Substrate Release Research Articles

Identification of a factor that accelerates substrate release from the signal recognition particle.

The eukaryotic signal recognition particle (SRP) cotranslationally recognizes the first hydrophobic segment of nascent secretory and membrane proteins and delivers them to a receptor at the endoplasmic reticulum (ER). How substrates are released from SRP at the ER to subsequently access translocation factors is not well understood. We found that TMEM208 can engage the substrate binding domain of SRP to accelerate release of its bound cargo. Without TMEM208, slow cargo release resulted in excessive synthesis of downstream polypeptide before engaging translocation factors. Delayed access to translocation machinery caused progressive loss of insertion competence, particularly for multipass membrane proteins, resulting in their impaired biogenesis. Thus, TMEM208 facilitates prompt cargo handover from the targeting to translocation machinery to minimize biogenesis errors and maintain protein homeostasis.

Allosteric substrate release by a sialic acid TRAP transporter substrate binding protein

The tripartite ATP-independent periplasmic (TRAP) transporters enable Vibrio cholerae and Haemophilus influenzae to acquire sialic acid, aiding their colonization of human hosts. This process depends on SiaP, a substrate-binding protein (SBP) that captures and delivers sialic acid to the transporter. We identified 11 nanobodies that bind specifically to the SiaP proteins from H. influenzae (HiSiaP) and V. cholerae (VcSiaP). Two nanobodies inhibited sialic acid binding. Detailed structural and biophysical studies of one nanobody-SBP complex revealed an allosteric inhibition mechanism, preventing ligand binding and releasing pre-bound sialic acid. A hydrophobic surface pocket of the SBP is crucial for the allosteric mechanism and for the conformational rearrangement that occurs upon binding of sialic acid to the SBP. Our findings provide new clues regarding the mechanism of TRAP transporters, as well as potential starting points for novel drug design approaches to starve these human pathogens of important host-derived molecules.

Methylisothiazolinone modulates community assembly and improves syntrophic cooperation via adaptive evolution during sludge anaerobic digestion

Various antimicrobials could pose multifaced threats to anaerobic digestion (AD) of waste-activated sludge (WAS), yet it is still unclear how antimicrobials would affect the metabolic activity and assembly process of anaerobic microbiome. In this study, a typical antimicrobial, methylisothiazolinone (MIT), was introduced in AD and induced an initial lag effect on methane production that was eventually recovered. Although MIT disintegrated WAS to promote the release of bioavailable substrates from extracellular polymeric substances (EPS), it also caused adverse stress to the microbiome. The initial microbial metabolic activities and syntrophic relationships associated with methane generation were distinctly inhibited. Because the self-adaptive systems of two-component systems (e.g., ddpF, and ddpD) and quorum sensing (e.g., cheR, and cydB) were activated to restore the syntrophic interaction among functional anaerobes, metabolic activities were gradually recovered for methane generation. Ultimately, the methane-generation species (e.g., Methanobacterium, and Methanomassiliicoccus) were driven as core species by deterministic processes, while non-functional species (e.g., Nitrospirota) were marginalized by stochastic processes under MIT stress. Overall, this finding indicated the intrinsic drive of evolutionary adaptation, unraveled the community assembly process at phylogenetic level under antimicrobial stress, and gave direction on the assessment and mitigation of external contaminant-related ecological risks in WAS treatment.

Structural dynamics of human deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase)

