Challenges and Strategies in the Development of Radiation Biodosimetry Tests for Patient Management.

The public health and medical response to a radiological or nuclear incident requires the capability to sort, assess, treat, triage and ultimately discharge, as well as to refer or transport people to their next step in medical care. The Public Health Emergency Medical Countermeasures Enterprise (PHEMCE), directed by the U.S. Department of Health and Human Services (HHS), facilitates a comprehensive, multi-agency effort to develop and deploy radiation biodosimetry tests. Within HHS, discovery and development of biodosimetry tests includes the National Institute of Allergy and Infectious Diseases (NIAID) National Institutes of Health (NIH), the Office of the Assistant Secretary of Preparedness and Response (ASPR), Biomedical Advanced Research and Development Authority (BARDA), and the Food and Drug Administration (FDA) as primary partners in this endeavor. The study of radiation biodosimetry has advanced significantly, with expansion into the fields of cytogenetics, genomics, proteomics, metabolomics, lipidomics and transcriptomics. In addition, expansion of traditional cytogenetic assessment methods using automated platforms, and development of laboratory surge capacity networks have helped to advance biodefense preparedness. This article describes various programs and coordinating efforts between NIAID, BARDA and FDA in the development of radiation biodosimetry approaches to respond to radiological and nuclear threats.

FDA Approval Summary: Alpelisib Plus Fulvestrant for Patients with HR-positive, HER2-negative, PIK3CA-mutated, Advanced or Metastatic Breast Cancer.

On May 24, 2019, the FDA granted regular approval to alpelisib in combination with fulvestrant for postmenopausal women, and men, with hormone receptor (HR)-positive, HER2-negative, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-mutated, advanced or metastatic breast cancer as detected by an FDA-approved test following progression on or after an endocrine-based regimen. Approval was based on the SOLAR-1 study, a randomized, double-blind, placebo-controlled trial of alpelisib plus fulvestrant versus placebo plus fulvestrant. The primary endpoint was investigator-assessed progression-free survival (PFS) per RECIST v1.1 in the cohort of trial participants whose tumors had a PIK3CA mutation. The estimated median PFS by investigator assessment in the alpelisib plus fulvestrant arm was 11 months [95% confidence interval (CI), 7.5-14.5] compared with 5.7 months (95% CI, 3.7-7.4) in the placebo plus fulvestrant arm (HR, 0.65; 95% CI, 0.50-0.85; two-sided P = 0.001). The median overall survival was not yet reached for the alpelisib plus fulvestrant arm (95% CI, 28.1-NE) and was 26.9 months (95% CI, 21.9-NE) for the fulvestrant control arm. No PFS benefit was observed in trial participants whose tumors did not have a PIK3CA mutation (HR, 0.85; 95% CI, 0.58-1.25). The most common adverse reactions, including laboratory abnormalities, on the alpelisib plus fulvestrant arm were increased glucose, increased creatinine, diarrhea, rash, decreased lymphocyte count, increased gamma glutamyl transferase, nausea, increased alanine aminotransferase, fatigue, decreased hemoglobin, increased lipase, decreased appetite, stomatitis, vomiting, decreased weight, decreased calcium, decreased glucose, prolonged activated partial thromboplastin time, and alopecia.

Mtr4-like protein coordinates nuclear RNA processing for heterochromatin assembly and for telomere maintenance.

The regulation of protein-coding and noncoding RNAs is linked to nuclear processes, including chromatin modifications and gene silencing. However, the mechanisms that distinguish RNAs and mediate their functions are poorly understood. We describe a nuclear RNA-processing network in fission yeast with a core module comprising the Mtr4-like protein, Mtl1, and the zinc-finger protein, Red1. The Mtl1-Red1 core promotes degradation of mRNAs and noncoding RNAs and associates with different proteins to assemble heterochromatin via distinct mechanisms. Mtl1 also forms Red1-independent interactions with evolutionarily conserved proteins named Nrl1 and Ctr1, which associate with splicing factors. Whereas Nrl1 targets transcripts with cryptic introns to form heterochromatin at developmental genes and retrotransposons, Ctr1 functions in processing intron-containing telomerase RNA. Together with our discovery of widespread cryptic introns, including in noncoding RNAs, these findings reveal unique cellular strategies for recognizing regulatory RNAs and coordinating their functions in response to developmental and environmental cues.

