Wiggert J. Altenburg

Bio: Wiggert J. Altenburg is an academic researcher from Harvard University. The author has contributed to research in topics: Eukaryote & Medicine. The author has an hindex of 2, co-authored 3 publications receiving 73 citations. Previous affiliations of Wiggert J. Altenburg include Wyss Institute for Biologically Inspired Engineering.

Prokaryotic nanocompartments form synthetic organelles in a eukaryote.

Abstract: Compartmentalization of proteins into organelles is a promising strategy for enhancing the productivity of engineered eukaryotic organisms. However, approaches that co-opt endogenous organelles may be limited by the potential for unwanted crosstalk and disruption of native metabolic functions. Here, we present the construction of synthetic non-endogenous organelles in the eukaryotic yeast Saccharomyces cerevisiae, based on the prokaryotic family of self-assembling proteins known as encapsulins. We establish that encapsulins self-assemble to form nanoscale compartments in yeast, and that heterologous proteins can be selectively targeted for compartmentalization. Housing destabilized proteins within encapsulin compartments afford protection against proteolytic degradation in vivo, while the interaction between split protein components is enhanced upon co-localization within the compartment interior. Furthermore, encapsulin compartments can support enzymatic catalysis, with substrate turnover observed for an encapsulated yeast enzyme. Encapsulin compartments therefore represent a modular platform, orthogonal to existing organelles, for programming synthetic compartmentalization in eukaryotes.

Prokaryotic nanocompartments form synthetic organelles in a eukaryote

Abstract: Compartmentalization of proteins into organelles is a promising strategy for enhancing the productivity of engineered eukaryotic organisms. However, approaches that co-opt endogenous organelles may be limited by the potential for unwanted crosstalk and disruption of native metabolic functions. Here, we present the construction of synthetic non-endogenous organelles in the eukaryotic yeast Saccharomyces cerevisiae, based on the prokaryotic family of self-assembling proteins known as encapsulins. We establish that encapsulins self-assemble to form nanoscale compartments in yeast, and that heterologous proteins can be selectively targeted for compartmentalization. Housing destabilized proteins within encapsulin compartments affords protection against proteolytic degradation in vivo, while the interaction between split protein components is enhanced upon co-localization within the compartment interior. Furthermore, encapsulin compartments can support enzymatic catalysis, with substrate turnover observed for an encapsulated yeast enzyme. Encapsulin compartments therefore represent a modular platform, orthogonal to existing organelles, for programming synthetic compartmentalization in eukaryotes.

Exploring targeting peptide-shell interactions in encapsulin nanocompartments.

Abstract: Encapsulins are recently discovered protein compartments able to specifically encapsulate cargo proteins in vivo. Encapsulation is dependent on C-terminal targeting peptides (TPs). Here, we characterize and engineer TP-shell interactions in the Thermotoga maritima and Myxococcus xanthus encapsulin systems. Using force-field modeling and particle fluorescence measurements we show that TPs vary in native specificity and binding strength, and that TP-shell interactions are determined by hydrophobic and ionic interactions as well as TP flexibility. We design a set of TPs with a variety of predicted binding strengths and experimentally characterize these designs. This yields a set of TPs with novel binding characteristics representing a potentially useful toolbox for future nanoreactor engineering aimed at controlling cargo loading efficiency and the relative stoichiometry of multiple concurrently loaded cargo proteins.

DNA‐Mediated Protein Shuttling between Coacervate‐Based Artificial Cells

Abstract: Abstract The regulation of protein uptake and secretion is crucial for (inter)cellular signaling. Mimicking these molecular events is essential when engineering synthetic cellular systems. A first step towards achieving this goal is obtaining control over the uptake and release of proteins from synthetic cells in response to an external trigger. Herein, we have developed an artificial cell that sequesters and releases proteinaceous cargo upon addition of a coded chemical signal: single‐stranded DNA oligos (ssDNA) were employed to independently control the localization of a set of three different ssDNA‐modified proteins. The molecular coded signal allows for multiple iterations of triggered uptake and release, regulation of the amount and rate of protein release and the sequential release of the three different proteins. This signaling concept was furthermore used to directionally transfer a protein between two artificial cell populations, providing novel directions for engineering lifelike communication pathways inside higher order (proto)cellular structures.

