Biology subjects

van Loo, B.

Publications and source records attributed to van Loo, B..

4 recordsLinked to original sources

bioRxiv2026

Intracellular crowding links dimensionality to cell fate through a mechano-metabolic signalling axis

Three-dimensional (3D) cell culture systems exhibit more native-like cell behaviour compared to conventional two-dimensional (2D) cell culture systems. However, it remains largely unknown how dimensionality alters cell behaviour. Here, we identify intracellular crowding as a key biophysical parameter altered by cell culture dimensionality, which directs cell fate over a mechano-metabolic axis. Specifically, culture dimensionality controlled intracellular crowding by altering cell volume, which was confirmed across multiple cell types and cell culture platforms. Using chondrogenesis as a model system, we demonstrated that dimensionality-induced intracellular changes lead to improved chondrogenic stem cell differentiation in microtissue culture via a FOXO1 signalling axis. Our findings highlight intracellular crowding as an important parameter of cell culture systems, and present a novel strategy for engineering biomimetic cell culture systems, which has implications for a multitude of applications including disease modelling, in vitro drug screening models, developmental biology, and cell-based therapies.

cell biology↗

bioRxiv2024

Ancestral aryl sulfatases in the alkaline phosphatase family allow for greater increases in catalytic efficiency than extant variants

Characterizing the dynamics and functional shifts during protein evolution is essential, both for understanding protein evolution and for rationalizing efficient strategies for e.g. enzymes with desired and effective functions. Most proteins organize in families, sets of divergent sequences which share a common ancestor and have a similar structural fold. We study aryl sulfatases (ASs), a subfamily of the large and evolutionary old alkaline phosphatase superfamily. In this manuscript we present how ultrahigh-throughput droplet micro-fluidics can be used for studying aryl sulfatases and their computationally reconstructed putative common ancestors. We compare the evolvability and robustness of three ancestors and four extant ASs which all exhibit catalytic promiscuity towards a range of substrate classes. Fourteen libraries with varying mutation rates were expressed in single-cell microdroplets. In general, higher mutation rates resulted in wider distribution of active variants but fewer improved variants overall. However, the impact of mutation rate differed between enzymes, with some benefiting from higher and others from lower mutation rate, underscoring the need to test diverse mutagenesis regimes.

evolutionary biology↗

bioRxiv2018

High-Throughput, Lysis-free Screening for sulfatase Activity Using Escherichia coli Autodisplay in Microdroplets

Directed evolution of enzymes toward improved catalytic performance has become a powerful tool in protein engineering. To be effective, a directed evolution campaign requires the use of high-throughput screening. In this study we describe the development of a high-throughput lysis-free procedure to screen for improved sulfatase activity by combining microdroplet-based single-variant activity sorting with E. coli autodisplay. For the first step in a 4-step screening procedure we quantitatively screened >105 variants of the homodimeric arylsulfatase from Silicibacter pomeroyi (SpAS1), displayed on the E. coli cell surface, for improved sulfatase activity using fluorescence activated droplet sorting. Display of the sulfatase variants on living E. coli cells ensured the continuous linkage of genotype and phenotype during droplet sorting and allowed for direct recovery by simple regrowth of the sorted cells. The use of autodisplay on living cells simplified and reduced the degree of liquid handling during all steps in the screening procedure to the single event of simply mixing substrate and cells. The percentage of apparent improved variants was enriched >10-fold as a result of droplet sorting. We ultimately identified 25 SpAS1-variants with improved performance toward 4-nitrophenyl sulfate (up to 6.2-fold) and/or fluorescein disulfate (up to 30-fold). In SpAS1 variants with improved performance toward the bulky fluorescein disulfate, many of the beneficial mutations occur in residues that form hydrogen bonds between -helices in the C-terminal oligomerization region, suggesting a non-trivial, previously unknown role for the dimer interface in shaping the substrate binding site of SpAS1.\n\nTable of contents graphic\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC=\"FIGDIR/small/479162v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (29K):\norg.highwire.dtl.DTLVardef@1f9211aorg.highwire.dtl.DTLVardef@ec3b2dorg.highwire.dtl.DTLVardef@1214667org.highwire.dtl.DTLVardef@1952a50_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2018

Transition State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present an investigation based on linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs wild type PAS has a much less negative Br0nsted coefficient ({beta}leaving groupobs-Enz = -0.33) than the uncatalyzed reaction ({beta}leavingroupobs = -1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the TS is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active-site lysine K375, in cooperation with histidine H211. 15N KIEs combined with an increased sensitivity to leaving group ability of the sulfatase activity in neat D2O ({Delta}{beta}leaving groupH-D = +0.06) suggest that the mechanism for S-Obridge bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions towards direct proton donation. 18Ononbridge KIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate in this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

biochemistry↗