A Mutant (Arg327→His) GPIIb Associated to Thrombasthenia Exerts a Dominant Negative Effect in Stably Transfected CHO Cells

This work reports the structural and functional characterization of the platelet glycoprotein complex GPIIb-IIIa (integrin αIIbβ3) in a patient of type II Glanzmann thrombasthenia, bearing a homozygous G→A base transition at position 1074 of GPIIb that results in an Arg327→His substitution. CHO cells stably transfected with cDNA encoding His327GPIIb showed a drastic reduction in the surface expression of αIIbβ3 complex relative to control cells transfected with wild type GPIIb. Immunopre-cipitation analysis demonstrated that GPIIb synthesis, heterodimeriza-tion, and short term maturation were not impeded, suggesting that conformational changes dependent on Arg327 of GPIIb may play an essential role in either the rate of maturation and/or transport of heterodimers to the cell surface. Cotransfection of CHO cells with equimolar amounts of cDNAs encoding wild type and mutant His327-GPIIb led to a marked reduction in the surface expression of αIIbβ3. This novel observation of a dominant-negative effect of the mutant His327αIIb subunit provides a molecular basis for the reduced platelet αIIbβ3 content observed in the heterozygous offspring.Peer reviewe

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field