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The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a key enzyme, which extensively digests CoV replicase polyproteins essential for viral replication and transcription, making it an attractive target for antiviral drug development. However, the molecular mechanism of how Mpro of SARS-CoV-2 digests replicase polyproteins, releasing the nonstructural proteins (nsps), and its substrate specificity remain largely unknown. Here, we determine the high-resolution structures of SARS-CoV-2 Mpro in its resting state, precleavage state, and postcleavage state, constituting a full cycle of substrate cleavage. The structures show the delicate conformational changes that occur during polyprotein processing. Further, we solve the structures of the SARS-CoV-2 Mpro mutant (H41A) in complex with six native cleavage substrates from replicase polyproteins, and demonstrate that SARS-CoV-2 Mpro can recognize sequences as long as 10 residues but only have special selectivity for four subsites. These structural data provide a basis to develop potent new inhibitors against SARS-CoV-2.

Background/Objective Insulin resistance is more prominent in men than women. If this involves adipose tissue is unknown and was presently examined. Subjects/Methods AdipoIR (in vivo adipose insulin resistance index) was measured in 2344 women and 787 men. In 259 of the women and 54 of the men, insulin induced inhibition of lipolysis (acylglycerol breakdown) and stimulation of lipogenesis (glucose conversion to acylglycerols) were determined in subcutaneous adipocytes; in addition, basal (spontaneous) lipolysis was also determined in the fat cells. In 234 women and 115 men, RNAseq expression of canonical insulin signal genes were measured in subcutaneous adipose tissue. Messenger RNA transcripts of the most discriminant genes were quantified in 175 women and 109 men. Results Men had higher AdipoIR values than women but only when obesity (body mass index 30 kg/m 2 or more) was present ( p  < 0.0001). The latter sex dimorphism was found among physically active and sedentary people, in those with and without cardiometabolic disease and in people using nicotine or not ( p  = 0.0003 or less). In obesity, adipocyte insulin sensitivity (half maximum effective hormone concentration) and maximal antilipolytic effect were tenfold and 10% lower, respectively, in men than women ( p  = 0.005 or less). Basal rate of lipolysis was two times higher in men than women ( p  > 0.0001). Sensitivity and maximum effect of insulin on lipogenesis were similar in both sexes ( p  = 0.26 and p = 0.18, respectively). When corrected for multiple comparison only RNAseq expression of insulin receptor substrate 1 ( IRS1 ) was lower in men than women ( p  < 0.0001). The mRNA transcript for IRS1 was 60% higher in women than men ( p  < 0.0001). Conclusions In obesity, adipose tissue insulin resistance is more pronounced in men than in women. The mechanism involves less efficient insulin-mediated inhibition of adipocyte lipolysis, increased basal rate of lipolysis and decreased adipose expression of a key element of insulin signaling, IRS1 .

The insulin receptor substrate proteins IRS1 and IRS2 are key targets of the insulin receptor tyrosine kinase and are required for hormonal control of metabolism. Tissues from insulin-resistant and diabetic humans exhibit defects in IRS-dependent signalling, implicating their dysregulation in the initiation and progression of metabolic disease. However, IRS1 and IRS2 are regulated through a complex mechanism involving phosphorylation of >50 serine/threonine residues (S/T) within their long, unstructured tail regions. In cultured cells, insulin-stimulated kinases (including atypical PKC, AKT, SIK2, mTOR, S6K1, ERK1/2 and ROCK1) mediate feedback (autologous) S/T phosphorylation of IRS, with both positive and negative effects on insulin sensitivity. Additionally, insulin-independent (heterologous) kinases can phosphorylate IRS1/2 under basal conditions (AMPK, GSK3) or in response to sympathetic activation and lipid/inflammatory mediators, which are present at elevated levels in metabolic disease (GRK2, novel and conventional PKCs, JNK, IKKβ, mPLK). An emerging view is that the positive/negative regulation of IRS by autologous pathways is subverted/co-opted in disease by increased basal and other temporally inappropriate S/T phosphorylation. Compensatory hyperinsulinaemia may contribute strongly to this dysregulation. Here, we examine the links between altered patterns of IRS S/T phosphorylation and the emergence of insulin resistance and diabetes.

