Full Activity Research Articles

Peri active site catalysis of proline isomerisation is the molecular basis of allomorphy in β-phosphoglucomutase

Metabolic regulation occurs through precise control of enzyme activity. Allomorphy is a post-translational fine control mechanism where the catalytic rate is governed by a conformational switch that shifts the enzyme population between forms with different activities. β-Phosphoglucomutase (βPGM) uses allomorphy in the catalysis of isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Herein, we describe structural and biophysical approaches to reveal its allomorphic regulatory mechanism. Binding of the full allomorphic activator β-glucose 1,6-bisphosphate stimulates enzyme closure, progressing through NAC I and NAC III conformers. Prior to phosphoryl transfer, loops positioned on the cap and core domains are brought into close proximity, modulating the environment of a key proline residue. Hence accelerated isomerisation, likely via a twisted anti/C4-endo transition state, leads to the rapid predominance of active cis-P βPGM. In contrast, binding of the partial allomorphic activator fructose 1,6-bisphosphate arrests βPGM at a NAC I conformation and phosphoryl transfer to both cis-P βPGM and trans-P βPGM occurs slowly. Thus, allomorphy allows a rapid response to changes in food supply while not otherwise impacting substantially on levels of important metabolites.

Local Means Binary Networks for Image Super-Resolution.

The success of modern single image super-resolution (SISR) algorithms is inspired by the development of deep convolutional neural networks (CNNs). However, these CNN-based methods require considerable computation and complexity, making it impossible for these methods to perform real-time calculations in edge devices. Thus, lightweight model design has become a development trend in the super-resolution field, including pruning, quantization, and other methods. The 1-bit quantization is an extreme lightweight method which can reduce the calculation amount of the model in an extreme manner and is friendly to hardware such as edge devices. Most existing binary quantization approaches lead to a large information loss during forward propagation, especially in detailed color information (e.g., edge, texture, and contrast). The loss of color information makes modern binary methods unsuitable for SISR tasks. We think the loss occurs because these methods typically utilize a uniform threshold to quantize the weights and activations. Thus, in this article, we thoroughly analyze the difference between normal classification tasks and SISR tasks, and present a binarization scheme based on local means. The proposed method can maintain more detailed information in feature maps using dynamic thresholds during quantization. Specifically, each value in the full precision activations has a corresponding threshold during the quantization process, and those thresholds are determined by the full precision values of the surroundings. In addition, a gradient approximator is introduced to adaptively optimize the gradient for updating binary weights. We then verify the effectiveness of our method for training binary networks on several SISR benchmarks including VDSR and SRResNet. Experimental results show that the proposed method can outperform the state-of-the-art algorithms to obtain binary networks for image super-resolution with better peak signal-to-noise ratio (PSNR) values and visual quality.

PRMT-7/PRMT7 activates HLH-30/TFEB to guard plasma membrane integrity compromised by bacterial pore-forming toxins

ABSTRACT Bacterial pore-forming toxins (PFTs) that disrupt host plasma membrane integrity (PMI) significantly contribute to the virulence of various pathogens. However, how host cells protect PMI in response to PFT perforation in vivo remains obscure. Previously, we demonstrated that the HLH-30/TFEB-dependent intrinsic cellular defense (INCED) is elicited by PFT to maintain PMI in Caenorhabditis elegans intestinal epithelium. Yet, the molecular mechanism for the full activation of HLH-30/TFEB by PFT remains elusive. Here, we reveal that PRMT-7 (protein arginine methyltransferase-7) is indispensable to the nuclear transactivation of HLH-30 elicited by PFTs. We demonstrate that PRMT-7 participates in the methylation of HLH-30 on its RAG complex binding domain to facilitate its nuclear localization and activation. Moreover, we showed that PRMT7 is evolutionarily conserved to regulate TFEB cellular localization and repair plasma damage caused by PFTs in human intestinal cells. Together, our observations not only unveil a novel PRMT-7/PRMT7-dependent post-translational regulation of HLH-30/TFEB but also shed insight on the evolutionarily conserved mechanism of the INCED against PFT in metazoans.