Structural- and functional heterogeneity, as well as allosteric regulation, in homo-monomeric enzymes is a highly active area of research. One such enzyme is human nuclear-associated deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase), which has emerged as an interesting drug target in combination therapy with traditional nucleotide analogue treatment of cancer. We report, for the first time, a full structural dynamics study of human dUTPase by NMR. dUTPase has been investigated in terms of structural dynamics in its apo form, in complex with the modified substrate resistant to hydrolysis, 2’-deoxyuridine 5’-α,β-imido-triphosphate (dUpNHpp), as well as the product, 2’-deoxy-uridine-monophosphate (dUMP). The apo form of the enzyme displayed slow dynamics in the milli- to microsecond regime in relaxation dispersion experiments, which was further slowed down to observable heterogeneity upon substrate-analogue binding. The results suggest that the non-hydrolysable substrate-analogue traps the enzyme in the conformational isomerization step that has been previously suggested to be part of the enzyme catalysis kinetics cycle. The observed heterogeneity fits well with the pattern expected to emerge from the suggested kinetic model, and no evidence for homotropic allosterism was found. The heatmaps of the slow dynamics, chemical shift perturbation upon substrate binding and conserved regions of the enzyme sequence all displayed a similar pattern, which suggests that the structural dynamics is finely tuned and important for the biological function of the enzyme for binding, conformational shift, catalysis and substrate release.

Enhanced nutritional profile of dehulled peas through fungal fermentation: Impacts on protein, starch, saponins, and total phenolic content

AbstractDehulled peas (DHP) are increasingly popular in food applications, but their integration in food products is limited due to high starch and fiber causing gelling and aggregation during cooking. Additionally, DHP contains saponins (secondary metabolite), contributing to bitter flavors, yet they also hold health benefits. We hypothesize that fungal fermentation could enhance DHP nutritional profile and integration into food products. This study evaluated the effects of six fungal organisms (Aspergillus niger [An], Aspergillus oryzae [Ao], Aureobasidium pullulans [Ap], Neurospora crassa [Nc], Rhizopus microspores var. oligosporus [Ro], Trichoderma reesei [Tr]) on DHP over 120 h of submerged fermentation, evaluating total phenolics, starch, saponins, crude proteins, and overall mass balance. Results from the study demonstrated notable changes post‐fermentation, including increased overall protein content and solubility, decreased starch content, reduced overall mass recovery, and elevated levels of total phenolics and saponins. Filamentous fungi exhibited a significant reduction in starch content, contributing to a substantial reduction in mass recovery (31%–60%) compared to the control. Unexpectedly, saponin concentrations increased (1.5 to 3 folds) during fermentation, possibly attributed to the breakdown of the substrate matrix and release of bound saponins. Total phenolic levels varied among microorganisms, with An and Nc demonstrating the highest increases (6 to 10 folds) as compared to the control. Overall, these findings point to fungal fermentation as a tool for adding value to yellow peas and other crops facing similar processing challenges. Further research is warranted to understand the health impacts and value of these enhancements.

Elucidating the synergic mechanism of sodium pyrophosphate assisted enzymolysis on the waste activated sludge fermentation enhancement: Insights from organic conversion and metagenomic analysis

Lysozyme has been identified as a promising approach for enhancing waste activated sludge (WAS) anaerobic fermentation. However, the extracellular polymeric substance (EPS), functioning as a ‘protective shell’ for microbial cells, hindered the lysozyme transfer and subsequent interaction with cell wall, resulting in excessive dosage and limited efficiency in practical engineering applications. This study innovatively proposed a sodium pyrophosphate (SP) assisted lysozyme treatment, aiming to overcome such limitations and enhance the solubilization and hydrolysis efficiency. As a result, 3583.5 mg COD/L short-chain fatty acids (SCFAs) and 23.1 % acidification rate were achieved after 2-day anaerobic fermentation within SP-lysozyme treatment. Mechanism investigation revealed that SP facilitated EPS disruption by inducing a loosening of the protein structure, leading to the enhanced solubilization of organic matter within the EPS and microbial cells. Consequently, the release of substantial substrates contributed to the enrichment of functional microbes associated with hydrolysis and acidogenesis, accompanied by the augmentation of corresponding metabolic functions. Besides, the key functional genes analysis demonstrated the amino acid metabolism, glycolysis, pyruvate metabolism and SCFAs transport were facilitated and the methanogenesis was inhibited on the contrary with SP-lysozyme treatment, which was undoubtedly responsible for the superior SCFAs accumulation in anaerobic fermentation process. Considering the carbon resource recovery in SCFAs form, such novel SP-lysozyme treatment exhibited 325.1 CNY /ton SS (45.5 USD / ton SS) economic benefit and avoided 0.12 CO2/ton SS carbon emission, suggesting its promising application potential in future WAS management.

