilizing. However, studies over the last three decades have shown that all 20 amino acids of the genetic code can act, in cognate sequence contexts, as destabilizing N-terminal residues. Advantages of the proposed terms include their brevity and semantic uniformity for N-degrons and C-degrons. In addition to being topologically analogous, N-degrons and C-degrons are related functionally. A proteolytic cleavage of a subunit in a multisubunit complex can create, at the same time, an N-degron (in a C-terminal fragment) and a spatially adjacent C-degron (in an N-terminal fragment). Consequently, both fragments of a subunit can be selectively destroyed through attacks by the N-degron and C-degron pathways

The N-end rule and regulation of apoptosis

The ubiquitin-dependent N-end rule pathway targets proteins for degradation through their destabilizing N-terminal residues. This pathway is known to control the import of peptides, chromosome stability and cardiovascular development. A new report identifies yet another function of the N-end rule pathway: the regulation of apoptosis through degradation of Drosophila melanogaster DIAP1

On the cause of sleep: Protein fragments, the concept of sentinels, and links to epilepsy

The molecular-level cause of sleep is unknown. In 2012, we suggested that the cause of sleep stems from cumulative effects of numerous intracellular and extracellular protein fragments. According to the fragment generation (FG) hypothesis, protein fragments (which are continually produced through nonprocessive cleavages by intracellular, intramembrane, and extracellular proteases) can be beneficial but toxic as well, and some fragments are eliminated slowly during wakefulness. We consider the FG hypothesis and propose that, during wakefulness, the degradation of accumulating fragments is delayed within natural protein aggregates such as postsynaptic densities (PSDs) in excitatory synapses and in other dense protein meshworks, owing to an impeded diffusion of the ∼3,000-kDa 26S proteasome. We also propose that a major function of sleep involves a partial and reversible expansion of PSDs, allowing an accelerated destruction of PSD-localized fragments by the ubiquitin/proteasome system. Expansion of PSDs would alter electrochemistry of synapses, thereby contributing to a decreased neuronal firing during sleep. If so, the loss of consciousness, a feature of sleep, would be the consequence of molecular processes (expansions of protein meshworks) that are required for degradation of protein fragments. We consider the concept of FG sentinels, which signal to sleep-regulating circuits that the levels of fragments are going up. Also discussed is the possibility that protein fragments, which are known to be overproduced during an epileptic seizure, may contribute to postictal sleep and termination of seizures. These and related suggestions, described in the paper, are compatible with current evidence about sleep and lead to testable predictions

Bivalent Inhibitor of the N-end Rule Pathway

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Ubr1p, the recognition (E3) component of the Saccharomyces cerevisiae N-end rule pathway, contains at least two substrate-binding sites. The type 1 site is specific for N-terminal basic residues Arg, Lys, and His. The type 2 site is specific for N-terminal bulky hydrophobic residues Phe, Leu, Trp, Tyr, and Ile. Previous work has shown that dipeptides bearing either type 1 or type 2 N-terminal residues act as weak but specific inhibitors of the N-end rule pathway. We took advantage of the two-site architecture of Ubr1p to explore the feasibility of bivalent N-end rule inhibitors, whose expected higher efficacy would result from higher affinity of the cooperative (bivalent) binding to Ubr1p. The inhibitor comprised mixed tetramers of beta-galactosidase that bore both N-terminal Arg (type 1 residue) and N-terminal Leu (type 2 residue) but that were resistant to proteolysis in vivo. Expression of these constructs in S. cerevisiae inhibited the N-end rule pathway much more strongly than the expression of otherwise identical beta-galactosidase tetramers whose N-terminal residues were exclusively Arg or exclusively Leu. In addition to demonstrating spatial proximity between the type 1 and type 2 substrate-binding sites of Ubr1p, these results provide a route to high affinity inhibitors of the N-end rule pathway