Abstract 4076: Interleukin-18 engineered for resistance to IL-18 binding protein (IL-18BP) and half-life extension to enhance its therapeutic potential

Abstract Interleukin-18 (IL-18) possesses a unique combination of innate and acquired immune functions with high potential to be transformative in cancer immunotherapy of solid tumors. In clinical studies, recombinant wild-type IL-18 was limited by rapid upregulation of IL-18 binding protein (IL-18BP), which serves as a negative feedback checkpoint of the IL-18 pathway. As its name implies, IL-18BP tightly binds to IL-18, thereby blocking its ability to activate the IL-18 receptor. We have focused our protein engineering efforts to obtain variants of IL-18 that are resistant to IL-18BP suppression while retaining IL-18 biological activity. To further capitalize on the immunological effects of IL-18, the IL-18BP resistant variants were fused to half-life extension protein scaffolds for enhanced in vivo exposure. Rational protein design and combinatorial approaches were used to generate a pool of IL-18 variants that were screened for potency and resistance to IL-18BP. Several IL-18 variants showed undetectable binding to IL-18BP (up to 1 mM IL-18BP) and displayed a spectrum of potencies relative to recombinant wild-type IL-18. Importantly, these variants retained their full biological activity in the presence of super-physiological levels of IL-18BP, as tested with in vitro cell-based IFN-γ release assays. In vivo pharmacokinetic studies in mice compared several IL-18BP resistant variants fused to a half-life extension scaffold with a non-half-life enhanced IL-18BP resistant variant. The half-lives for the fusion proteins ranged from 15 - 30h, while the non-fused variant showed a half-live of 0.6h. Furthermore, the IL-18 variants displayed a more durable, Th1 immune response compared to a non-half-life enhanced variant. Thus, IL-18 variants with strong resistance to IL-18BP that are fused to a half-life extension protein scaffold displayed enhanced pharmacokinetic and pharmacodynamic properties in preclinical mouse models. These protein engineering modifications will enable us to select an appropriate development candidate with enhanced pharmaceutical properties to achieve the full therapeutic potential of IL-18 to assess in patients with cancer. Citation Format: Jean Chamoun, Clifford DiLea, Kristiana Dreaden, Pinar Gurel, Joshua Heiber, Robert G. Newman, Su-Ping Pearson, Chunhua Wang, Yanchun Zhao, Mark Whitmore. Interleukin-18 engineered for resistance to IL-18 binding protein (IL-18BP) and half-life extension to enhance its therapeutic potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4076.

Redefining Wound Healing Using Near-Infrared Spectroscopy.

No standard definition for a completely healed wound currently exists; it is recommended that providers use a reliable wound assessment tool to determine healing. The objective of this feasibility study was to determine if a point-of-care, noncontact, near-infrared (NIR) imaging device could provide an objective measure of wound resolution and guide clinical decision-making for the optimal time to transition from protective wound dressings and gradual return to full activity. In this single-center feasibility study, adult patients 18 years and older with a lower extremity wound of any size and etiology were seen weekly for wound assessment and standard-of-care treatment. The researchers performed serial imaging with a point-of-care, noncontact, NIR imaging device (Snapshot NIR ; Kent Imaging Inc) to assess the wound and surrounding skin and evaluated the difference in time to 100% reepithelialization on visual inspection and homogeneous tissue oxygen saturation levels at the wound site and surrounding closed skin envelope. An average time difference of 13.5 ± 10 days (median, 12 days; range, 0-35 days) was observed between 100% reepithelialization on visual wound inspection and imaging assessment. Further, NIR imaging could determine when a patient was at risk for recurrent wound breakdown. The addition of point-of-care, noncontact, NIR imaging may help guide clinical decision-making for the optimal time to transition from protective wound dressings with gradual return to full activity and minimize wound recurrence.

New Methodology to Evaluate and Optimize Indoor Ventilation Based on Rapid Response Sensors.

