Cerliponase alfa (Brineura™) is a recombinant human tripeptidyl peptidase-1 (TPP1) being developed by BioMarin Pharmaceutical Inc. for use in patients with neuronal ceroid lipofuscinosis type 2 ...(CLN2), a paediatric neurodegenerative disease caused by a deficiency in TPP1. CLN2 is characterised by progressive impairment of motor function, language deficiencies, seizures, ataxia, blindness and early death, and intracerebroventricular infusion of cerliponase alfa has been shown to reduce the progression of functional decline. This article summarizes the milestones in the development of cerliponase alfa leading to its first global approval in the USA for the treatment of motor function loss in paediatric patients ≥3 years of age with CLN2, and subsequent approval in the EU for CLN2 in all ages.
Due to its properties, such as biodegradability, low density, excellent biocompatibility and unique mechanics, spider silk has been used as a natural biomaterial for a myriad of applications. First ...clinical applications of spider silk as suture material go back to the 18th century. Nowadays, since natural production using spiders is limited due to problems with farming spiders, recombinant production of spider silk proteins seems to be the best way to produce material in sufficient quantities. The availability of recombinantly produced spider silk proteins, as well as their good processability has opened the path towards modern biomedical applications. Here, we highlight the research on spider silk-based materials in the field of tissue engineering and summarize various two-dimensional (2D) and three-dimensional (3D) scaffolds made of spider silk. Finally, different applications of spider silk-based materials are reviewed in the field of tissue engineering in vitro and in vivo.
The α7 nicotinic acetylcholine receptor plays critical roles in the central nervous system and in the cholinergic inflammatory pathway. This ligand-gated ion channel assembles as a homopentamer, is ...exceptionally permeable to Ca2+, and desensitizes faster than any other Cys-loop receptor. The α7 receptor has served as a prototype for the Cys-loop superfamily yet has proven refractory to structural analysis. We present cryo-EM structures of the human α7 nicotinic receptor in a lipidic environment in resting, activated, and desensitized states, illuminating the principal steps in the gating cycle. The structures also reveal elements that contribute to its function, including a C-terminal latch that is permissive for channel opening, and an anionic ring in the extracellular vestibule that contributes to its high conductance and calcium permeability. Comparisons among the α7 structures provide a foundation for mapping the gating cycle and reveal divergence in gating mechanisms in the Cys-loop receptor superfamily.
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•Structures of the human α7 nicotinic acetylcholine receptor•Structures of the three major conformations of the gating cycle•Identification of a C-terminal substructure involved in gating•Identification of an extracellular residue key to calcium permeability
Cryo-EM structures of the α7 nicotinic acetylcholine receptor in three major conformational states of the gating cycle—resting, activated, and desensitized—along with functional studies reveal components involved in calcium conductance and allosteric coupling of ligand binding to channel opening.
The methylotrophic yeast
Komagataella
(
Pichia
)
pastoris
has become one of the most utilized cell factories for the production of recombinant proteins over the last three decades. This success story ...is linked to its specific physiological traits, i.e., the ability to grow at high cell density in inexpensive culture medium and to secrete proteins at high yield. Exploiting methanol metabolism is at the core of most
P. pastoris
-based processes but comes with its own challenges. Co-feeding cultures with glycerol/sorbitol and methanol is a promising approach, which can benefit from improved understanding and prediction of metabolic response. The development of profitable processes relies on the construction and selection of efficient producing strains from less efficient ones but also depends on the ability to master the bioreactor process itself. More specifically, how a bioreactor processes could be monitored and controlled to obtain high yield of production. In this review, new perspectives are detailed regarding a multi-faceted approach to recombinant protein production processes by
P. pastoris
; including gaining improved understanding of the metabolic pathways involved, accounting for variations in transcriptional and translational efficiency at the single cell level and efficient monitoring and control of methanol levels at the bioreactor level.
The recent development of chemical and bio-conjugation techniques allows for the engineering of various protein polymers. However, most of the polymerization process is difficult to control. To meet ...this challenge, we develop an enzymatic procedure to build polyprotein using the combination of a strict protein ligase OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases 1) and a protease TEV (tobacco etch virus). We firstly demonstrate the use of OaAEP1-alone to build a sequence-uncontrolled ubiquitin polyprotein and covalently immobilize the coupled protein on the surface. Then, we construct a poly-metalloprotein, rubredoxin, from the purified monomer. Lastly, we show the feasibility of synthesizing protein polymers with rationally-controlled sequences by the synergy of the ligase and protease, which are verified by protein unfolding using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides a strategy for polyprotein engineering and immobilization.
Growth and differentiation factor 15 (GDF15) is an inflammation-associated hormone with poorly defined biology. Here, we investigated the role of GDF15 in bacterial and viral infections. We found ...that inflammation induced GDF15, and that GDF15 was necessary for surviving both bacterial and viral infections, as well as sepsis. The protective effects of GDF15 were largely independent of pathogen control or the magnitude of inflammatory response, suggesting a role in disease tolerance. Indeed, we found that GDF15 was required for hepatic sympathetic outflow and triglyceride metabolism. Failure to defend the lower limit of plasma triglyceride levels was associated with impaired cardiac function and maintenance of body temperature, effects that could be rescued by exogenous administration of lipids. Together, we show that GDF15 coordinates tolerance to inflammatory damage through regulation of triglyceride metabolism.
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•GDF15 is induced upon bacterial and viral inflammation•GDF15 promotes metabolic adaptation to systemic inflammation•GDF15 stimulates hepatic triglyceride export via beta-adrenergic signaling•Maintenance of triglyceride levels during acute inflammation is cardioprotective
The hormone GDF15 promotes tolerance and survival during conditions of infection and sepsis by modulating systemic levels of plasma triglycerides.
Most neutralizing antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) target the receptor-binding domain (RBD) of the spike glycoprotein and block its binding to the cellular ...receptor dipeptidyl peptidase 4 (DPP4). The epitopes and mechanisms of mAbs targeting non-RBD regions have not been well characterized yet. Here we report the monoclonal antibody 7D10 that binds to the N-terminal domain (NTD) of the spike glycoprotein and inhibits the cell entry of MERS-CoV with high potency. Structure determination and mutagenesis experiments reveal the epitope and critical residues on the NTD for 7D10 binding and neutralization. Further experiments indicate that the neutralization by 7D10 is not solely dependent on the inhibition of DPP4 binding, but also acts after viral cell attachment, inhibiting the pre-fusion to post-fusion conformational change of the spike. These properties give 7D10 a wide neutralization breadth and help explain its synergistic effects with several RBD-targeting antibodies.
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•Carboxylate reductases (CARs) catalyze the selective one step reduction of an acid to an aldehyde.•The substrate scope of CARs is broad.•CARs have been implemented into in vitro and ...in vivo reaction cascades.•Mutational studies and sequence analyses provided deeper insight into the function and phylogeny of CARs.•The number of new CARs and their application is growing rapidly.
Carboxylate reductases (CARs) are emerging as valuable catalysts for the selective one-step reduction of carboxylic acids to their corresponding aldehydes. The substrate scope of CARs is exceptionally broad and offers potential for their application in diverse synthetic processes. Two major fields of application are the preparation of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates. The latest applications of CARs are dominated by in vivo cascades and chemo-enzymatic reaction sequences. The challenge to fully exploit product selectivity is discussed. Recent developments in the characterization of CARs are summarized, with a focus on aspects related to the domain architecture and protein sequences of CAR enzymes.
SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related ...bat virus, RaTG13. We determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.
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