Early steps of protein disaggregation by Hsp70 chaperone and class B J-domain proteins are shaped by Hsp110

Hsp70 is a key cellular system counteracting protein misfolding and aggregation, associated with stress, ageing, and disease. Hsp70 solubilises aggregates and aids protein refolding through substrate binding and release cycles regulated by co-chaperones: J-domain proteins (JDPs) and nucleotide exchange factors (NEFs). Here, we elucidate the collaborative impact of Hsp110 NEFs and different JDP classes throughout Hsp70-dependent aggregate processing. We show that Hsp110 plays a major role at initial stages of disaggregation, determining its final efficacy. The NEF catalyses the recruitment of thick Hsp70 assemblies onto aggregate surface, which modifies aggregates into smaller species more readily processed by chaperones. Hsp70 stimulation by Hsp110 is much stronger with class B than class A JDPs and requires the auxiliary interaction between class B JDP and the Hsp70 EEVD motif. Furthermore, we demonstrate for the first time that Hsp110 disrupts the JDP-Hsp70 interaction. Such destabilisation of chaperone complexes at the aggregate surface might improve disaggregation, but also lead to the inhibition above the sub-stoichiometric Hsp110 optimum. Thus, balanced interplay between the co-chaperones and Hsp70 is critical to unlock its disaggregating potential.

Early steps of protein disaggregation by Hsp70 chaperone and class B J-domain proteins are shaped by Hsp110.

Hsp70 is a key cellular system counteracting protein misfolding and aggregation, associated with stress, ageing, and disease. Hsp70 solubilises aggregates and aids protein refolding through substrate binding and release cycles regulated by co-chaperones: J-domain proteins (JDPs) and nucleotide exchange factors (NEFs). Here, we elucidate the collaborative impact of Hsp110 NEFs and different JDP classes throughout Hsp70-dependent aggregate processing. We show that Hsp110 plays a major role at initial stages of disaggregation, determining its final efficacy. The NEF catalyses the recruitment of thick Hsp70 assemblies onto aggregate surface, which modifies aggregates into smaller species more readily processed by chaperones. Hsp70 stimulation by Hsp110 is much stronger with class B than class A JDPs and requires the auxiliary interaction between class B JDP and the Hsp70 EEVD motif. Furthermore, we demonstrate for the first time that Hsp110 disrupts the JDP-Hsp70 interaction. Such destabilisation of chaperone complexes at the aggregate surface might improve disaggregation, but also lead to the inhibition above the sub-stoichiometric Hsp110 optimum. Thus, balanced interplay between the co-chaperones and Hsp70 is critical to unlock its disaggregating potential.

SUMOylation of protein phosphatase 5 regulates phosphatase activity and substrate release

The serine/threonine protein phosphatase 5 (PP5) regulates hormone and stress-induced signaling networks. Unlike other phosphoprotein phosphatases, PP5 contains both regulatory and catalytic domains and is further regulated through post-translational modifications (PTMs). Here we identify that SUMOylation of K430 in the catalytic domain of PP5 regulates phosphatase activity. Additionally, phosphorylation of PP5-T362 is pre-requisite for SUMOylation, suggesting the ordered addition of PTMs regulates PP5 function in cells. Using the glucocorticoid receptor, a well known substrate for PP5, we demonstrate that SUMOylation results in substrate release from PP5. We harness this information to create a non-SUMOylatable K430R mutant as a ‘substrate trap’ and globally identified novel PP5 substrate candidates. Lastly, we generated a consensus dephosphorylation motif using known substrates, and verified its presence in the new candidate substrates. This study unravels the impact of cross talk of SUMOylation and phosphorylation on PP5 phosphatase activity and substrate release in cells.

Additive Effects in Metal/Lewis Acid Cooperativity Assessed in a Tetrahedral Copper Hydrazine Complex Featuring an Appended Borane.