RNAi triggered by specialized machinery silences developmental genes and retrotransposons.

RNA interference (RNAi) is a conserved mechanism in which small interfering RNAs (siRNAs) guide the degradation of cognate RNAs, but also promote heterochromatin assembly at repetitive DNA elements such as centromeric repeats. However, the full extent of RNAi functions and its endogenous targets have not been explored. Here we show that, in the fission yeast Schizosaccharomyces pombe, RNAi and heterochromatin factors cooperate to silence diverse loci, including sexual differentiation genes, genes encoding transmembrane proteins, and retrotransposons that are also targeted by the exosome RNA degradation machinery. In the absence of the exosome, transcripts are processed preferentially by the RNAi machinery, revealing siRNA clusters and a corresponding increase in heterochromatin modifications across large domains containing genes and retrotransposons. We show that the generation of siRNAs and heterochromatin assembly by RNAi is triggered by a mechanism involving the canonical poly(A) polymerase Pla1 and an associated RNA surveillance factor Red1, which also activate the exosome. Notably, siRNA production and heterochromatin modifications at these target loci are regulated by environmental growth conditions, and by developmental signals that induce gene expression during sexual differentiation. Our analyses uncover an interaction between RNAi and the exosome that is conserved in Drosophila, and show that differentiation signals modulate RNAi silencing to regulate developmental genes.

Different means, same end-heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission yeast.

The assembly of heterochromatin in eukaryotic genomes is critical for diverse chromosomal events including regulation of gene expression, silencing of repetitive DNA elements, proper segregation of chromosomes and maintenance of genomic integrity. Previous studies have shown that noncoding RNAs and the RNA interference (RNAi) machinery promote the assembly of heterochromatin that serves as a multipurpose platform for targeting effectors involved in various chromosomal processes. Recent work has revealed that RNAi-independent mechanisms, involving RNA processing activities that utilize both noncoding and coding RNAs, operate in the assembly of heterochromatin. These findings have established that, in addition to coding for proteins, mRNAs also function as signaling molecules that modify chromatin structure by targeting heterochromatin assembly factors.

RNA elimination machinery targeting meiotic mRNAs promotes facultative heterochromatin formation.

Facultative heterochromatin that changes during cellular differentiation coordinates regulated gene expression, but its assembly is poorly understood. Here, we describe facultative heterochromatin islands in fission yeast and show that their formation at meiotic genes requires factors that eliminate meiotic messenger RNAs (mRNAs) during vegetative growth. Blocking production of meiotic mRNA or loss of RNA elimination factors, including Mmi1 and Red1 proteins, abolishes heterochromatin islands. RNA elimination machinery is enriched at meiotic loci and interacts with Clr4/SUV39h, a methyltransferase involved in heterochromatin assembly. Heterochromatin islands disassemble in response to nutritional signals that induce sexual differentiation. This process involves the antisilencing factor Epe1, the loss of which causes dramatic increase in heterochromatic loci. Our analyses uncover unexpected regulatory roles for mRNA-processing factors that assemble dynamic heterochromatin to modulate gene expression.

Defects in RNA quality control factors reveal RNAi-independent nucleation of heterochromatin.

