ABSTRACT
Technologies to improve the throughput for screening protein formulations are continuously evolving. The purpose of this article is to highlight novel applications of a molecular rotor dye, 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (DASPMI) in screening for the conformational stability, colloidal stability, and subtle pretransition dynamics of protein structures during early formulation development. The measurement of the apparent unfolding temperature (Tm) for a monoclonal antibody in the presence of Tween 80 was conducted and data were compared to the results of differential scanning calorimetry (DSC) measurements. Additionally, measuring the fluorescence intensity of DASPMI as a function of protein concentration shows consistent correlation to the diffusion interaction parameter (kD) for two distinct monoclonal antibody formulations measured by DLS. Lastly, due to the sensitivity of the molecular rotor dye to changes in microviscosity (ηmicro), subtle pretransition dynamics were discernable for two monoclonal antibody formulations that correlate with findings by red-edge excitation shift (REES) experiments. This novel application of molecular rotor dyes offers a valuable and promising approach for streamlining the early formulation development process due to low material consumption and rapid analysis time in a 96-well plate format.
Subject(s)
High-Throughput Screening Assays/methods , Protein Conformation/drug effects , Protein Stability/drug effects , Pyridinium Compounds/chemistry , Calorimetry, Differential Scanning/methods , Colloids/chemistry , Fluorescent Dyes/chemistry , Hydrogen-Ion Concentration , Spectrometry, Fluorescence/methods , TemperatureABSTRACT
Endoplasmic reticulum (ER)-Golgi membrane transport and autophagy are intersecting trafficking pathways that are tightly regulated and crucial for homeostasis, development and disease. Here, we identify UVRAG, a beclin-1-binding autophagic factor, as a phosphatidylinositol-3-phosphate (PtdIns(3)P)-binding protein that depends on PtdIns(3)P for its ER localization. We further show that UVRAG interacts with RINT-1, and acts as an integral component of the RINT-1-containing ER tethering complex, which couples phosphoinositide metabolism to COPI-vesicle tethering. Displacement or knockdown of UVRAG profoundly disrupted COPI cargo transfer to the ER and Golgi integrity. Intriguingly, autophagy caused the dissociation of UVRAG from the ER tether, which in turn worked in concert with the Bif-1-beclin-1-PI(3)KC3 complex to mobilize Atg9 translocation for autophagosome formation. These findings identify a regulatory mechanism that coordinates Golgi-ER retrograde and autophagy-related vesicular trafficking events through physical and functional interactions between UVRAG, phosphoinositide and their regulatory factors, thereby ensuring spatiotemporal fidelity of membrane trafficking and maintenance of organelle homeostasis.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Membrane Proteins/metabolism , Phosphatidylinositols/metabolism , Tumor Suppressor Proteins/metabolism , Amino Acid Sequence , Animals , Autophagy , Autophagy-Related Proteins , Beclin-1 , Biological Transport , COS Cells , Cell Line , Cells, Cultured , HEK293 Cells , HeLa Cells , Humans , Mice , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , Vesicular Transport ProteinsABSTRACT
UVRAG is a promoter of the autophagy pathway, and its deficiency may fuel the development of cancers. Intriguingly, our recent study has demonstrated that this protein also mediates the repair of damaged DNA and patrols centrosome stability, mechanisms that commonly prevent cancer progression, in a manner independent of its role in autophagy signaling. Given the central role of UVRAG in genomic stability and autophagic cleaning, it is speculated that UVRAG is a bona fide genome protector and that the decrease in UVRAG seen in some cancers may render these cells vulnerable to chromosomal damage, making UVRAG an appealing target for cancer therapy.
Subject(s)
Autophagy/genetics , Genomic Instability/genetics , Tumor Suppressor Proteins/metabolism , Animals , Centrosome/metabolism , DNA Damage , DNA Repair , MiceABSTRACT
Autophagy defects have recently been associated with chromosomal instability, a hallmark of human cancer. However, the functional specificity and mechanism of action of autophagy-related factors in genome stability remain elusive. Here we report that UVRAG, an autophagic tumor suppressor, plays a dual role in chromosomal stability, surprisingly independent of autophagy. We establish that UVRAG promotes DNA double-strand-break repair by directly binding and activating DNA-PK in nonhomologous end joining. Disruption of UVRAG increases genetic instability and sensitivity of cells to irradiation. Furthermore, UVRAG was also found to be localized at centrosomes and physically associated with CEP63, an integral component of centrosomes. Disruption of the association of UVRAG with centrosomes causes centrosome instability and aneuploidy. UVRAG thus represents an autophagy-related molecular factor that also has a convergent role in patrolling both the structural integrity and proper segregation of chromosomes, which may confer autophagy-independent tumor suppressor activity.
