Between 8 to 14 calcium-calmodulin (Ca(2+)/CaM) dependent protein kinase-II (CaMKII) subunits form a complex that modulates synaptic activity. In living cells, the autoinhibited holoenzyme is organized as catalytic-domain pairs distributed around a central oligomerization-domain core. The functional significance of catalytic-domain pairing is not known. In a provocative model, catalytic-domain pairing was hypothesized to prevent ATP access to catalytic sites. If correct, kinase-activity would require catalytic-domain pair separation. Simultaneous homo-FRET and fluorescence correlation spectroscopy was used to detect structural changes correlated with kinase activation under physiological conditions. Saturating Ca(2+)/CaM triggered Threonine-286 autophosphorylation and a large increase in CaMKII holoenzyme hydrodynamic volume without any appreciable change in catalytic-domain pair proximity or subunit stoichiometry. An alternative hypothesis is that two appropriately positioned Threonine-286 interaction-sites (T-sites), each located on the catalytic-domain of a pair, are required for holoenzyme interactions with target proteins. Addition of a T-site ligand, in the presence of Ca(2+)/CaM, elicited a large decrease in catalytic-domain homo-FRET, which was blocked by mutating the T-site (I205K). Apparently catalytic-domain pairing is altered to allow T-site interactions.

The rotavirus nonstructural protein NSP1 repurposes cullin-RING E3 ubiquitin ligases (CRLs) to antagonize innate immune responses. By functioning as substrate adaptors of hijacked CRLs, NSP1 causes ubiquitination and proteasomal degradation of host proteins that are essential for expression of interferon (IFN) and IFN-stimulated gene products. The target of most human and porcine rotaviruses is the β-transducin repeat-containing protein (β-TrCP), a regulator of NF-κB activation. β-TrCP recognizes a phosphorylated degron (DSGΦXS) present in the inhibitor of NF-κB (IκB); phosphorylation of the IκB degron is mediated by IκB kinase (IKK). Because NSP1 contains a C-terminal IκB-like degron (ILD; DSGXS) that recruits β-TrCP, we investigated whether the NSP1 ILD is similarly activated by phosphorylation and whether this modification is required to trigger the incorporation of NSP1 into CRLs. Based on mutagenesis and phosphatase treatment studies, we found that both serine residues of the NSP1 ILD are phosphorylated, a pattern mimicking phosphorylation of IκB. A three-pronged approach using small-molecule inhibitors, small interfering RNAs, and mutagenesis demonstrated that NSP1 phosphorylation is mediated by the constitutively active casein kinase II (CKII), rather than IKK. In coimmunoprecipitation assays, we found that this modification was essential for NSP1 recruitment of β-TrCP and induced changes involving the NSP1 N-terminal RING motif that allowed formation of Cul3-NSP1 complexes. Taken together, our results indicate a highly regulated stepwise process in the formation of NSP1-Cul3 CRLs that is initiated by CKII phosphorylation of NSP1, followed by NSP1 recruitment of β-TrCP and ending with incorporation of the NSP1–β-TrCP complex into the CRL via interactions dependent on the highly conserved NSP1 RING motif.

While kinases are typically composed of one or two subunits, calcium-calmodulin (CaM)-dependent protein kinase II (CaMKII) is composed of 8-14 subunits arranged as pairs around a central core. It is not clear if the CaMKII holoenzyme functions as an assembly of independent subunits, as catalytic pairs, or as a single unit. One strategy to address this question is to genetically engineer monomeric and dimeric CaMKII and evaluate how their activity compares to the wild-type (WT) holoenzyme. Here a technique that combines fluorescence correlation spectroscopy and homo-FRET analysis was used to characterize assembly mutants of Venus-tagged CaMKIIα to identify a dimeric CaMKII. Spectroscopy was then used to compare how holoenzyme structure and function changes in response to activation with CaM in the dimeric mutant, WT-holoenzyme, and a monomeric CaMKII oligomerization-domain deletion mutant control. CaM triggered an increase in hydrodynamic volume in both WT and dimeric CaMKII without altering subunit stoichiometry or the net homo-FRET between Venus-tagged catalytic domains. Biochemical analysis revealed that the dimeric mutant also functioned like WT holoenzyme in terms of its kinase activity with an exogenous substrate, and for endogenous T286 autophosphorylation. We conclude that the fundamental functional units of CaMKII holoenzyme are paired catalytic-domains.