Heterochromatin assembly at Schizosaccharomyces pombe centromeres involves a self-reinforcing loop mechanism wherein chromatin-bound RNAi factors facilitate targeting of Clr4-Rik1 methyltransferase. However, the initial nucleation of heterochromatin has remained elusive. We show that cells lacking Mlo3, a protein involved in mRNP biogenesis and RNA quality control, assemble functional heterochromatin in RNAi-deficient cells. Heterochromatin restoration is linked to RNA surveillance because loss of Mlo3-associated TRAMP also rescues heterochromatin defects of RNAi mutants. mlo3Δ, which causes accumulation of bidirectional repeat-transcripts, restores Rik1 enrichment at repeats and triggers de novo heterochromatin formation in the absence of RNAi. RNAi-independent heterochromatin nucleation occurs at selected euchromatic loci that show upregulation of antisense RNAs in mlo3Δ cells. We find that the exosome RNA degradation machinery acts parallel to RNAi to promote heterochromatin formation at centromeres. These results suggest that RNAi-independent mechanisms exploit transcription and non-coding RNAs to nucleate heterochromatin.

Polyubiquitin binding and cross-reactivity in the USP domain deubiquitinase USP21.

Modification of proteins by ubiquitin (Ub) and Ub-like (Ubl) modifiers regulates a variety of cellular functions. The ability of Ub to form chains of eight structurally and functionally distinct types adds further complexity to the system. Ub-specific proteases (USPs) hydrolyse polyUb chains, and some have been suggested to be cross-reactive with Ubl modifiers, such as neural precursor cell expressed, developmentally downregulated 8 (NEDD8) and interferon-stimulated gene 15 (ISG15). Here, we report that USP21 cleaves Ub polymers, and with reduced activity also targets ISG15, but is inactive against NEDD8. A crystal structure of USP21 in complex with linear diUb aldehyde shows how USP21 interacts with polyUb through a previously unidentified second Ub- and ISG15-binding surface on the USP domain core. We also rationalize the inability of USP21 to target NEDD8 and identify differences that allow USPs to distinguish between structurally related modifications.

Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes.

Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin or ubiquitin-like gene products, reverse the modification of proteins by a single ubiquitin(-like) protein, and remodel polyubiquitin(-like) chains on target proteins. The human genome encodes nearly 100 DUBs with specificity for ubiquitin in five gene families. Most DUB activity is cryptic, and conformational rearrangements often occur during the binding of ubiquitin and/or scaffold proteins. DUBs with specificity for ubiquitin contain insertions and extensions modulating DUB substrate specificity, protein-protein interactions, and cellular localization. Binding partners and multiprotein complexes with which DUBs associate modulate DUB activity and substrate specificity. Quantitative studies of activity and protein-protein interactions, together with genetic studies and the advent of RNAi, have led to new insights into the function of yeast and human DUBs. This review discusses ubiquitin-specific DUBs, some of the generalizations emerging from recent studies of the regulation of DUB activity, and their roles in various cellular processes.

Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains.

At least eight types of ubiquitin chain exist, and individual linkages affect distinct cellular processes. The only distinguishing feature of differently linked ubiquitin chains is their structure, as polymers of the same unit are chemically identical. Here, we have crystallized Lys 63-linked and linear ubiquitin dimers, revealing that both adopt equivalent open conformations, forming no contacts between ubiquitin molecules and thereby differing significantly from Lys 48-linked ubiquitin chains. We also examined the specificity of various deubiquitinases (DUBs) and ubiquitin-binding domains (UBDs). All analysed DUBs, except CYLD, cleave linear chains less efficiently compared with other chain types, or not at all. Likewise, UBDs can show chain specificity, and are able to select distinct linkages from a ubiquitin chain mixture. We found that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO (NF-kappaB essential modifier) binds to linear chains exclusively, whereas the NZF (Npl4 zinc finger) domain of TAB2 (TAK1 binding protein 2) is Lys 63 specific. Our results highlight remarkable specificity determinants within the ubiquitin system.

Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes).