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo–electron microscopy of apo human LAT2/CD98hc and a multi-disciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.

   The NAD + -dependent aldehyde dehydrogenase AldB from Aromatoleum aromaticum was recombinantly produced in Escherichia coli and biochemically characterized. As suggested by its substrate spectrum, the most probably physiological function of AldB is the oxidation of short aliphatic aldehydes such as acetaldehyde, which occur as intermediates in the degradation of the corresponding alcohols. In addition, we generated some mutant variants in residue Tyr460, which is located at the neck region of the substrate channel and analyzed their effects on the catalytic parameters for different substrates. Single amino acid exchanges at this position revealed profound changes in substrate preference and substrate inhibition of the variants. Key points • Small aliphatic aldehydes show the best catalytic efficiency with aldehyde dehydrogenase AldB • Amino acid exchanges at Y460 results in changed catalytic efficiencies and substrate inhibition • AldB is a member of a new clade of the aldehyde dehydrogenase superfamily

Surface‐enhanced Raman scattering (SERS) is a powerful sensing technique. However, the employment of SERS sensors in practical applications is hindered by high fabrication costs from processes with limited scalability, poor batch‐to‐batch reproducibility, substrate stability, and uniformity. Here, highly scalable and reproducible flame aerosol technology is employed to rapidly self‐assemble uniform SERS sensing films. Plasmonic Ag nanoparticles are deposited on substrates as nanoaggregates with fine control of their interparticle distance. The interparticle distance is tuned by adding a dielectric spacer during nanoparticle synthesis that separates the individual Ag nanoparticles within each nanoaggregate. The dielectric spacer thickness dictates the plasmonic coupling extinction of the deposited nanoaggregates and finely tunes the Raman hotspots. By systematically studying the optical and morphological properties of the developed SERS surfaces, structure–performance relationships are established and the optimal hot‐spots occur for interparticle distance of 1 to 1.5 nm among the individual Ag nanoparticles, as also validated by computational modeling, are identified for the highest signal enhancement of a molecular Raman reporter. Finally, the superior stability and batch‐to‐batch reproducibility of the developed SERS sensors are demonstrated and their potential with a proof‐of‐concept practical application in food‐safety diagnostics for pesticide detection on fruit surfaces is explored. Robust surface‐enhanced Raman scattering (SERS) sensing surfaces are fabricated using one‐step flame nanoparticle deposition. The sensing surfaces exhibit superior stability and high batch‐to‐batch reproducibility, highlighting their potential in practical (bio)chemical sensing. The detection of pesticides on fruit surfaces demonstrates a proof‐of‐concept practical application in food safety diagnostics at the point of consumption.

Substrate inhibition of enzymes can be a major obstacle to the production of valuable chemicals in engineered microorganisms. Here, we show substrate inhibition of lycopene cyclase as the main limitation in carotenoid biosynthesis in Yarrowia lipolytica . To overcome this bottleneck, we exploit two independent approaches. Structure-guided protein engineering yields a variant, Y27R, characterized by complete loss of substrate inhibition without reduction of enzymatic activity. Alternatively, establishing a geranylgeranyl pyrophosphate synthase-mediated flux flow restrictor also prevents the onset of substrate inhibition by diverting metabolic flux away from the inhibitory metabolite while maintaining sufficient flux towards product formation. Both approaches result in high levels of near-exclusive β-carotene production. Ultimately, we construct strains capable of producing 39.5 g/L β-carotene at a productivity of 0.165 g/L/h in bioreactor fermentations (a 1441-fold improvement over the initial strain). Our findings provide effective approaches for removing substrate inhibition in engineering pathways for efficient synthesis of natural products. Substrate inhibition has not been widely studied in the context of synthetic biology and metabolic engineering. Here, the authors report removal of lycopene substrate inhibition by two different strategies and enable high carotenoid productivity in Yarrowia lipolytica .