The recent pandemic increased attention to the need for appropriated ventilation and good air quality as efficient measures to achieve safe and healthy indoor air. This work provides a novel methodology for continuously evaluating ventilation in public areas using modern rapid response sensors (RRS). This methodology innovatively assesses the ventilation of a space by combining a quantitative estimation of the real air exchange in the space-obtained from CO2 experimental RRS measurements and the characteristics of and activity in the space-and indoor and outdoor RRS measurements of other pollutants, with healthy recommendations from different organisations. The methodology allows space managers to easily evaluate, in a continuous form, the appropriateness of their ventilation strategy, thanks to modern RRS measurements and direct calculations (implemented here in a web app), even in situations of full activity. The methodology improves on the existing standards, which imply the release of tracer gases and expert intervention, and could also be used to set a control system that measures continuously and adapts the ventilation to changes in indoor occupancy and activity, guaranteeing safe and healthy air in an energy-efficient way. Sample public concurrence spaces with different conditions are used to illustrate the methodology.

The Influence of Insurance Status on Extensor Tendon Repair Outcomes

Background: Little is known regarding how patient insurance status influences outcomes after extensor tendon-injury repair. We aim to establish a relationship between the outcomes of primarily repaired extensor tendon injuries and patient insurance status. We hypothesize that commercially insured patients will achieve superior outcomes due to more facile access to postoperative hand therapy and fewer barriers to appropriate postoperative care. Methods: A retrospective chart review was conducted of patients who underwent primary extensor tendon repair in any zone, excluding the thumb, at a single large safety-net hospital. Inclusion criteria included a minimum of eight weeks of follow-up, complete data available for review, and an extensor tendon injury requiring primary surgical repair. Four cohorts were examined: patients with commercial insurance, patients with Medicare/Medicaid, patients with county hospital-sponsored insurance, and uninsured patients. Statistical analysis was performed using Chi-Square and ANOVA analyses, with significance defined as p ≤ 0.05. Results: Of the 62 patients (100 digits) included, 20 had commercial insurance, 12 had Medicare/Medicaid, 13 had hospital-sponsored insurance, and 17 were uninsured. Except for mean age, there were no significant differences between groups in terms of demographic data, medical comorbidities, or digit characteristics. There were also no significant differences in mean follow-up, time to return to full activity, or surgical complications among groups. Procedure duration differed significantly between groups, with procedures lasting longer in uninsured patients. Postoperative final flexion total arc of motion (TAM) and extension measurements were similar across all groups. Additionally, hand therapy visits did not differ significantly between groups. Conclusions: Following extensor tendon repair, patient insurance status did not affect outcomes in terms of final range of motion, return to full activity, or postoperative complications.

Plitidepsin as an Immunomodulator against Respiratory Viral Infections.

Plitidepsin is a host-targeted compound known for inducing a strong anti-SARS-CoV-2 activity, as well as for having the capacity of reducing lung inflammation. Because IL-6 is one of the main cytokines involved in acute respiratory distress syndrome, the effect of plitidepsin in IL-6 secretion in different invitro and invivo experimental models was studied. A strong plitidepsin-mediated reduction of IL-6 was found in human monocyte-derived macrophages exposed to nonproductive SARS-CoV-2. In resiquimod (a ligand of TLR7/8)-stimulated THP1 human monocytes, plitidepsin-mediated reductions of IL-6 mRNA and IL-6 levels were also noticed. Additionally, although resiquimod-induced binding to DNA of NF-κB family members was unaffected by plitidepsin, a decrease in the regulated transcription by NF-κB (a key transcription factor involved in the inflammatory cascade) was observed. Furthermore, the phosphorylation of p65 that is required for full transcriptional NF-κB activity was significantly reduced by plitidepsin. Moreover, decreases of IL-6 levels and other proinflammatory cytokines were also seen in either SARS-CoV-2 or H1N1 influenza virus-infected mice, which were treated at low enough plitidepsin doses to not induce antiviral effects. In summary, plitidepsin is a promising therapeutic agent for the treatment of viral infections, not only because of its host-targeted antiviral effect, but also for its immunomodulatory effect, both of which were evidenced invitro and invivo by the decrease of proinflammatory cytokines.

NCP, a dual kappa and mu opioid receptor agonist, is a potent analgesic against inflammatory pain without reinforcing or aversive properties.

