The ubiquitous microtubule-associated protein 4 (MAP4) controls organelle distribution by regulating the activity of the kinesin motor.

Regulation of organelle transport by molecular motors along the cytoskeletal microtubules is central to maintaining cellular functions. Here, we show that the ubiquitous tau-related microtubule-associated protein 4 (MAP4) can bias the bidirectional transport of organelles toward the microtubule minus-ends. This is concurrent with MAP4 phosphorylation, mediated by the kinase GSK3ß. We demonstrate that MAP4 achieves this bias by tethering the cargo to the microtubules, allowing it to impair the force generation of the plus-end motor kinesin-1. Consistent with this mechanism, MAP4 physically interacts with dynein and dynactin and, when phosphorylated, associates with the cargo-motor complex through its projection domain. Its phosphorylation coincides with the perinuclear accumulation of organelles, a phenotype that is rescued by abolishing the cargo-microtubule MAP4 tether or by the pharmacological inhibition of dynein, confirming the ability of kinesin to inch along, albeit inefficiently, in the presence of phosphorylated MAP4. These findings have broad biological significance because of the ubiquity of MAP4 and the involvement of GSK3ß in multiple diseases, more specifically in cancer, where the MAP4-dependent redistribution of organelles may be prevalent in cancer cells, as we demonstrate here for mitochondria in lung carcinoma epithelial cells.

Dimethyl sulfoxide reduces the stability but enhances catalytic activity of the main SARS-CoV-2 protease 3CLpro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), one of the most challenging global pandemics of the modern era. Potential treatment strategies against COVID-19 are yet to be devised. It is crucial that antivirals that interfere with the SARS-CoV-2 life cycle be identified and developed. 3-Chymotrypsin-like protease (3CLpro) is an attractive antiviral drug target against SARS-CoV-2, and coronaviruses in general, because of its role in the processing of viral polyproteins. Inhibitors of 3CLpro activity are screened in enzyme assays before further development of the most promising leads. Dimethyl sulfoxide (DMSO) is a common additive used in such assays and enhances the solubility of assay components. However, it may also potentially affect the stability and efficiency of 3CLpro but, to date, this effect had not been analyzed in detail. Here, we investigated the effect of DMSO on 3CLpro-catalyzed reaction. While DMSO (5%-20%) decreased the optimum temperature of catalysis and thermodynamic stability of 3CLpro, it only marginally affected the kinetic stability of the enzyme. Increasing the DMSO concentration up to 20% improved the catalytic efficiency and peptide-binding affinity of 3CLpro. At such high DMSO concentration, the solubility and stability of peptide substrate were improved because of reduced aggregation. In conclusion, we recommend 20% DMSO as the minimum concentration to be used in screens of 3CLpro inhibitors as lead compounds for the development of antiviral drugs against COVID-19.

Mesoporous γ-Iron Oxide Nanoparticles for Magnetically Triggered Release of Doxorubicin and Hyperthermia Treatment.

Mesoporous iron-oxide nanoparticles (mNPs) were prepared by using a modified nanocasting approach with mesoporous carbon as a hard template. mNPs were first loaded with doxorubicin (Dox), an anticancer drug, and then coated with the thermosensitive polymer Pluronic F108 to prevent the leakage of Dox molecules from the pores that would otherwise occur under physiological conditions. The Dox-loaded, Pluronic F108-coated system (Dox@F108-mNPs) was stable at room temperature and physiological pH and released its Dox cargo slowly under acidic conditions or in a sudden burst with magnetic heating. No significant toxicity was observed in vitro when Dox@F108-mNPs were incubated with noncancerous cells, a result consistent with the minimal internalization of the particles that occurs with normal cells. On the other hand, the drug-loaded particles significantly reduced the viability of cervical cancer cells (HeLa, IC50 =0.70 µm), wild-type ovarian cancer cells (A2780, IC50 =0.50 µm) and Dox-resistant ovarian cancer cells (A2780/AD, IC50 =0.53 µm). In addition, the treatment of HeLa cells with both Dox@F108-mNPs and subsequent alternating magnetic-field-induced hyperthermia was significantly more effective at reducing cell viability than either Dox or Dox@F108-mNP treatment alone. Thus, Dox@F108-mNPs constitute a novel soft/hard hybrid nanocarrier system that is highly stable under physiological conditions, temperature-responsive, and has chemo- and thermotherapeutic modes of action.