Within metal/ligand cooperative systems employing acidic groups, studies that empirically assess distance relationships are needed to maximize cooperative interactions with substrates. We report the formation of two Cu(I)-N2H4 complexes using 1,4,7-triazacyclononane ligand frameworks bearing two tert-butyl groups and either a Lewis acidic trialkylborane or an inert alkyl group. Metal/Lewis acid cooperativity imparts heightened acidification of the hydrazine substrate and plays a key role in the release of substrate to a competitive Lewis acidic group.

Vps4 substrate binding and coupled mechanisms of Vps4p substrate recruitment and release from autoinhibition.

The ESCRT pathway's AAA+ ATPase, Vps4p, remodels ESCRT-III complexes to drive membrane fission. Here, we use peptide binding assays to further the understanding of substrate specificity and the mechanism of autoinhibition. Our results reveal unexpected sequence preference to the substrate binding groove and an elegant mechanism of regulation that couples localization to substrate with release from autoinhibition.

Direct detection of the chloride release and uptake reactions of Natronomonas pharaonis halorhodopsin

Membrane transport proteins undergo multistep conformational changes to fulfill the transport of substrates across biological membranes. Substrate release and uptake are the most important events of these multistep reactions that accompany significant conformational changes. Thus, their relevant structural intermediates should be identified to better understand the molecular mechanism. However, their identifications have not been achieved for most transporters due to the difficulty of detecting the intermediates. Herein, we report the success of these identifications for a light-driven chloride transporter halorhodopsin (HR). We compared the time course of two flash-induced signals during a single transport cycle. One is a potential change of Cl--selective membrane, which enabled us to detect tiny Cl--concentration changes due to the Cl- release and the subsequent Cl--uptake reactions by HR. The other is the absorbance change of HR reflecting the sequential formations and decays of structural intermediates. Their comparison revealed not only the intermediates associated with the key reactions but also the presence of two additional Cl--binding sites on the Cl--transport pathways. The subsequent mutation studies identified one of the sites locating the protein surface on the releasing side. Thus, this determination also clarified the Cl--transport pathway from the initial binding site until the release to the medium.

New insight into process pH regulation on caproate production from waste activated sludge acidogenic fermentation liquid by cathodic electro-fermentation

Cathodic electro-fermentation (CEF) has been proven to enhance the production of caproate from biowaste by improving chain elongation metabolism. However, the impact of process pH control in catholyte to avoid pH changes caused by redox reactions under polarized cathode on chain elongation has been rarely reported. Here the caproate production performances from waste activated sludge acidogenic fermentation liquid were evaluated and compared between different process pHs of 5.5, 6.0, and 6.5 with control group at 0.9 V. Results demonstrated the maximum caproate concentration was reached 4.1 g/L at pH 6.0, which was significantly higher than the 2.7 g/L of the control group, indicating the benefits of pH control on chain elongation metabolism in CEF. Mechanisms analysis revealed that the enhancement of pH regulation was attributed to the enrichment of organic degraders (Pseudomonas) in catholyte and the increased metabolic activity (like acetyl-CoA carboxylase) of chain elongator (Clostridium_sensus_stricto_12) on biofilm. These results were evidenced by the improved release and conversion performance of substrates (especially the amino acids) in catholyte and the enhanced biofilm activity. Hence, this study contributes novel insights to the pH regulation of chain elongation for enhancing caproate production from biowaste by electro-fermentation.

Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.

Janus and Amphiphilic MoS2 2D Sheets for Surface-Directed Orientational Assemblies toward Ex Vivo Dual Substrate Release.