DUBs (deubiquitinating enzymes) are a family of proteases responsible for the specific removal of ubiquitin attached to target proteins and thus control the free cellular pools of this molecule. DUB activity is usually assayed using full-length ubiquitin, and these enzymes generally show low activity towards small substrates that constitute the P4-P1 LRGG (Lys-Arg-Gly-Gly) C-terminal motif of ubiquitin. To gain insight into the C-terminal recognition region of ubiquitin by DUBs, we synthesized positional scanning libraries of fluorigenic tetrapeptides and tested them on three examples of human DUBs [OTU-1 (ovarian tumour 1), Iso-T (isopeptidase T) and UCH-L3 (ubiquitin C-terminal hydrolase L3)] and one viral ubiquitin-specific protease, namely PLpro (papain-like protease) from SARS (severe acute respiratory syndrome) virus. In most cases the results show flexibility in the P4 position, very high specificity for arginine in the P3 position and glycine in the P2 position, in accord with the sequence of the natural substrate, ubiquitin. Surprisingly, screening of the P2 position revealed that UCH-L3, in contrast with all the other tested DUBs, demonstrates substantial tolerance of alanine and valine at P2, and a parallel analysis using the appropriate mutation of the full-length ubiquitin confirms this. We have also used an optimal tetrapeptide substrate, acetyl-Lys-Arg-Gly-Gly-7-amino-4-methylcoumarin, to investigate the activation mechanism of DUBs by ubiquitin and elevated salt concentration. Together, our results reveal the importance of the dual features of (1) substrate specificity and (2) the mechanism of ubiquitin binding in determining deubiquitination by this group of proteases.

Recognition of polyubiquitin isoforms by the multiple ubiquitin binding modules of isopeptidase T.

The conjugation of polyubiquitin to target proteins acts as a signal that regulates target stability, localization, and function. Several ubiquitin binding domains have been described, and while much is known about ubiquitin binding to the isolated domains, little is known with regard to how the domains interact with polyubiquitin in the context of full-length proteins. Isopeptidase T (IsoT/USP5) is a deubiquitinating enzyme that is largely responsible for the disassembly of unanchored polyubiquitin in the cell. IsoT has four ubiquitin binding domains: a zinc finger domain (ZnF UBP), which binds the proximal ubiquitin, a UBP domain that forms the active site, and two ubiquitin-associated (UBA) domains whose roles are unknown. Here, we show that the UBA domains are involved in binding two different polyubiquitin isoforms, linear and K48-linked. Using isothermal titration calorimetry, we show that IsoT has at least four ubiquitin binding sites for both polyubiquitin isoforms. The thermodynamics of the interactions reveal that the binding is enthalpy-driven. Mutation of the UBA domains suggests that UBA1 and UBA2 domains of IsoT interact with the third and fourth ubiquitins in both polyubiquitin isoforms, respectively. These data suggest that recognition of the polyubiquitin isoforms by IsoT involves considerable conformational mobility in the polyubiquitin ligand, in the enzyme, or in both.

Structural basis for ubiquitin recognition by the Otu1 ovarian tumor domain protein.

Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of approximately 130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys(48) linkages, having little or no activity on Lys(63)- and Lys(29)-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.

The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin.

Ubiquitin binding proteins regulate the stability, function, and/or localization of ubiquitinated proteins. Here we report the crystal structures of the zinc-finger ubiquitin binding domain (ZnF UBP) from the deubiquitinating enzyme isopeptidase T (IsoT, or USP5) alone and in complex with ubiquitin. Unlike other ubiquitin binding domains, this domain contains a deep binding pocket where the C-terminal diglycine motif of ubiquitin is inserted, thus explaining the specificity of IsoT for an unmodified C terminus on the proximal subunit of polyubiquitin. Mutations in the domain demonstrate that it is required for optimal catalytic activation of IsoT. This domain is present in several other protein families, and the ZnF UBP domain from an E3 ligase also requires the C terminus of ubiquitin for binding. These data suggest that binding the ubiquitin C terminus may be necessary for the function of other proteins.