Precise modulation of hepatic glucose metabolism is crucial during the fasting and feeding cycle and is controlled by the actions of circulating insulin and glucagon. The insulin-signaling pathway requires insulin receptor substrate 1 (IRS1) and IRS2, which are found to be dysregulated in diabetes and obesity. The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1A) is a fasting-induced transcriptional coactivator. In nonalcoholic fatty liver disease and in patients with type 2 diabetes, low hepatic PGC1A levels are associated with insulin resistance. However, how PGC1A activity impacts the hepatic insulin-signaling pathway is still unclear. We used gain- and loss-of-function models in mouse primary hepatocytes and measured hepatocyte insulin response by gene and protein expression and ex vivo glucose production. We found that the PGC1A level determines the relative ratio of IRS1 and IRS2 in hepatocytes, impacting insulin receptor signaling via protein kinase B/AKT (AKT). PGC1A drove the expression of IRS2 downstream of glucagon signaling while simultaneously reducing IRS1 expression. We illustrate that glucagon- or PGC1A-induced IRS2 expression was dependent on cAMP Response Element Binding Protein activity and that this was essential for suppression of hepatocyte gluconeogenesis in response to insulin in vitro. We also show that increased hepatic PGC1A improves glucose homeostasis in vivo, revealing a counterregulatory role for PGC1A in repressing uncontrolled glucose production in response to insulin signaling. These data highlight a mechanism by which PGC1A plays dual roles in the control of gluconeogenesis during the fasting-to-fed transition through regulated balance between IRS1 and IRS2 expression.

ABCB1, also known as P-glycoprotein, actively extrudes xenobiotic compounds across the plasma membrane of diverse cells, which contributes to cellular drug resistance and interferes with therapeutic drug delivery. We determined the 3.5-angstrom cryo–electron microscopy structure of substrate-bound human ABCB1 reconstituted in lipidic nanodiscs, revealing a single molecule of the chemotherapeutic compound paclitaxel (Taxol) bound in a central, occluded pocket. A second structure of inhibited, human-mouse chimeric ABCB1 revealed two molecules of zosuquidar occupying the same drug-binding pocket. Minor structural differences between substrate- and inhibitor-bound ABCB1 sites are amplified toward the nucleotide-binding domains (NBDs), revealing how the plasticity of the drug-binding site controls the dynamics of the adenosine triphosphate–hydrolyzing NBDs. Ordered cholesterol and phospholipid molecules suggest how the membrane modulates the conformational changes associated with drug binding and transport.

The 26S proteasome is involved in degrading and regulating the majority of proteins in eukaryotic cells, which requires a sophisticated balance of specificity and promiscuity. In this Review, we discuss the principles that underly substrate recognition and ATP-dependent degradation by the proteasome. We focus on recent insights into the mechanisms of conventional ubiquitin-dependent and ubiquitin-independent protein turnover, and discuss the plethora of modulators for proteasome function, including substrate-delivering cofactors, ubiquitin ligases and deubiquitinases that enable the targeting of a highly diverse substrate pool. Furthermore, we summarize recent progress in our understanding of substrate processing upstream of the 26S proteasome by the p97 protein unfoldase. The advances in our knowledge of proteasome structure, function and regulation also inform new strategies for specific inhibition or harnessing the degradation capabilities of the proteasome for the treatment of human diseases, for instance, by using proteolysis targeting chimera molecules or molecular glues. Most proteins are degraded and regulated by the 26S proteasome. A balance of specificity and promiscuity underlies substrate recognition and ATP-dependent degradation, which can occur in a ubiquitin-dependent or ubiquitin-independent manner. Recent insights into the mechanisms and regulation of substrate delivery, processing and degradation will inform new strategies for the treatment of human diseases.