Background: Only 5% of circulating cortisol is active and unbound to carrier proteins. Because cortisol levels vary rapidly due to the pulsatile nature of cortisol secretion, the dynamics of cortisol ...binding are critical determinants of tissue levels of free cortisol and consequent hormonal signaling. The major glucocorticoid carrier protein is corticosteroid binding globulin (CBG), a member of the serpin family that undergoes conformational changes to bind and release hormones. This mechanism has been noted to be temperature responsive, and we have now investigated the effects of temperature on the binding of human CBG to both cortisol and progesterone.
Methods: Recombinant human CBG was synthesized and used for binding studies with cortisol and progesterone between 34 and 43 C. Binding was monitored by recording the change in intrinsic protein fluorescence. Binding of the steroids to the other major carrier, serum albumin, was measured in a similar manner.
Results: There was no effect of temperature on the interaction between human serum albumin and either cortisol or progesterone. The association of both cortisol and progesterone with CBG is more than three orders of magnitude greater than that with HSA, and this interaction was extremely responsive to changes in temperature. The affinity of both cortisol and progesterone for CBG drops approximately 16-fold as temperature increases from 35 to 42 C.
Conclusions: This study clearly shows that even within the clinically relevant range of temperatures found in humans, CBG acts as a protein thermocouple that is exquisitely sensitive to temperature change and will release cortisol in response to fever or external sources of heat. This has major implications for our understanding of cortisol regulation in febrile patients.
Corticosteroid-binding globulin acts as a protein thermocouple that is exquisitely sensitive to temperature change and releases cortisol in response to fever or external sources of heat.
The enzyme protochlorophyllide oxidoreductase (POR) catalyses a light-dependent step in chlorophyll biosynthesis that is essential to photosynthesis and, ultimately, all life on Earth
. POR, which is ...one of three known light-dependent enzymes
, catalyses reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, the structural basis for POR photocatalysis has remained unknown. Here we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with the nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide-NADPH-POR complex identify multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in driving POR photochemistry in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways.
Magnetic microfluidics has been gradually recognized as an area of its own. Both conventional microfluidic platforms have incorporated magnetic actuation for microfluidic operation and microscale ...object manipulation. Nonetheless, there is still much room for improvement after decades of development. In this Perspective, we first provide a quick review of existing magnetic microfluidic platforms with a focus on the magnetic tools and actuation mechanisms. Next, we discuss several emerging technologies, including magnetic microrobots, additive manufacture, and artificial intelligence, and their potential application in the future development of magnetic microfluidics. We believe that these technologies can eventually inspire highly functional magnetic tools for microfluidic manipulation and coordinated microfluidic control at the system level, which eventually drives magnetic microfluidics into an intelligent system for automated experimentation.
The nucleolus has been recently described as a stress sensor. The nucleoplasmic translocation of nucleolar protein nucleophosmin (NPM1) is a hallmark of nucleolar stress; however, the causes of this ...translocation and its connection to p53 activation are unclear. Using single live-cell imaging and the redox biosensors, we demonstrate that nucleolar oxidation is a general response to various cellular stresses. During nucleolar oxidation, NPM1 undergoes S-glutathionylation on cysteine 275, which triggers the dissociation of NPM1 from nucleolar nucleic acids. The C275S mutant NPM1, unable to be glutathionylated, remains in the nucleolus under nucleolar stress. Compared with wild-type NPM1 that can disrupt the p53-HDM2 interaction, the C275S mutant greatly compromises the activation of p53, highlighting that nucleoplasmic translocation of NPM1 is a prerequisite for stress-induced activation of p53. This study elucidates a redox mechanism for the nucleolar stress sensing and may help the development of therapeutic strategies.
Hunter syndrome is a rare but devastating childhood disease caused by mutations in the IDS gene encoding iduronate-2-sulfatase, a crucial enzyme in the lysosomal degradation pathway of dermatan ...sulfate and heparan sulfate. These complex glycosaminoglycans have important roles in cell adhesion, growth, proliferation and repair, and their degradation and recycling in the lysosome is essential for cellular maintenance. A variety of disease-causing mutations have been identified throughout the IDS gene. However, understanding the molecular basis of the disease has been impaired by the lack of structural data. Here, we present the crystal structure of human IDS with a covalently bound sulfate ion in the active site. This structure provides essential insight into multiple mechanisms by which pathogenic mutations interfere with enzyme function, and a compelling explanation for severe Hunter syndrome phenotypes. Understanding the structural consequences of disease-associated mutations will facilitate the identification of patients that may benefit from specific tailored therapies.
Context:
Recent studies of corticosteroid-binding globulin (CBG) indicate that it does not merely transport cortisol passively but also actively regulates its release in the circulation. We show how ...CBG binding affinity can vary to give changes in free cortisol concentration in a physiologically relevant range.
Objective:
The objective was to determine how the binding affinity of plasma CBG is affected by glycosylation, changes in body temperature, and the conformational change induced by proteases at sites of inflammation.
Design:
Binding assays were performed over a range of temperatures with plasma and recombinant CBG to determine the contribution of glycosylation. The role of conformational change was assessed by measuring binding affinities of plasma CBG before and after reactive loop cleavage by neutrophil elastase.
Main Outcome Measures:
Determination of binding constants allows calculation of clinically relevant changes in CBG saturation and free cortisol concentrations.