While agonists of mu (MOR) and kappa (KOR) opioid receptors have analgesic effects, they produce euphoria and dysphoria, respectively. Other side effects include respiratory depression and addiction for MOR agonists and sedation for KOR agonists. We reported that 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-{[4'-(2'-cyanopyridyl)]carboxamido}cmorphinan (NCP) displayed potent KOR full agonist and MOR partial agonist activities (58%) with 6.5x KOR-over-MOR selectivity in vitro Herein, we characterized pharmacological effects of NCP in rodents. In mice, NCP exerted analgesic effects against inflammatory pain in both the formalin test and the acetic acid writhing test, with A50 values of 47.6 and 14.4 microg/kg (s.c.), respectively. The analgesic effects in the acetic acid writhing test were mediated by the KOR. NCP at doses much higher than those effective in reducing inflammatory pain did not produce antinociception in the hot plate and tail flick tests, inhibit compound 48/80-induced scratching, cause conditioned place aversion (CPA) or preference, impair rotarod performance, inhibit locomotor activity, cause respiratory depression, or precipitate morphine withdrawal. However, NCP (10~100 microg/kg) inhibited gastrointestinal transit with a maximum of ~40% inhibition. In MOR knockout mice, NCP caused CPA, demonstrating that its lack of CPA is due to combined actions on the MOR and KOR. Following s.c. injection, NCP penetrated into the mouse brain. In rats trained to self-administer heroin, NCP (1~320 microg/kg/infusion) did not function as a reinforcer. Thus, NCP produces potent analgesic effects via KOR without side effects except constipation. Therefore, dual full KOR/partial MOR agonists with moderate KOR-over-MOR selectivity may be promising as non-addictive analgesics for inflammatory pain. Significance Statement Developing non-addictive analgesics is crucial for reducing opioid overdose deaths, minimizing drug misuse, and promoting safer pain management practices. Herein, pharmacology of a potential non-addictive analgesic, NCP, is reported. NCP has full KOR agonist / partial MOR agonist activities with a 6.5 x selectivity for KOR over MOR. Unlike MOR agonists, analgesic doses of NCP do not lead to self-administration or respiratory depression. Furthermore, NCP does not produce aversion, hypolocomotion, or motor incoordination, side effects typically associated with KOR activation.

Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress.

Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.

A Quality Improvement Project to Reduce Rapid Response System Inequities for Patients with Limited English Proficiency at a Quaternary Academic Medical Center.

Recognition of clinically deteriorating hospitalized patients with activation of rapid response (RR) systems can prevent patient harm. Patients with limited English proficiency (LEP), however, experience less benefit from RR systems than do their English-speaking counterparts. To improve outcomes among hospitalized LEP patients experiencing clinical deteriorations. Quasi-experimental pre-post design using quality improvement (QI) statistics. All adult hospitalized non-intensive care patients with LEP who were admitted to a large academic medical center from May 2021 through March 2023 and experienced RR system activation were included in the evaluation. All patients included after May 2022 were exposed to the intervention. Implementation of a modified RR system for LEP patients in May 2022 that included electronic dashboard monitoring of early warning scores (EWSs) based on electronic medical record data; RR nurse initiation of consults or full RR system activation; and systematic engagement of interpreters. Process of care measures included monthly rates of RR system activation, critical response nurse consultations, and disease severity scores prior to activation. Main outcomes included average post-RR system activation length of stay, escalation of care, and in-hospital mortality. Analyses used QI statistics to identify special cause variation in pre-post control charts based on monthly data aggregates. In total, 222 patients experienced at least one RR system activation during the study period. We saw no special cause variation for process measures, or for length of hospitalization or escalation of care. There was, however, special cause variation in mortality rates with an overall pre-post decrease in average monthly mortality from 7.42% (n = 8/107) to 6.09% (n = 7/115). In this pilot study, prioritized tracking, utilization of EWS-triggered evaluations, and interpreter integration into the RR system for LEP patients were feasible to implement and showed promise for reducing post-RR system activation mortality.

