Biology subjects

Steinauer, A.

Publications and source records attributed to Steinauer, A..

3 recordsLinked to original sources

bioRxiv2025

An engineered closed-shell, two-component, 480-subunit nucleocapsid

Self-assembling protein cages are powerful nanoscale containers for biotechnology and medical applications. Two-component systems are especially attractive due to their potential for functional complexity. In this study, we demonstrate that the subunits of the 240mer nucleocapsid NC-4, which was previously evolved to package and protect its encoding mRNA, can be split into two fragments without disrupting cage assembly or structure, generating a two-component, 480-subunit capsid. This modification introduces additional termini on the cages exterior surface, creating new opportunities for functionalization. We exploited these new sites by genetically appending peptide and protein tags to the exterior surface of split NC-4 (spNC-4), enabling site-specific glycosylation via post-translational modification and cell-specific delivery by targeted antibody recruitment. Our findings broaden the utility of the NC-4 nucleocapsid. By extension, splitting related protein compartments that bind diverse cargoes could offer a robust platform for biotechnological applications requiring simultaneous encapsulation and customizable surface modification. SIGNIFICANCE STATEMENTNatural protein shells such as viral capsids and bacterial microcompartments have inspired efforts to design synthetic compartments that protect and deliver functional molecules. Here we show that a nonviral, artificially evolved nucleocapsid can be split into two fragments that reassemble into a closed, two-component, 480-subunit cage that packages its own mRNA. This redesign preserves the original architecture and selective RNA packaging while creating new engineerable sites on both the interior and exterior surfaces, enabling simultaneous control of internal cargo binding and external modification. The resulting two-component architecture highlights the structural plasticity of synthetic nucleocapsids and provides a general strategy for constructing modular, evolvable protein containers for biotechnology and synthetic biology.

biochemistry↗

bioRxiv2020

Evolution of a virus-like architecture and packaging mechanism in arepurposed bacterial protein

Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme lacking affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding mRNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid impermeable to nucleases, while emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing non-viral carriers for diverse vaccine and delivery applications.

biochemistry↗

bioRxiv2018

HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins

Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered that cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine motif traffic readily to the cytosol and nucleus with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs are released efficiently from endosomes while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using a broad spectrum of techniques, including an RNA interference (RNAi) screen, fluorescence correlation spectroscopy (FCS), and confocal imaging, we identify VPS39--a gene encoding a subunit of the homotypic fusion and protein sorting (HOPS) complex--as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. Subsequent multi-color confocal imaging studies identify CPMPs within the endosomal lumen, particularly within the intraluminal vesicles (ILVs) of Rab7+ and Lamp1+ endosomes that are the products of HOPS-mediated fusion. These results suggest that CPMPs require HOPS to reach ILVs--an environment that serves as a prerequisite for efficient endosomal escape.

biochemistry↗