Oral enzyme therapy for maple syrup urine disease (MSUD) suppresses plasma leucine levels in intermediate MSUD mice and healthy nonhuman primates.

Maple syrup urine disease (MSUD) is an inborn error of branched-chain amino acid metabolism affecting several thousand individuals worldwide. MSUD patients have elevated levels of plasma leucine and its metabolic product α-ketoisocaproate (KIC), which can lead to severe neurotoxicity, coma, and death. Patients must maintain a strict diet of protein restriction and medical formula, and periods of noncompliance or illness can lead to acute metabolic decompensation or cumulative neurological impairment. Given the lack of therapeutic options for MSUD patients, we sought to develop an oral enzyme therapy that can degrade leucine within the gastrointestinal tract prior to its systemic absorption and thus enable patients to maintain acceptable plasma leucine levels while broadening their access to natural protein. We identified a highly active leucine decarboxylase enzyme from Planctomycetaceae bacterium and used directed evolution to engineer the enzyme for stability to gastric and intestinal conditions. Following high-throughput screening of over 12 000 enzyme variants over 9 iterative rounds of evolution, we identified a lead variant, LDCv10, which retains activity following simulated gastric or intestinal conditions in vitro. In intermediate MSUD mice or healthy nonhuman primates given a whey protein meal, oral treatment with LDCv10 suppressed the spike in plasma leucine and KIC and reduced the leucine area under the curve in a dose-dependent manner. Reduction in plasma leucine correlated with decreased brain leucine levels following oral LDCv10 treatment. Collectively, these data support further development of LDCv10 as a potential new therapy for MSUD patients.

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription.

Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates.1-3 The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle,4-7 together with variations in substrate docking to sites on associated cyclin and Cks subunits.3,6,8-10 Many substrates are modified at multiple sites to provide more complex regulation.10-14 Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of budding yeast genes required for mitotic progression.11,12 As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback.13-16 Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that when high Cdk1 activity accumulates in a mitotic arrest, CLB2 expression is inhibited. Inhibition is accompanied by Ndd1 degradation, and we present evidence that degradation is triggered by multisite Ndd1 phosphorylation by high mitotic Cdk1-Clb2 activity. Complete Ndd1 phosphorylation by Clb2-Cdk1-Cks1 requires the phosphothreonine-binding site of Cks1, as well as a recently identified phosphate-binding pocket on the cyclin Clb2.17 We therefore propose that initial phosphorylation by Cdk1 primes Ndd1 for delayed secondary phosphorylation at suboptimal sites that promote degradation. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression.

Phosphoregulation of Phase Separation by the SARS-CoV-2 N Protein Suggests a Biophysical Basis for its Dual Functions.

The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription. It is not clear how the N protein mediates such distinct functions. The N protein contains two RNA-binding domains surrounded by regions of intrinsic disorder. Phosphorylation of the central disordered region promotes the protein's transcriptional function, but the underlying mechanism is not known. Here, we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates. Unmodified N protein forms partially ordered gel-like condensates and discrete 15-nm particles based on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces these interactions, generating a more liquid-like droplet. We propose that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing.

Phosphorylation modulates liquid-liquid phase separation of the SARS-CoV-2 N protein.

The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription in the infected cell 1-3 . The N protein contains two globular RNA-binding domains surrounded by regions of intrinsic disorder 4 . Phosphorylation of the central disordered region is required for normal viral genome transcription 5,6 , which occurs in a cytoplasmic structure called the replication transcription complex (RTC) 7-11 . It is not known how phosphorylation controls N protein function. Here we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates 12-15 . Unmodified N protein forms partially ordered gel-like structures that depend on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces a subset of these interactions, generating a more liquid-like droplet. We speculate that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing. Inhibitors of N protein phosphorylation could therefore serve as antiviral therapy.

Cohesin cleavage by separase is enhanced by a substrate motif distinct from the cleavage site.

Chromosome segregation begins when the cysteine protease, separase, cleaves the Scc1 subunit of cohesin at the metaphase-to-anaphase transition. Separase is inhibited prior to metaphase by the tightly bound securin protein, which contains a pseudosubstrate motif that blocks the separase active site. To investigate separase substrate specificity and regulation, here we develop a system for producing recombinant, securin-free human separase. Using this enzyme, we identify an LPE motif on the Scc1 substrate that is distinct from the cleavage site and is required for rapid and specific substrate cleavage. Securin also contains a conserved LPE motif, and we provide evidence that this sequence blocks separase engagement of the Scc1 LPE motif. Our results suggest that rapid cohesin cleavage by separase requires a substrate docking interaction outside the active site. This interaction is blocked by securin, providing a second mechanism by which securin inhibits cohesin cleavage.

Editor's Highlight: High-Throughput Functional Genomics Identifies Modulators of TCE Metabolite Genotoxicity and Candidate Susceptibility Genes.

Trichloroethylene (TCE), an industrial chemical and environmental contaminant, is a human carcinogen. Reactive metabolites are implicated in renal carcinogenesis associated with TCE exposure, yet the toxicity mechanisms of these metabolites and their contribution to cancer and other adverse effects remain unclear. We employed an integrated functional genomics approach that combined functional profiling studies in yeast and avian DT40 cell models to provide new insights into the specific mechanisms contributing to toxicity associated with TCE metabolites. Genome-wide profiling studies in yeast identified the error-prone translesion synthesis (TLS) pathway as an import mechanism in response to TCE metabolites. The role of TLS DNA repair was further confirmed by functional profiling in DT40 avian cell lines, but also revealed that TLS and homologous recombination DNA repair likely play competing roles in cellular susceptibility to TCE metabolites in higher eukaryotes. These DNA repair pathways are highly conserved between yeast, DT40, and humans. We propose that in humans, mutagenic TLS is favored over homologous recombination repair in response to TCE metabolites. The results of these studies contribute to the body of evidence supporting a mutagenic mode of action for TCE-induced renal carcinogenesis mediated by reactive metabolites in humans. Our approach illustrates the potential for high-throughput in vitro functional profiling in yeast to elucidate toxicity pathways (molecular initiating events, key events) and candidate susceptibility genes for focused study.