Troponin Structural Dynamics in the Native Cardiac Thin Filament Revealed by Cryo Electron Microscopy

Cardiac muscle contraction occurs due to repetitive interactions between myosin thick and actin thin filaments (TF) regulated by Ca2+ levels, active cross-bridges, and cardiac myosin-binding protein C (cMyBP-C). The cardiac TF (cTF) has two nonequivalent strands, each comprised of actin, tropomyosin (Tm), and troponin (Tn). Tn shifts Tm away from myosin-binding sites on actin at elevated Ca2+ levels to allow formation of force-producing actomyosin cross-bridges. The Tn complex is comprised of three distinct polypeptides – Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. The molecular mechanism of their collective action is unresolved due to lack of comprehensive structural information on Tn region of cTF. C1 domain of cMyBP-C activates cTF in the absence of Ca2+ to the same extent as rigor myosin. Here we used cryo-EM of native cTFs to show that cTF Tn core adopts multiple structural conformations at high and low Ca2+ levels and that the two strands are structurally distinct. At high Ca2+ levels, cTF is not entirely activated by Ca2+ but exists in either partially or fully activated state. Complete dissociation of TnI C-terminus is required for full activation. In presence of cMyBP-C C1 domain, Tn core adopts a fully activated conformation, even in absence of Ca2+. Our data provide a structural description for the requirement of myosin to fully activate cTFs and explain increased affinity of TnC to Ca2+ in presence of active cross-bridges. We suggest that allosteric coupling between Tn subunits and Tm is required to control actomyosin interactions.

Arthroscopic Modified Lasso-Loop Stitch Technique for Chronic Lateral Ankle Instability in Skeletally Immature vs Mature Patients.

In recent years, arthroscopic lateral ankle ligament repair has become increasingly popular. However, reports on the clinical outcomes of arthroscopic ankle stabilization for skeletally immature patients remain scarce. This study investigated the clinical outcomes of arthroscopic lateral ankle ligaments repair in skeletally immature patients compared to skeletally mature patients. Our retrospective analysis compared skeletally immature patients and skeletally mature adults who underwent arthroscopic repair of the ankle lateral ligaments with a modified lasso-loop stitch using a suture anchor. Skeletal immaturity was defined as patients whose epiphyseal line of the distal fibula remained open on plain radiography. Clinical outcomes were evaluated using the Self-Administered Foot Evaluation Questionnaire (SAFE-Q) at 2 years after surgery. The time to postoperative walking, jogging, and return to full sports activities were also evaluated. Sixty-four skeletally immature patients (IM group) and 103 skeletally mature adults (M group) were included. No significant differences were observed during both walking and jogging after surgery between the groups; however, return to full athletic activities was significantly earlier in IM group (P = .05). The mean scores in all SAFE-Q subscales significantly improved in both groups after surgery (P < .001). There were also no statistically significant differences between the groups in the mean postoperative SAFE-Q scores for all subscales. All patients in the IM group returned to playing sports at their preinjury levels postoperatively. We found that skeletally immature patients with chronic lateral ankle instability had generally similar responses to arthroscopic lateral ankle as skeletally mature adult patients at minimally 2 years' follow-up with a high rate of successful return to sport. Level III, retrospective case control study.

Activation of zinc uptake regulator by zinc binding to three regulatory sites.

Zur is a Fur-family metalloregulator that is widely used to control zinc homeostasis in bacteria. In Streptomyces coelicolor, Zur (ScZur) acts as both a repressor for zinc uptake (znuA) gene and an activator for zinc exporter (zitB) gene. Previous structural studies revealed three zinc ions specifically bound per ScZur monomer; a structural one to allow dimeric architecture and two regulatory ones for DNA-binding activity. In this study, we present evidence that Zur contains a fourth specific zinc-binding site with a key histidine residue (H36), widely conserved among actinobacteria, for regulatory function. Biochemical, genetic, and calorimetric data revealed that H36 is critical for hexameric binding of Zur to the zitB zurbox and further binding to its upstream region required for full activation. A comprehensive thermodynamic model demonstrated that the DNA-binding affinity of Zur to both znuA and zitB zurboxes is remarkably enhanced upon saturation of all three regulatory zinc sites. The model also predicts that the strong coupling between zinc binding and DNA binding equilibria of Zur drives a biphasic activation of the zitB gene in response to a wide concentration change of zinc. Similar mechanisms may be pertinent to other metalloproteins, expanding their response spectrum through binding multiple regulatory metals.

