Members of the family of small GTPases regulate a variety of important cellular functions. In order to accomplish this, tight temporal and spatial regulation is absolutely necessary. The two most important factors for this regulation are GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), the latter being responsible for the activation of the GTPase downstream pathways at the correct location and time. Although a large number of exchange factors have been identified, it is likely that a similarly large number remains unidentified. We have therefore developed a procedure to specifically enrich GEF proteins from biological samples making use of the high affinity binding of GEFs to nucleotide-free GTPases. In order to verify the results of these pull-down experiments, we have additionally developed two simple validation procedures: An in vitro transcription/translation system coupled with a GEF activity assay and a yeast two-hybrid screen for detection of GEFs. Although the procedures were established and tested using the Rab protein Sec4, the similar basic principle of action of all nucleotide exchange factors will allow the method to be used for identification of unknown GEFs of small GTPases in general.
Adaptor protein complex-3 (AP-3) mediates cargo sorting from endosomes to lysosomes and lysosome-related organelles. Recently, it was shown that AP-3 adopts a constitutively open conformation compared to the related AP-1 and AP-2 coat complexes, which are inactive until undergoing large conformational changes upon membrane recruitment. How AP-3 is regulated is therefore an open question.
Fluorescence correlation spectroscopy (FCS) enables the measurement of fluctuations at fast timescales (typically few nanoseconds) and with high spatial resolution (tens of nanometers). This single-molecule measurement has been used to characterize single-molecule transport and flexibility of polymers and biomolecules such as DNA and RNA. Here, we apply this technique as dual-color fluorescence cross-correlation spectroscopy (dcFCCS) to identify the motor function of the tethering protein EEA1 and the small GTPase Rab5 by probing the flexibility changes through end-monomer fluctuations.
KRAS mutations are frequent in various human cancers. The development of selective inhibitors targeting KRAS mutations has opened a new era for targeted therapy. However, intrinsic and acquired resistance to these inhibitors remains a major challenge.
Renal Division, Department of Medicine, Peking University First Hospital, Peking University Institute of Nephrology, Key Laboratory of Renal Disease, Ministry of Health of China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment (Peking University), Ministry of Education, State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing, China.
Objectives: Podocyte bridging may be a key initial event occurring early in crescent formation. This study aims to probe the underlying mechanism of atypical protein kinase C (aPKC)/protease-activated receptor 3(Par3)/Par6 polarity complexes on podocyte motility and crescent formation during the progression of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).
Methods: The effects of anti-TNF-α monoclonal antibody (mAb) on the crescent formation, localization and expression of aPKC/Par3/Par6 polarity complexes, and activities of small GTPases (Rho/Rac1/Cdc42) were explored in an AAV mouse model.
Objective: This study investigated absorption, metabolism, and excretion of adagrasib after a single oral 600 mg dose (1 µCi [C]-adagrasib) in 7 healthy subjects and compared the metabolite profile to the profile at steady-state in 4 patients dosed at 600 mg twice daily.
Methods: Plasma, urine, and feces were collected post [C]-adagrasib administration and total radioactivity and pooled sample metabolite profiles were determined. Adagrasib pharmacokinetics were determined in plasma and urine.
