Biology subjects

Schwartze, T. A.

Publications and source records attributed to Schwartze, T. A..

4 recordsLinked to original sources

bioRxiv2024

Molecular basis of interchain disulfide-bond formation in BMP-9 and BMP-10

BMP-9 and BMP-10 are TGF-{beta} family signaling ligands naturally secreted into blood. They act on endothelial cells and are required for proper development and maintenance of the vasculature. In hereditary hemorrhagic telangiectasia, regulation is disrupted due to mutations in the BMP-9/10 pathway, namely in the type I receptor ALK1 or the co-receptor endoglin. It has been demonstrated that BMP-9/10 heterodimers are the most abundant signaling species in the blood, but it is unclear how they form. Unlike other ligands of the TGF-{beta} family, BMP-9 and -10 are secreted as a mixture of monomers and disulfide-linked dimers. Here, we show that the monomers are secreted in a cysteinylated form that crystallizes as a noncovalent dimer. Despite this, monomers do not self-associate at micromolar or lower concentrations and have reduced signaling potency compared to dimers. We further show using protein crystallography that the interchain disulfide of the BMP-9 homodimer adopts a highly strained syn-periplanar conformation. Hence, geometric strain across the interchain disulfide is responsible for the reduced propensity to dimerize, not the cysteinylation. Additionally, we show that the dimerization propensity of BMP-9 is lower than BMP-10 and these propensities can be reversed by swapping residues near the interchain disulfide that form attractive interactions with the opposing monomer. Finally, we discuss the implications of these observations on BMP-9/10 heterodimer formation.

molecular biology↗

bioRxiv2024

Design of High Affinity Binders to Convex Protein Target Sites

While there has been progress in the de novo design of small globular miniproteins (50-65 residues) to bind to primarily concave regions of a target protein surface, computational design of minibinders to convex binding sites remains an outstanding challenge due to low level of overall shape complementarity. Here, we describe a general approach to generate computationally designed proteins which bind to convex target sites that employ geometrically matching concave scaffolds. We used this approach to design proteins binding to TGF{beta}RII, CTLA-4 and PD-L1 which following experimental optimization have low nanomolar to picomolar affinities and potent biological activity. Co-crystal structures of the TGF{beta}RII and CTLA-4 binders in complex with the receptors are in close agreement with the design models. Our approach provides a general route to generating very high affinity binders to convex protein target sites.

biochemistry↗

bioRxiv2023

TGM6, a helminth secretory product, mimics TGF-β binding to TβRII to antagonize TGF-β signaling in fibroblasts

The murine helminth parasite Heligmosomoides polygyrus expresses a family of proteins structurally related to TGF-{beta} Mimic 1 (TGM1), a secreted five domain protein that activates the TGF-{beta} pathway and converts naive T lymphocytes to immunosuppressive Tregs. TGM1 signals through the TGF-{beta} type I and type II receptors, T{beta}RI and T{beta}RII, with domains 1-2 and 3 binding T{beta}RI and T{beta}RII, respectively, and domains 4-5 binding CD44, a co-receptor abundant on T cells. TGM6 is a homologue of TGM1 that is co-expressed with TGM1, but lacks domains 1 and 2. Herein, we show that TGM6 binds T{beta}RII through domain 3, but does not bind T{beta}RI, or other type I or type II receptors of the TGF-{beta} family. In TGF-{beta} reporter assays in fibroblasts, TGM6, but not truncated TGM6 lacking domains 4 and 5, potently inhibits TGF-{beta}- and TGM1-induced signaling, consistent with its ability to bind T{beta}RII but not T{beta}RI or other receptors of the TGF-{beta} family. However, TGM6 does not bind CD44 and is unable to inhibit TGF-{beta} and TGM1 signaling in T cells. To understand how TGM6 binds T{beta}RII, the X-ray crystal structure of the TGM6 domain 3 bound to T{beta}RII was determined at 1.4 [A]. This showed that TGM6 domain 3 binds T{beta}RII through an interface remarkably similar to the TGF-{beta}:T{beta}RII interface. These results suggest that TGM6 has adapted its domain structure and sequence to mimic TGF-{beta} binding to T{beta}RII and function as a potent TGF-{beta} and TGM1 antagonist in fibroblasts. The coexpression of TGM6, along with the immunosuppressive TGMs that activate the TGF-{beta} pathway, may prevent tissue damage caused by the parasite as it progresses through its life cycle from the intestinal lumen to submucosal tissues and back again.

biochemistry↗

bioRxiv2023

Shear stress and very low levels of ligand synergize toactivate ALK1 signaling in endothelial cells

Endothelial cells (ECs) respond to concurrent stimulation by biochemical fac-tors and wall shear stress (SS) exerted by blood flow. Disruptions in flow-induced responses can result in remodeling issues and cardiovascular diseases, but the detailed mechanisms linking flow-mechanical cues and biochemical signaling remain unclear. Activin receptor-like kinase 1 (ALK1) integrates SS and ALK1-ligand cues in ECs; ALK1 mutations cause hereditary hemorrhagic telangiectasia (HHT), marked by arteriovenous malformation (AVM) development. However, the mechanistic underpinnings of ALK1 signaling modulation by fluid flow and the link to AVMs remain uncertain. We recorded EC responses under varying SS magnitudes and ALK1 ligand concentrations by assaying pSMAD1/5/9 nuclear localization using a custom multi-SS microfluidic device and a custom image analysis pipeline. We extended the previously reported syn-ergy between SS and BMP9, to include BMP10 and BMP9/10 . Moreover, we demonstrated this synergy is effective even at extremely low SS magnitudes (0.4 dyn/cm2) and ALK1 ligand range (femtogram/mL). The synergistic response to ALK1 ligands and SS requires the kinase activity of ALK1. Moreover, ALK1s basal activity and response to minimal ligand levels depend on endo-cytosis, distinct from cell-cell junctions, cytoskeleton-mediated mechanosensing, or cholesterol-enriched microdomains. Yet, an in-depth comprehension of ALK1 receptor traffickings molecular mechanisms requires further investigation.

cell biology↗