SPINT2 mutations in the Kunitz domain 2 found in SCSD patients inactivate HAI-2 as prostasin inhibitor via abnormal protein folding and N-glycosylation.

Mutations in the Kunitz-type serine protease inhibitor HAI-2, encoded by SPINT2, are responsible for the pathogenesis of syndromic congenital sodium diarrhea (SCSD), an intractable secretory diarrhea of infancy. Some of the mutations cause defects in the functionally required Kunitz domain 1 and/or subcellular targeting signals. Almost all SCSD patients, however, harbor SPINT2 missense mutations that affect the functionally less important Kunitz domain 2. How theses single amino acid substitutions inactivate HAI-2 was, here, investigated by the doxycycline-inducible expression of three of these mutants in HAI-2-knockout Caco-2 human colorectal adenocarcinoma cells. Examining protein expressed from these HAI-2 mutants reveals that roughly 50% of the protein is synthesized as disulfide-linked oligomers that lose protease inhibitory activity due to the distortion of the Kunitz domains by disarrayed disulfide bonding. Although the remaining protein is synthesized as monomers, its glycosylation status suggests that the HAI-2 monomer remains in the immature, lightly glycosylated form, and is not converted to the heavily glycosylated mature form. Heavily glycosylated HAI-2 possesses full anti-protease activity and appropriate subcellular targeting signals, including the one embedded in the complex-type N-glycan. As predicted, these HAI-2 mutants cannot suppress the excessive prostasin proteolysis caused by HAI-2 deletion. The oligomerization and glycosylation defects have also been observed in a colorectal adenocarcinoma line that harbors one of these SPINT2 missense mutations. Our study reveals that the abnormal protein folding and N-glycosylation can cause widespread HAI-2 inactivation in SCSD patents.

Thermophilic PHP Protein Tyrosine Phosphatases (Cap8C and Wzb) from Mesophilic Bacteria.

Protein tyrosine phosphatases (PTPs) of the polymerase and histidinol phosphatase (PHP) superfamily with characteristic phosphatase activity dependent on divalent metal ions are found in many Gram-positive bacteria. Although members of this family are co-purified with metal ions, they still require the exogenous supply of metal ions for full activation. However, the specific roles these metal ions play during catalysis are yet to be well understood. Here, we report the metal ion requirement for phosphatase activities of S. aureus Cap8C and L. rhamnosus Wzb. AlphaFold-predicted structures of the two PTPs suggest that they are members of the PHP family. Like other PHP phosphatases, the two enzymes have a catalytic preference for Mn2+, Co2+ and Ni2+ ions. Cap8C and Wzb show an unusual thermophilic property with optimum activities over 75 °C. Consistent with this model, the activity-temperature profiles of the two enzymes are dependent on the divalent metal ion activating the enzyme.

0D/2D Bi2WO6:Yb3+,Tm3+/Bi12O17Cl2 S-scheme heterojunction photocatalyst with enhanced full spectrum light activity by upconversion luminescence

To effectively utilize near-infrared light in the solar spectrum, expanding the spectral response range of photocatalysts is one of the main issues at present. Herein, a novel 0D/2D Bi2WO6:Yb3+, Tm3+/Bi12O17Cl2 S-scheme heterostructure with full spectral response is successfully synthesized through a hydrothermal method. The optimized Bi2WO6:Yb3+,Tm3+/Bi12O17Cl2 (1:1) photocatalyst degraded 68.2% and 59.1% of methylene blue (MB) and Rhodamine B (RhB) at 980 nm NIR light irradiation for 240 min, and the degradation of MB and RhB was 96.1% and 92.3% under visible light irradiation for 40 min, respectively. The enhanced photocatalytic activity could be attributed to these synergistic effects including the unique 0D/2D contact interface, the expanded photoresponse range, enhanced upconversion luminescence, as well as efficient energy transfer process between Yb3+/Tm3+ and Bi2WO6. Based on characterization and photocatalytic experiments, the introduction of Bi2WO6:Yb3+, Tm3+ can construct S-scheme heterojunctions with Bi12O17Cl2 and produce a full-spectrum response to sunlight. The result, the Bi2WO6:Yb3+, Tm3+/Bi12O17Cl2 photocatalysts possessed effective charge distribution and migration and maintained the excellent redox capacity of semiconductor materials. The hydroxyl radicals (·OH), superoxide radicals (·O2), and holes (h+) as active species played crucial roles in the degradation process. This work describes a potential method to construct S-scheme photocatalyst with great upconversion activity that possesses full spectral response for water pollution control.

