Emergence and Enhancement of Ultrasensitivity through Posttranslational Modulation of Protein Stability

Signal amplification in biomolecular networks converts a linear input to a steeply sigmoid output and is central to a number of cellular functions including proliferation, differentiation, homeostasis, adaptation, and biological rhythms. One canonical signal amplifying motif is zero-order ultrasensitivity that is mediated through the posttranslational modification (PTM) cycle of signaling proteins. The functionality of this signaling motif has been examined conventionally by supposing that the total amount of the protein substrates remains constant, as by the classical Koshland–Goldbeter model. However, covalent modification of signaling proteins often results in changes in their stability, which affects the abundance of the protein substrates. Here, we use mathematical models to explore the signal amplification properties in such scenarios and report some novel aspects. Our analyses indicate that PTM-induced protein stabilization brings the enzymes closer to saturation. As a result, ultrasensitivity may emerge or is greatly enhanced, with a steeper sigmoidal response, higher magnitude, and generally longer response time. In cases where PTM destabilizes the protein, ultrasensitivity can be regained through changes in the activities of the involved enzymes or from increased protein synthesis. Importantly, ultrasensitivity is not limited to modified or unmodified protein substrates—when protein turnover is considered, the total free protein substrate can also exhibit ultrasensitivity under several conditions. When full enzymatic reactions are used instead of Michaelis–Menten kinetics for the modeling, the total free protein substrate can even exhibit nonmonotonic dose–response patterns. It is conceivable that cells use inducible protein stabilization as a strategy in the signaling network to boost signal amplification while saving energy by keeping the protein substrate levels low at basal conditions.

Emergence and Enhancement of Ultrasensitivity through Posttranslational Modulation of Protein Stability

Signal amplification converts a linear input to a steeply sigmoid output and is central to cellular functions. One canonical signal amplifying motif is zero-order ultrasensitivity through the posttranslational modification (PTM) cycle signaling proteins. The functionality of this signaling motif has been examined conventionally by supposing that the total amount of the protein substrates remains constant. However, covalent modification of signaling proteins often results in changes in their stability, which affects the abundance of the protein substrates. Here we use a mathematical model to explore the signal amplification properties in such scenarios. Our simulations indicate that PTM-induced protein stabilization brings the enzymes closer to saturation, and as a result, ultrasensitivity may emerge or is greatly enhanced, with a steeper sigmoidal response of higher magnitude and generally longer response time. In cases where PTM destabilizes the protein, ultrasensitivity can be regained through changes in the activities of the involved enzymes or from increased protein synthesis. Interestingly, ultrasensitivity is not limited to modified or unmodified protein substrates; the total protein substrate can also exhibit ultrasensitivity. It is conceivable that cells use inducible protein stabilization as a way to boost signal amplification while saving energy by keeping the protein substrate at low basal conditions.

Discovery of aryl aminothiazole γ-secretase modulators with novel effects on amyloid β-peptide production

A series of analogs based on a prototype aryl aminothiazole γ-secretase modulator (GSM) were synthesized and tested for their effects on the profile of 37-to-42-residue amyloid β-peptides (Aβ) generated through processive proteolysis of precursor protein substrate by γ-secretase. Certain substitutions on the terminal aryl D ring resulted in an altered profile of Aβ production compared to that seen with the parent molecule. Small structural changes led to concentration-dependent increases in Aβ37 and Aβ38 production without parallel decreases in their precursors Aβ40 and Aβ42, respectively. The new compounds therefore apparently also stimulate carboxypeptidase trimming of Aβ peptides > 43 residues, providing novel chemical tools for mechanistic studies of processive proteolysis by γ-secretase.

Effect of Macronutrient Combination on Survivorship, Growth, and Nutritional Content of Black Soldier Fly Larvae (Hermetia illucens)