The two-dimensional (2-D) Janus and amphiphilic molybdenum disulfide (MoS2) nanosheet with opposite optical activities on each side (amphichiral) is synthesized by modifying sandwich-like bulk MoS2 with tannic acid and cholesterol through biphasic emulsion method. This new type of amphichiral Janus MoS2 nanosheet consists of a hydrophilic and positive optical activity tannic acid side as well as a hydrophobic and negative optical activity cholesterol side thereby characterized by circular dichroism. Surface-directed orientational differentiation assemblies are performed for the as-synthesized 2D material and are characterized by contact angle, infrared spectroscopy, X-ray photoelectron, and circular dichroism spectroscopies. The amphiphilic nature of the materials is demonstrated by the pre-organization of the nanosheets on either hydrophobic or hydrophilic surfaces, providing unprecedented properties of circular dichroism signal enhancement and wettability. Selective detachment of the surface organic groups (cholesterol and tannic acid fragments) is realized by matrix-assisted laser desorption/ionisation - time-of-flight(MALDI-TOF) mass spectrometry, and the dual substrate release in tissue is detected by ex vivo mass spectrometry imaging.

Natural deep eutectic solvents pretreatments for sludge dewaterability improvement and subsequent volatile fatty acids production: Insights of sludge disintegration and metagenomic characterization

Higher economic returns and carbon–neutral benefits drive research shifting to volatile fatty acids (VFAs) recovery from sludge treatment. The novel natural deep eutectic solvents (NADES) based pretreatments of waste activated sludge (WAS) were proposed, focusing on sludge dewaterability, organics dissolution, and subsequent production of VFAs via anaerobic fermentation. Three different pretreatment methods, i.e., NADES treatment alone, NADES with 80 °C thermal treatment, and NADES with 180 °C hydrothermal treatment were systematically compared. The dewaterability of sludge significantly improved only when NADES assisted by thermal treatments, which was interpreted by a substantial decline in capillary suction time (CST) and water-holding capacity. All three pretreatments boosted the VFAs production from sludge (1.40–2.33 times compared with control), and increased the proportion of acetic acid by about 10%. Results showed that the highest VFAs yield of 2728.71 mg COD/L was obtained from NADES combined with 180 °C hydrothermal treatment, which was increased by 133 % compared with the control. Such performance is much higher than a surfactant assisted hydrothermal treatment recently reported in the literature (increased by 27 %). Mechanistic studies elucidated that NADES stimulated sludge solubilization, enhancing the release of organic substrates for VFAs production. Further metagenomic analysis demonstrated that the hydrolytic-acidogenic bacteria were enriched after NADES treatments. Moreover, glycosyl transferases (GTs) and carbohydrate esterases (CEs) were the primary carbohydrate-active enzymes (CAZymes) enhanced by NADES addition. This study provides new insights into green solvents simultaneously enhanced sludge dewatering and resource recovery.

Control of Physiologic Glucose Homeostasis via the Hypothalamic Modulation of Gluconeogenic Substrate Availability.

The brain augments glucose production during fasting, but the mechanisms are poorly understood. Here, we show that Cckbr-expressing neurons in the ventromedial hypothalamic nucleus (VMNCckbr cells) prevent low blood glucose during fasting through sympathetic nervous system (SNS)-mediated augmentation of adipose tissue lipolysis and substrate release. Activating VMNCckbr neurons mobilized gluconeogenic substrates without altering glycogenolysis or gluconeogenic enzyme expression. Silencing these cells (CckbrTetTox animals) reduced fasting blood glucose, impaired lipolysis, and decreased circulating glycerol (but not other gluconeogenic substrates) despite normal insulin, counterregulatory hormones, liver glycogen, and liver gluconeogenic gene expression. Furthermore, β3-adrenergic adipose tissue stimulation in CckbrTetTox animals restored lipolysis and blood glucose. Hence, VMNCckbr neurons impact blood glucose not by controlling islet or liver physiology, but rather by mobilizing gluconeogenic substrates. These findings establish a central role for hypothalamic and SNS signaling during normal glucose homeostasis and highlight the importance of gluconeogenic substrate mobilization during physiologic fasting.

