Autophagy is activated in response to a variety of cellular stresses including metabolic stress. While elegant genetic studies in yeast have identified the core autophagy machinery, the signaling pathways that regulate this process are less understood. AMPK is an energy sensing kinase and several studies have suggested that AMPK is required for autophagy. The biochemical connections between AMPK and autophagy, however, have not been elucidated. In this report, we identify a biochemical connection between a critical regulator of autophagy, ULK1, and the energy sensing kinase, AMPK. ULK1 forms a complex with AMPK, and AMPK activation results in ULK1 phosphorylation. Moreover, we demonstrate that the immediate effect of AMPK-dependent phosphorylation of ULK1 results in enhanced binding of the adaptor protein YWHAZ/14-3-3ζ; and this binding alters ULK1 phosphorylation in vitro. Finally, we provide evidence that both AMPK and ULK1 regulate localization of a critical component of the phagophore, ATG9, and that some of the AMPK phosphorylation sites on ULK1 are important for regulating ATG9 localization. Taken together these data identify an ULK1-AMPK signaling cassette involved in regulation of the autophagy machinery.

Chemical conjugation of small recombinant proteins with polyethylene glycol (PEG) is an established strategy to extend their typically short circulation times to a therapeutically useful range. We have investigated the production of a genetic fusion with a glycine-rich homo-amino-acid polymer (HAP) as an alternative way to attach a solvated random chain with large hydrodynamic volume. The anti-HER2 Fab fragment 4D5 was used as a model system and fused with either 100 or 200 residue polymers of the repetitive sequence (Gly₄Ser)_n to its light chain. Both fusion proteins were successfully produced in the periplasm of Escherichia coli and obtained as homogeneous preparations after two-step affinity chromatography via the His₆ tag fused to the heavy chain and the Strep-tag II fused to the extended light chain. Both modified Fab fragments showed binding activity towards the HER2 antigen indistinguishable from the conventional recombinant Fab fragment. When compared with the unfused Fab fragment, a significantly increased hydrodynamic volume, by ca. 120%, was observed during gel filtration for the 200 residue HAP fusion protein and, to a lesser extent, in the case of the 100 residue HAP. Difference CD measurements revealed a characteristic random coil spectrum for the 100 and 200 residue HAP fusion moieties. Finally, pharmacokinetic experiments were carried out in mice after radioiodination of the recombinant Fab fragments. Although the 100 residue HAP fusion showed a behavior very similar to the unfused Fab fragment, with a terminal plasma half-life of ca. 2 h, the 200 residue HAPylated Fab fragment gave rise to a significantly prolonged half-life of ca. 6 h. While this moderate effect may so far be most beneficial for specialized medical applications, such as in vivo imaging, the genetic engineering of optimized HAP sequences should yield pharmacokinetic properties similar to PEGylation, yet without necessitating in vitro modification steps.

Key discoveries in aging research have been made possible with the use of model organisms. Caenorhabditis elegans is a short-lived nematode that has become a well-established system to study aging. The practicality and powerful genetic manipulations associated with this metazoan have revolutionized our ability to understand how organisms age. 25 years after the publication of the discovery of the daf-2 gene as a genetic modifier of lifespan, C. elegans remains as relevant as ever in the quest to understand the process of aging. Nematode aging research has proven useful in identifying transcriptional regulators, small molecule signals, cellular mechanisms, epigenetic modifications associated with stress resistance and longevity, and lifespan-extending compounds. Here, we review recent discoveries and selected topics that have emerged in aging research using this incredible little worm.

Glyoxalase I (GloI) catalyzes the glutathione-dependent conversion of 2-oxoaldehydes to S-2-hydroxyacylglutathione derivatives. Studies on GloI from diverse organisms such as man, bacteria, yeast, and different parasites show striking differences among these potentially isofunctional enzymes as far as metal content and the number of active sites per subunit are concerned. So far, it is not known whether this structural variability is linked to catalytic or regulatory features in vivo. Here we show that recombinant GloI from the malaria parasite Plasmodium falciparum has a high- and a low-affinity binding site for the diastereomeric hemithioacetals formed by addition of glutathione to methylglyoxal. Both active sites of the monomeric enzyme are functional and have similar k(cat)(app) values. Proteolytic susceptibility studies and detailed analyses of the steady-state kinetics of active-site mutants suggest that both reaction centers can adopt two discrete conformations and are allosterically coupled. As a result of the positive homotropic allosteric coupling, P. falciparum GloI has an increased affinity at low substrate concentrations and an increased activity at higher substrate concentrations. This could also be the case for GloI from yeast and other organisms. Potential physiologically relevant differences between monomeric GloI and homodimeric GloI are discussed. Our results provide a strong basis for drug development strategies and significantly enhance our understanding of GloI kinetics and structure-function relationships. Furthermore, they extend the current knowledge on allosteric regulation of monomeric proteins in general.

Background: Increasing knowledge of cancer biology and an expanding spectrum of molecularly targeted therapies provide the basis for precision oncology. Despite extensive gene diagnostics, previous reports indicate that less than 10% of patients benefit from this concept. Methods: We retrospectively analyzed all patients referred to our center’s Molecular Tumor Board (MTB) from 2018 to 2021. Molecular testing by next-generation sequencing (NGS) included a 67-gene panel for the detection of short-sequence variants and copy-number alterations, a 53- or 137-gene fusion panel and an ultra-low-coverage whole-genome sequencing for the detection of additional copy-number alterations outside the panel’s target regions. Immunohistochemistry for microsatellite instability and PD-L1 expression complemented NGS. Results: A total of 109 patients were referred to the MTB. In all, 78 patients received therapeutic proposals (70 based on NGS) and 33 were treated accordingly. Evaluable patients treated with MTB-recommended therapy (n = 30) had significantly longer progression-free survival than patients treated with other therapies (n = 17) (4.3 vs. 1.9 months, p = 0.0094). Seven patients treated with off-label regimens experienced major clinical benefits. Conclusion: The combined focused sequencing assays detected targetable alterations in the majority of patients. Patient benefits appeared to lie in the same range as with large-scale sequencing approaches.