Enzymatic Regulation of Protein–Protein Interactions in Artificial Cells

Abstract: Membraneless organelles are important for spatial organization of proteins and regulation of intracellular processes. Proteins can be recruited to these condensates by specific protein–protein or protein–nucleic acid interactions, which are often regulated by post‐translational modifications. However, the mechanisms behind these dynamic, affinity‐based protein recruitment events are not well understood. Here, a coacervate system that incorporates the 14‐3‐3 scaffold protein to study enzymatically regulated recruitment of 14‐3‐3‐binding proteins is presented, which mostly bind in a phosphorylation‐dependent manner. Synthetic coacervates are efficiently loaded with 14‐3‐3, and phosphorylated binding partners, such as the c‐Raf pS233/pS259 peptide (c‐Raf), show 14‐3‐3‐dependent sequestration with up to 161‐fold increase in local concentration. The c‐Raf domain is fused to green fluorescent protein (GFP‐c‐Raf) to demonstrate recruitment of proteins. In situ phosphorylation of GFP‐c‐Raf by a kinase leads to enzymatically regulated uptake. The introduction of a phosphatase into coacervates preloaded with the phosphorylated 14‐3‐3‐GFP‐c‐Raf complex results in a significant cargo efflux mediated by dephosphorylation. Finally, the general applicability of this platform to study protein–protein interactions is demonstrated by the phosphorylation‐dependent and 14‐3‐3‐mediated active reconstitution of a split‐luciferase inside artificial cells. This work presents an approach to study dynamically regulated protein recruitment in condensates, using native interaction domains.

Light-based control of metabolic flux through assembly of synthetic organelles.

Abstract: To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.

Formation and function of bacterial organelles.

Abstract: Advances in imaging technologies have revealed that many bacteria possess organelles with a proteomically defined lumen and a macromolecular boundary. Some are bound by a lipid bilayer (such as thylakoids, magnetosomes and anammoxosomes), whereas others are defined by a lipid monolayer (such as lipid bodies), a proteinaceous coat (such as carboxysomes) or have a phase-defined boundary (such as nucleolus-like compartments). These diverse organelles have various metabolic and physiological functions, facilitating adaptation to different environments and driving the evolution of cellular complexity. This Review highlights that, despite the diversity of reported organelles, some unifying concepts underlie their formation, structure and function. Bacteria have fundamental mechanisms of organelle formation, through which conserved processes can form distinct organelles in different species depending on the proteins recruited to the luminal space and the boundary of the organelle. These complex subcellular compartments provide evolutionary advantages as well as enabling metabolic specialization, biogeochemical processes and biotechnological advances. Growing evidence suggests that the presence of organelles is the rule, rather than the exception, in bacterial cells. Advances in imaging techniques have revealed an unexpected abundance and diversity of organelles in bacteria. In this Review, Greening and Lithgow outline the different types of bacterial organelles and discuss common themes in their formation and function.

Genetically engineered magnetic nanocages for cancer magneto-catalytic theranostics

Abstract: The clinical applications of magnetic hyperthermia therapy (MHT) have been largely hindered by the poor magnetic-to-thermal conversion efficiency of MHT agents. Herein, we develop a facile and efficient strategy for engineering encapsulin-produced magnetic iron oxide nanocomposites (eMIONs) via a green biomineralization procedure. We demonstrate that eMIONs have excellent magnetic saturation and remnant magnetization properties, featuring superior magnetic-to-thermal conversion efficiency with an ultrahigh specific absorption rate of 2390 W/g to overcome the critical issues of MHT. We also show that eMIONs act as a nanozyme and have enhanced catalase-like activity in the presence of an alternative magnetic field, leading to tumor angiogenesis inhibition with a corresponding sharp decrease in the expression of HIF-1α. The inherent excellent magnetic-heat capability, coupled with catalysis-triggered tumor suppression, allows eMIONs to provide an MRI-guided magneto-catalytic combination therapy, which may open up a new avenue for bench-to-bed translational research of MHT. The clinical application of magnetic hyperthermia therapy (MHT) is limited by the poor magnetic-to-thermal conversion efficiency of MHT agents. Here, the authors develop encapsulin-produced magnetic iron oxide nanocomposites (eMIONs) with excellent magnetic-heat capability and catalysis-triggered tumor suppression ability to overcome the critical issues of MHT.