The molecular mechanisms that define the specificity of flavivirus RNA encapsulation are poorly understood. Virions composed of the structural proteins of one flavivirus and the genomic RNA of a heterologous strain can be assembled and have been developed as live attenuated vaccine candidates for several flaviviruses. In this study, we discovered that not all combinations of flavivirus components are possible. While a West Nile virus (WNV) subgenomic RNA could readily be packaged by structural proteins of the DENV2 strain 16681, production of infectious virions with DENV2 strain New Guinea C (NGC) structural proteins was not possible, despite the very high amino acid identity between these viruses. Mutagenesis studies identified a single residue (position 101) of the DENV capsid (C) protein as the determinant for heterologous virus production. C101 is located at the P1= position of the NS2B/3 protease cleavage site at the carboxy terminus of the C protein. WNV NS2B/3 cleavage of the DENV structural polyprotein was possible when a threonine (Thr101 in strain 16681) but not a serine (Ser101 in strain NGC) occupied the P1= position, a finding not predicted by in vitro protease specificity studies. Critically, both serine and threonine were tolerated at the P1= position of WNV capsid. More extensive mutagenesis revealed the importance of flanking residues within the polyprotein in defining the cleavage specificity of the WNV protease. A more detailed understanding of the context dependence of viral protease specificity may aid the development of new protease inhibitors and provide insight into associated patterns of drug resistance. West Nile virus (WNV) and the four serotypes of dengue virus (DENV1 to -4) are mosquito-borne viruses of the Flavivirus genus that significantly impact public health (1, 2). Despite a clear need, neither vaccines nor therapeutics for WNV or DENV have been licensed for use in humans. The flavivirus genome is an ϳ11-kb, single-stranded, positive-sense RNA that encodes a single open reading frame flanked by 5= and 3= untranslated regions. The viral genome is translated on endoplasmic reticulum (ER)-derived membranes into a single polyprotein that undergoes co-and posttranslational cleavage by the viral protease NS2B/3 and host proteases into 10 functionally distinct proteins, including the structural proteins capsid (C), premembrane (prM), and envelope (E) that form the virus particle. During assembly, membrane-anchored prM and E glycoproteins are incorporated into virions as they bud into the ER lumen. The C protein associates with the viral genome in the cytoplasm to form an unstructured nucleocapsid that is incorporated into the budding particle via unknown mechanisms (3). The carboxy terminus (C terminus) of the C protein includes a signal sequence, flanked by protease cleavage sites, that directs the translocation of prM into the ER lumen and tethers C to the cytosolic face of the ER membrane.Cleavage at both sites is essential for virion morphogenesis and occurs in a sequenti...

Viruses have evolved a number of mechanisms to combat host antiviral responses in order to establish a pro-viral cellular environment. Many host antiviral responses rely on signaling cascades initiated by the production of IFN. Rotavirus, a pathogen known to infect nearly all known mammalian and avian animal species, employs NSP1 to counter IFN production. NSP1 proteins encoded by various rotavirus strains share little sequence conservation except for the presence of a putative N-terminal RING domain and a C-terminal substrate-targeting domain. While the targeting domain of most human and porcine rotavirus NSP1 proteins mediates the recruitment of β-TrCP, the targeting domain of many animal strains (simian, murine, equine, etc.) mediates the recruitment of IFN-regulatory factors (e.g., IRF-3/-7). NSP1 binding to β-TrCP or IRF proteins is correlated with proteasomal degradation of these targets in the infected cell.There is a growing body of data to suggest that NSP1 triggers the degradation of targets by hijacking a subset of E3 ubiquitin ligases: the cullin-RING ligases (CRLs). Hijacked CRLs are presumed to direct the ubiquitination and proteasomal degradation of NSP1-bound targets. CRLs are large modular complexes that are minimally comprised of a cullin scaffold protein (Cul1, 2, 3, 4a, 4b, 5, 7), a RING-domain containing protein (Rbx1, 2), and a substrate adaptor that directs the CRL to the target protein. Through multiple

Barophilic bacteria are microorganisms that grow preferentially (facultative barophiles) or exclusively (obligate barophiles) under elevated hydrostatic pressure. Barophilic bacteria have been isolated from a variety of deep-sea environments. Attempts to characterize these organisms have been hampered by a lack of appropriate methodologies. A colorimetric method for the detection of 19 constitutively expressed enzymes under in situ conditions of pressure and temperature has been devised, using a simple modification of the commercially available API ZYME enzyme assay kit. By using this method, enzyme profiles of 11 barophilic isolates, including an obligate barophile, were determined. Nine of the 10 facultatively barophilic isolates examined exhibited a change of phenotype in at least one enzyme reaction when tested at 1 atm (1 atm = 101.29 kPa), compared with results obtained under in situ pressure. The assay is simple and rapid and allows for direct determination of enzyme activity under conditions of high pressure and low temperature.

Calcium/calmodulin dependent protein kinase II (CaMKII) is a family of multifunctional Ser/Thr kinases that play a key role in calcium signaling in many cell types, including neurons, where it performs both structural and signaling roles in learning and memory. CaMKII exists as a dodecameric holoenzyme.

Objective: Single ventricle (SV) heart disease is a set of rare conditions occurring in 5 out of 100,000 births. Forty years ago, such diagnoses were universally fatal, but surgical advances have enabled most patients to live into their thirties. However, SV patients face a lifetime of expensive, invasive clinical care for complications and co-morbidities and decreased life expectancy and quality of life. The cause of SV heart disease is still unknown; believed to be both Mendelian and non-Mendelian, multigenic and multifactorial. To date, four genes have been linked to SV disease; however, the penetrance is low, and none are causal. Project Singular aims to uncover the cause(s) of SV by sequencing at least 5,000 SV patients and immediate family members, in conjunction with collecting important medical data, creating the largest genetic dataset available to date in this disease community and make it available free of charge to all qualified researchers. Methods: Subjects will be recruited and consented virtually through an online portal and will receive a biospecimen collection kit (saliva or buccal) by mail to return to the Broad Institute of MIT and Harvard. Samples will be processed, quality-controlled, and whole genome-sequenced using Illumina HiSeq at 30X coverage, with methylation arrays conducted on a subset. Sequences will be aligned to the human genome, annotated, deidentified, and securely deposited into the Terra.bio platform. Participants will fill out a survey on demographics and medical history, and patients will provide medical records. Deidentified phenotypic and genotypic data will be made available for free to qualified researchers globally. Integration with other SV-related registries will be undertaken where possible to increase the power of the study. Conclusion: Project Singular is the first genetics sequencing initiative of its size and depth focused specifically on SV, the most complex of all congenital heart defects. By enabling patients and families to participate from their homes, Project Singular will reduce barriers of travel and cost to increase diversity of participants. Data democratization of the dataset will fuel discovery in single ventricle etiology and lead to new targeted treatments and potentially curative solutions.