Results:
On reactive loop cleavage at inflammation sites, CBG can continue to act as a buffered source of cortisol, although with a much reduced affinity, to give a potential quadrupling of free cortisol. Predicted increases in systemic free cortisol resulting from elevated body temperatures, previously reported based on affinity measurements using nonglycosylated recombinant CBG, were shown here to be considerably increased using glycosylated plasma CBG, with a doubling for every 2°C rise in body temperature.
Conclusions:
The ability of CBG to modulate free cortisol levels in blood must be considered in the understanding and management of disease processes, as illustrated here with predictable changes in inflammation and fever.
The N‐terminal somatomedin B domain (SMB) of vitronectin binds PAI‐1 and the urokinase receptor with high affinity and regulates tumor cell adhesion and migration. We have shown previously in the ...crystal structure of the PAI‐1/SMB complex that SMB, a peptide of 51 residues, is folded as a compact cysteine knot of four pairs of crossed disulfide bonds. However, the physiological significance of this structure was questioned by other groups, who disputed the disulfide bonding shown in the crystal structure (Cys5–Cys21, Cys9–Cys39, Cys19–Cys32, Cys25–Cys31), notably claiming that the first disulfide is Cys5–Cys9 rather than the Cys5–Cys21 bonding shown in the structure. To test if the claimed Cys5–Cys9 bond does exist in the SMB domain of plasma vitronectin, we purified mouse and rat plasma vitronectin that have a Met (hence cleavable by cyanogen bromide) at residue 14, and also prepared recombinant human SMB variants from insect cells with residues Asn14 or Leu24 mutated to Met. HPLC and mass spectrometry analysis showed that, after cyanogen bromide digestion, all the fragments of the SMB derived from mouse or rat vitronectin or the recombinant SMB mutants are still linked together by disulfides, and the N‐terminal peptide (residue 1–14 or 1–24) can only be released when the disulfide bonds are broken. This clearly demonstrates that Cys5 and Cys9 of SMB do not form a disulfide bond in vivo, and together with other structural evidence confirms that the only functional structure of the SMB domain of plasma vitronectin is that seen in its crystallographic complex with PAI‐1.
Blood pressure is critically controlled by angiotensins, which are vasopressor peptides specifically released by the enzyme renin from the tail of angiotensinogen-a non-inhibitory member of the ...serpin family of protease inhibitors. Although angiotensinogen has long been regarded as a passive substrate, the crystal structures solved here to 2.1 Å resolution show that the angiotensin cleavage site is inaccessibly buried in its amino-terminal tail. The conformational rearrangement that makes this site accessible for proteolysis is revealed in our 4.4 Å structure of the complex of human angiotensinogen with renin. The co-ordinated changes involved are seen to be critically linked by a conserved but labile disulphide bridge. Here we show that the reduced unbridged form of angiotensinogen is present in the circulation in a near 40:60 ratio with the oxidized sulphydryl-bridged form, which preferentially interacts with receptor-bound renin. We propose that this redox-responsive transition of angiotensinogen to a form that will more effectively release angiotensin at a cellular level contributes to the modulation of blood pressure. Specifically, we demonstrate the oxidative switch of angiotensinogen to its more active sulphydryl-bridged form in the maternal circulation in pre-eclampsia-the hypertensive crisis of pregnancy that threatens the health and survival of both mother and child.
The use of antibodies to target immune checkpoints, particularly PD-1/PD-L1, has made a profound impact in the field of cancer immunotherapy. Here, we identified KN035, an anti-PD-L1 nanobody that ...can strongly induce T-cell responses and inhibit tumor growth. The crystal structures of KN035 complexed with PD-L1 and free PD-L1, solved here at 1.7 and 2.7 Å resolution, respectively, show that KN035 competes with PD-1 (programmed death protein 1) for the same flat surface on PD-L1, mainly through a single surface loop of 21 amino acids. This loop forms two short helices and develops key hydrophobic and ionic interactions with PD-L1 residues, such as Ile54, Tyr56 and Arg113, which are also involved in PD-1 binding. The detailed mutagenesis study identified the hotspot residues of the PD-L1 surface and provides an explanation for the stronger (~1 000-fold) binding of KN035 to PD-L1 than PD-1 and its lack of binding to PD-L2. Overall, this study reveals how a single immunoglobulin-variable scaffold of KN035 or PD-1 can bind to a flat protein surface through either a single surface loop or beta-sheet strands; and provides a basis for designing new immune checkpoint blockers and generating bi-specific antibodies for combination therapy.
4D printing provides a means to create dynamic structures for self-actuating devices fabricated by additive manufacturing techniques. Most existing 4D printing processes are only capable of one-way ...shape morphing using a single mechanism for stimulation. In the limited successful reversible 4D-printing studies, most only demonstrate 2D-to-3D shape morphing. This study introduces a contactless shape setting mechanism to empower reversible 4D printing using a combination of stimuli. The forward shape programming is realised by asymmetric swelling with heated ethanol, and the shape recovery is accomplished by dry heating. The shape morphing can be predicted using a simulation model. Several designs are 4D-printed to achieve different forms of morphing to obtain 3D-to-3D shape morphing. The entire shape morphing cycle could be completed within as short as 3 min. The capability of 3D-to-3D shape morphing and reversibility of our 4D-printing process promise great potential for different applications such as biomimetics and soft robotics.
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