Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein EHD1. We have demonstrated this interaction by GST-pulldown, yeast two-hybrid assay, isothermal calorimetry, and co-immunoprecipitation and found that the binding occurs between the EH-domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 co-localizes and interacts with components of the retromer complex such as Vps26, suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor (M6PR) transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as over-expression of a wild-type Rank-5-siRNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of VSV-G. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion.

Pseudoknots have diverse and important roles in many biological functions. We used a combination of UV spectroscopy and differential scanning calorimetry to investigate the effect of the loop length on the unfolding thermodynamics of three sets of DNA stem-loop motifs with the following sequences: (a) d(GCGCTnGCGC), where n = 3, 5, 7, 9; (b) d(CGCGCGT4GAAATTCGCGCGTnAATTTC), where n = 4, 6, and 8; and (c) d(TCTCTTnAAAAAAAAGAGAT5TTTTTTT), where n = 5, 7, 9, and 11. The increase in loop length of the first set of hairpins yielded decreasing TM's and constant unfolding enthalpies, resulting in an entropy driven decrease in the stability of the hairpin (ΔG° = -7.5 to -6.1 kcal/mol). In the second set, the increase in the length of the loops yielded similar TM's and slight increases in the unfolding enthalpies. This translated into more stable pseudoknots with an increasing ΔG° from -13.2 to -17.1 kcal/mol. This effect can be rationalized in terms of the increased flexibility of the pseudoknot with larger loops optimizing base-pair stacking interactions. In the last set of molecules, the increase in the length of one of the loops yielded an increase in the TM's and larger increases in the enthalpies, which stabilize the pseudoknot significantly increasing the ΔG° from -8.5 to -16.6 kcal/mol. In this set, the thymine loop is complementary to the stem of A·T base pairs and the longer loops are able to form T*A·T base triplets due to the partial folding of the thymine loop into the ceiling of the major groove of the duplex, thus yielding a net formation of 1-3 T*AT/T*AT base-triplet stacks at the middle of its stem, depending on the loop length.

Aim: Direct analytical comparison of two major drug-linkers in the antibody-drug conjugate (ADC) field was conducted. Methods: Four different analytical methods [AlogP calculation, reverse phase (RP) high-performance liquid chromatography (HPLC; RP-HPLC), size exclusion chromatography HPLC (SEC-HPLC), and differential scanning calorimetry (DSC)] were tested for this comparison. Results: Maytansinoid-based ADCs showed less hydrophobicity than auristatin-based ADCs. Regardless of the drug-linker and drug-to-antibody ratios (DARs), the stability detected by DSC was decreased by conjugation. Conclusions: The cost and time-efficient analytical comparison described in this manuscript may be useful information for an initial characterization of ADCs prior to detailed biological studies.

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