Larvae of black soldier fly (Hermetia illucens) has been widely applied as a biological agent for biodegradable wastes upcycling through bioconversion process. However, most of the biodegradable wastes produced from economic activities other than industrial is heterogenous. This may cause some physiological change which may alter the survivorship, growth, and efficiency of the bioconversion process. In this study, the substrate combination of macronutrients provided to black soldier fly larvae were observed to understand the larvae ability to degrade organic waste from economic activities. The substrat proportion consist of three major macronutrients (carbohydrate, protein, and lipid) and made of a mixture of decayed cabbage (Brassica oleracea) (source of carbohydrate), shark catfish (Pangasius sp.) (source of protein), and avocado (Persea americana) (source of lipid) which consisted of four types of substrate namely high fiber, high protein, high lipid, and balance. The feeding rate was 100 mg/larvae/day which provides every three days until 50% of larvae metamorphosed into prepupae. Mortality rate, the weight of larvae, and weight of residue (undigested substrate) were measured during substrate replacement and used to calculated survivorship rate, ECD (Efficiency of Conversion Digested-feed), AD (Approximate digestibility), and WRI (Waste Reduction Index). The proximate analysis also conducted on the harvested larvae biomass. The larvae group fed on high protein substrate showed best survivorship (64,75±2,60%), growth rate (2,97±0,166 mg/larvae/day), and AD (57,39±3,39) while the highest WRI recorded for larvae group fed on high fiber substrate and the highest ECD recorded for larvae group fed on high lipid substrate. The proximate analysis showed the best nutritional content of prepupae of larvae group fed on high protein substrate. It can be concluded that the proportion of macronutrients of substrate effect the growth and bioconversion performance of black soldier fly larvae. Some strategies related to the optimization of the bioconversion process for heterogeny substrate are discussed.Keywords: biodegradable wastes, black soldier fly, heterogeneity, growth, nutritional content, survivorship.

Structure and Mechanism of Hedgehog Acyl Transferase

The iconic Sonic Hedgehog (SHH) morphogen pathway is a fundamental orchestrator of embryonic development and stem cell maintenance, and is implicated in cancers in various organs. A key step in signalling is transfer of a palmitate group to the N-terminal cysteine residue of SHH, catalysed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT) resident in the endoplasmic reticulum (ER). Here, we present the high-resolution cryo-EM structure of HHAT bound to substrate analogue palmityl-coenzyme A and a SHH mimetic megabody. Surprisingly, we identified a heme group bound to an HHAT cysteine residue and show that this modification is essential for HHAT structure and function. A structure of HHAT bound to potent small molecule inhibitor IMP-1575 revealed conformational changes in the active site which occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the novel mechanism by which HHAT adapts the membrane environment to transfer a long chain fatty acid across the ER membrane from cytosolic acyl-CoA to a luminal protein substrate. This structure of a member of the protein-substrate membrane-bound O-acyltransferase (MBOAT) superfamily provides a blueprint for other protein substrate MBOATs, such as WNT morphogen acyltransferase Porcupine and ghrelin O-acyltransferase GOAT, and a template for future drug discovery.

BacPROTACs mediate targeted protein degradation in bacteria

Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis targeting chimeras (PROTACs). Despite their superior properties over classical inhibitors, it has so far not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, highlighting the potential of the technology to provide next generation antibiotics.

Protein Substrate Alters Cell Physiology in Primary Culture of Vocal Fold Epithelial Cells

The basement membrane interacts directly with the vocal fold epithelium. Signaling between the basement membrane and the epithelium modulates gene regulation, differentiation, and proliferation. The purpose of this study was to identify an appropriate simple single-protein substrate for growth of rabbit vocal fold epithelial cells. Vocal folds from 3 New Zealand white rabbits (<i>Oryctolagus cuniculus</i>) were treated to isolate epithelial cells, and cells were seeded onto cell culture inserts coated with collagen I, collagen IV, laminin, or fibronectin. Transepithelial electrical resistance (TEER) was measured, and phase contrast microscopy, PanCK, CK14, and E-cadherin immunofluorescence were utilized to assess for epithelial cell-type characteristics. Further investigation via immunofluorescence labeling was conducted to assess proliferation (Ki67) and differentiation (Vimentin). There was a significant main effect of substrate on TEER, with collagen IV eliciting the highest, and laminin the lowest resistance. Assessment of relative TEER across cell lines identified a larger range of TEER in collagen I and laminin. Phase contrast imaging identified altered morphology in the laminin condition, but cell layer depth did not appear to be related to TEER, differentiation, or morphology. Ki67 staining additionally showed no significant difference in proliferation. All conditions had confluent epithelial cells and dispersed mesenchymal cells, with increased mesenchymal cell numbers over time; however, a higher proportion of mesenchymal cells was observed in the laminin condition. The results suggest collagen IV is a preferable basement membrane substrate for in vitro vocal fold epithelial primary cell culture, providing consistent TEER and characteristic cell morphology, and that laminin is an unsuitable substrate for vocal fold epithelial cells and may promote mesenchymal cell proliferation.