Comparative analysis of two Caenorhabditis elegans kinesins KLP-6 and UNC-104 reveals a common and distinct activation mechanism in kinesin-3

Kinesin-3 is a family of microtubule-dependent motor proteins that transport various cargos within the cell. However, the mechanism underlying kinesin-3 activations remains largely elusive. In this study, we compared the biochemical properties of two Caenorhabditis elegans kinesin-3 family proteins, KLP-6 and UNC-104. Both KLP-6 and UNC-104 are predominantly monomeric in solution. As previously shown for UNC-104, non-processive KLP-6 monomer is converted to a processive motor when artificially dimerized. We present evidence that releasing the autoinhibition is sufficient to trigger dimerization of monomeric UNC-104 at nanomolar concentrations, which results in processive movement of UNC-104 on microtubules, although it has long been thought that enrichment in the phospholipid microdomain on cargo vesicles is required for the dimerization and processive movement of UNC-104. In contrast, KLP-6 remains to be a non-processive monomer even when its autoinhibition is unlocked, suggesting a requirement of other factors for full activation. By examining the differences between KLP-6 and UNC-104, we identified a coiled-coil domain called coiled-coil 2 (CC2) that is required for the efficient dimerization and processive movement of UNC-104. Our results suggest a common activation mechanism for kinesin-3 family members, while also highlighting their diversification.

Comparative analysis of two Caenorhabditis elegans kinesins KLP-6 and UNC-104 reveals a common and distinct activation mechanism in kinesin-3.

Kinesin-3 is a family of microtubule-dependent motor proteins that transport various cargos within the cell. However, the mechanism underlying kinesin-3 activations remains largely elusive. In this study, we compared the biochemical properties of two Caenorhabditis elegans kinesin-3 family proteins, KLP-6 and UNC-104. Both KLP-6 and UNC-104 are predominantly monomeric in solution. As previously shown for UNC-104, non-processive KLP-6 monomer is converted to a processive motor when artificially dimerized. We present evidence that releasing the autoinhibition is sufficient to trigger dimerization of monomeric UNC-104 at nanomolar concentrations, which results in processive movement of UNC-104 on microtubules, although it has long been thought that enrichment in the phospholipid microdomain on cargo vesicles is required for the dimerization and processive movement of UNC-104. In contrast, KLP-6 remains to be a non-processive monomer even when its autoinhibition is unlocked, suggesting a requirement of other factors for full activation. By examining the differences between KLP-6 and UNC-104, we identified a coiled-coil domain called coiled-coil 2 (CC2) that is required for the efficient dimerization and processive movement of UNC-104. Our results suggest a common activation mechanism for kinesin-3 family members, while also highlighting their diversification.

Thermal Stabilization of a Bacterial Zn(II)-Dependent Phospholipase C through Consensus Sequence Design.

Proteins' extraordinary performance in recognition and catalysis has led to their use in a range of applications. However, proteins obtained from natural sources are oftentimes not suitable for direct use in industrial or diagnostic setups. Natural proteins, evolved to optimally perform a task in physiological conditions, usually lack the stability required to be used in harsher conditions. Therefore, the alteration of the stability of proteins is commonly pursued in protein engineering studies. Here, we achieved a substantial thermal stabilization of a bacterial Zn(II)-dependent phospholipase C by consensus sequence design. We retrieved and analyzed sequenced homologues from different sources, selecting a subset of examples for expression and characterization. A non-natural consensus sequence showed the highest stability and activity among those tested. Comparison of the stability parameters of this stabilized mutant and other natural variants bearing similar mutations allows us to pinpoint the sites most likely to be responsible for the enhancement. Point mutations in these sites alter the unfolding process of the consensus sequence. We show that the stabilized version of the protein retains full activity even in harsh oil degumming conditions, making it suitable for industrial applications.