Highly basic clusters in the HSV-1 nuclear egress complex drive membrane budding by inducing lipid ordering

During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and also promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC/membrane interactions and inhibit NEC-mediated budding in vitro, providing a biophysical explanation for the in-vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an "off" switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding.

Diagnostic and Prognostic Value of Circulating CircRNAs in Cancer

Cancer has been regarded as one of the leading causes of mortality worldwide. Diagnostic and prognostic biomarkers with high sensitivity and specificity for cancer play a crucial role in preventing or treating cancer. Circular RNAs (circRNAs), which hold great potential for the management of cancer patients due to their abundance, stable property, and high specificity in serum, plasma, and other body fluids, can be used as non-invasive and blood-based biomarkers in cancer diagnosis and prognosis. There are four types of circRNAs including exonic circRNAs (ecircRNA), intronic circRNAs, exon-intron circRNAs (EIciRNA), and intergenic circRNAs. CircRNAs can act as miRNA sponges, affect protein translation, interplay with RNA binding proteins, regulate protein recruitment, and modulate protein scaffolding and assembly. Therefore, the multifunctionalities of circRNAs make them ideal for detecting and predicting cancer. Indeed, circRNAs manifest high sensitivity and specificity in more than ten types of cancer. This review aims to consolidate the types and functions of circRNAs, as well as discuss the diagnostic and prognostic value of circulating circRNAs in cancer.

The role of Ca2+and protein scaffolding in the formation of nature’s water oxidizing complex

Photosynthetic O2evolution is catalyzed by the Mn4CaO5cluster of the water oxidation complex of the photosystem II (PSII) complex. The photooxidative self-assembly of the Mn4CaO5cluster, termed photoactivation, utilizes the same highly oxidizing species that drive the water oxidation in order to drive the incorporation of Mn2+into the high-valence Mn4CaO5cluster. This multistep process proceeds with low quantum efficiency, involves a molecular rearrangement between light-activated steps, and is prone to photoinactivation and misassembly. A sensitive polarographic technique was used to track the assembly process under flash illumination as a function of the constituent Mn2+and Ca2+ions in genetically engineered membranes of the cyanobacteriumSynechocystissp. PCC6803 to elucidate the action of Ca2+and peripheral proteins. We show that the protein scaffolding organizing this process is allosterically modulated by the assembly protein Psb27, which together with Ca2+stabilizes the intermediates of photoactivation, a feature especially evident at long intervals between photoactivating flashes. The results indicate three critical metal-binding sites: two Mn and one Ca, with occupation of the Ca site by Ca2+critical for the suppression of photoinactivation. The long-observed competition between Mn2+and Ca2+occurs at the second Mn site, and its occupation by competing Ca2+slows the rearrangement. The relatively low overall quantum efficiency of photoactivation is explained by the requirement of correct occupancy of these metal-binding sites coupled to a slow restructuring of the protein ligation environment, which are jointly necessary for the photooxidative trapping of the first stable assembly intermediate.

The role of Ca2+ and protein scaffolding in the formation in nature’s water oxidizing complex

The photosystem II (PSII) complex catalyzing the H2O-oxidation reaction of photosynthesis is highly prone to photodamage. Nature has evolved synthesis and repair mechanisms that include the photooxidative self-assembly, termed photoactivation, of the Mn4CaO5 metal cluster responsible for H2O-oxidation. Assembly is a multi-step light-driven process that proceeds with low quantum yield, involves a critical molecular rearrangement between light-activated steps, and is prone to photoinactivation and mis-assembly. A sensitive polarographic technique was used to track the assembly process under flash illumination as a function of the constituent Mn2+ and Ca2+ ions in genetically engineered samples to elucidate the action of Ca2+ and peripheral proteins. We show that the protein scaffolding that organizes this process is modulated allosterically by the assembly protein Psb27, which together with Ca2+, stabilizes the intermediates of photoactivation, a feature especially evident at long intervals between photoactivating flashes. Besides stabilizing intermediates, the Ca2+ ion is also critical to prevent photoinactivation due to inappropriate binding of Mn2+. Overexpression of Psb27, deletion of extrinsic protein PsbO, and excess Ca2+ characteristically modify these processes and retard the dark rearrangement. The results suggest the involvement of three metal binding sites, two Mn and one Ca with occupation of the Ca site by Ca2+ critical for the suppression of inactivation and the long-observed competition between Mn2+ and Ca2+ occurring at the second Mn site necessary for trapping the first stable assembly intermediates.Significance StatementThe oxidation of water by the photosystem II is the foundation of bioproductivity on Earth and represents a blueprint for sustainable, carbon neutral technologies. Water oxidation is catalyzed by a metal cluster containing of 4 Mn and 1 Ca atoms linked via oxo bridges. The initial assembly is a complex sequential reaction harnessing the photochemical reaction center to photooxidatively incorporate Mn2+ and Ca2+ ions into the catalytic unit embedded in the protein matrix. This photoassembly is crucial for both de novo biosynthesis and as part of the ‘self-healing’ mechanism to cope with incessant photodamage that photosynthetic organisms experience. The results have implications for the natural mechanism as well as the highly desirable biomimetic devices currently envisioned for solar energy production.

Dynamic spatial and structural organization in artificial cells regulates signal processing by protein scaffolding

Engineered artificial cells respond to environmental cues through a pre-programmed enzymatic machinery that induces spatio-structural organization and activation of effector proteins at the lipid membrane.

Conformational dynamics of FERM-mediated autoinhibition in Pyk2 tyrosine kinase

Pyk2 is a non-receptor tyrosine kinase that evolved from gene duplication of focal adhesion kinase (FAK) and subsequent functional specialization in the brain and hemopoietic cells. Pyk2 shares a domain organization with FAK, with an N-terminal regulatory FERM domain adjoining the kinase domain. FAK regulation involves integrin-mediated membrane clustering to relieve autoinhibitory interactions between FERM and kinase domains. Pyk2 regulation remains cryptic, involving Ca2+ influx and protein scaffolding. While the mechanism of the FAK FERM domain in autoinhibition is well-established, the regulatory role of the Pyk2 FERM is ambiguous. We probed the mechanisms of FERM-mediated autoinhibition of Pyk2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and kinase activity profiling. The results reveal FERM-kinase interfaces responsible for autoinhibition. Pyk2 autoinhibition impacts activation loop conformation. In addition, the autoinhibitory FERM-kinase interface exhibits allosteric linkage with the FERM basic patch conserved in both FAK and Pyk2.Table of Contents graphic