Nanoplastics enhance the denitrification process and microbial interaction network in wetland soils

With the widespread presence of plastic waste in ecosystems, it is imperative to understand the response of natural processes to micro- and nanoplastic pollution pressures. However, the effects of nanoplastics on biogeochemical cycles are still overlooked and controversial. This study investigated the effects of three particle sizes (100 μm, 7 μm, and 80 nm) of polystyrene (PS) micro/nanoplastics (0.08 % of mass concentration) on denitrification processes and nirS/nirK denitrifying bacterial communities in wetland soils. The results indicated that PS nanoplastics were found to significantly enhance denitrification rates from 21.30 to 54.73 μmol N2·h−1·kg−1, increasing by 1.57 times compared to the control. Exposure to nanoplastics caused shifts in the composition and structure of the nirS-type denitrifier community. LEfSe analysis, random forest, and Mantel tests revealed that nirS denitrifying bacteria, especially Sideroxydans, played a pivotal role in driving denitrification rates (Mantel's R = 0.24, p = 0.002), likely due to the faster release of organic substrates from nanoplastics. Microbial co-occurrence networks demonstrated that nanoplastic amendments fostered a denser denitrifier network and led to shifts in keystone species. Sideroxydans appeared more likely to cooperate with other bacteria, such as Burkholderiales, to complete denitrification processes. This study suggests that nanoplastics are a potentially stronger driver of denitrification than microplastics, providing insight into the impact of plastic pollutants on biogeochemical cycling in natural wetland ecosystems. Given the widespread distribution of wetlands, the potential increase in gaseous nitrogen emissions due to nanoplastics pollution warrants attention.

Photoswitchable Cascades for Allosteric and Bidirectional Control over Covalent Bonds and Assemblies.

Studies of complex systems and emerging properties to mimic biosystems are at the forefront of chemical research. Dynamic multistep cascades, especially those exhibiting allosteric regulation, are challenging. Herein, we demonstrate a versatile platform of photoswitchable covalent cascades toward remote and bidirectional control of reversible covalent bonds and ensuing assemblies. The relay of a photochromic switch, keto-enol equilibrium, and ring-chain equilibrium allows light-mediated reversible allosteric structural changes. The accompanying distinct reactivity further enables photoswitchable dynamic covalent bonding and release of substrates bidirectionally through alternating two wavelengths of light, essentially realizing light-mediated signaling cycles. The downfall of energy by covalent bond formation/scission upon photochemical reactions offers the driving force for the controlled direction of the cascade. To show the molecular diversity, photoswitchable on-demand assembly/disassembly of covalent polymers, including structurally reconfigurable polymers, was realized. This work achieves photoswitchable allosteric regulation of covalent architectures within dynamic multistep cascades, which has rarely been reported before. The results resemble allosteric control within biological signaling networks and should set the stage for many endeavors, such as dynamic assemblies, molecular motors, responsive polymers, and intelligent materials.

Enabling one-step regeneration of LiBH4 with self-sustaining hydrogen in its spent fuel – One pathway to storing renewable hydrogen

LiBH4 (LB in short) with a hydrogen capacity as high as 18.5 wt% and a low molecular weight (21.78 g mol−1) is among the most promising candidates for the hydrogen-based economy. However, current major technologies in (re)generation of LB rely heavily on energy-intensive processes, which greatly prohibits practical scaling-up of applications. Here we report a sustainable and effective approach to (re)generate LB via converting renewable H+ in crystalline water into H-, achieving a desirable yield of ∼50%. This one-step synthesis method relies on the reaction between spent fuels, specifically LiBO2·xH2O, and Mg-based alloys to form LB under ambient atmosphere. Notably, our approach surpasses the efficiency of other conventional method, such as LiH-B-H2 and MgH2-LiBO2 systems, which not only bypasses the energy-intensive dehydration procedure of LiBO2·xH2O (∼470 ℃) but also eliminates the use of costly hydrides or high-pressure H2. Our findings indicate that Mg participated in the regeneration process prior to Al in Mg-Al alloys and [BH4]- is gradually evolved from other intermediate species [BHx(OH)4-x]- (x = 0, 1, 2, 3). By combining hydrogen release and efficient storage of hydrogen-rich substrate in a closed materials cycle, this study may shed light on a promising step toward application of renewable hydrogen in a fuel cell-based hydrogen economy.