Biology subjects

Petrovic, I.

Publications and source records attributed to Petrovic, I..

5 recordsLinked to original sources

bioRxiv2024

A high-resolution analysis of arrestin2 interactions responsible for CCR5 endocytosis

Clathrin-mediated endocytosis (CME) is crucial for regulating G protein-coupled receptors (GPCRs) via phosphorylation-dependent arrestin interactions. Despite detailed structural knowledge on the arrestin interactions with phosphorylated tails of GPCRs, the interplay between receptor phosphorylation and arrestin coupling to the CME machinery is not well understood, in particular due to the weakness and dynamics of the individual molecular interactions. Here we have characterized the interactions of arrestin2, which is activated by the phosphorylated C-terminus of the human chemokine receptor 5 (CCR5), with the main protein constituents of CME, namely clathrin and AP2 by solution NMR spectroscopy, biochemical and cellular assays. The NMR analysis revealed that arrestin2 interacts weakly with clathrin through a single binding site, independent of arrestin2 activation. In contrast, the arrestin2-AP2 interaction is stronger, requires arrestin2 activation by the CCR5 phospho-tail, and depends quantitatively on its degree of phosphorylation. These in vitro results are corroborated by cellular assays, which show that the chemokine-induced formation of a long-lived CCR5-arrestin2 internalization complex depends strongly on the interaction of arrestin2 with AP2, but not with clathrin. Taken together, these findings provide quantitative, atom-scale insights on the first steps of CCR5 endocytosis.

biochemistry↗

bioRxiv2024

Biased agonism of carvedilol in the beta1-adrenergic receptor is governed by conformational exclusion

Only two non-visual arrestins recognize many hundreds of different, intracellularly phosphorylated G protein-coupled receptors (GPCRs). Due to the highly dynamic nature of GPCR*arrestin complexes, the critical determinants of GPCR-arrestin recognition have remained largely unclear. We show here that arrestin2 recruitment to the {beta}1-adrenergic receptor ({beta}1AR) can be induced by an arrestin-activating phosphopeptide that is not covalently linked to the receptor and that the recruitment is independent of the presence and type of the orthosteric receptor ligand. Apparently, the arrestin-receptor interaction is driven by the conformational switch within arrestin induced by the phosphopeptide, whereas the electrostatic attraction towards the receptor phosphosites may only play an auxiliary role. Extensive NMR observations show that in contrast to previous static GPCR*arrestin complex structures, the {beta}1AR complex with the beta-blocker carvedilol and arrestin2 is in a G protein-inactive conformation. The insensitivity to the specific receptor conformation provides a rationale for arrestins promiscuous recognition of GPCRs and explains the arrestin-biased agonism of carvedilol, which largely blocks G protein binding, while still enabling arrestin engagement. Significance statementG protein-coupled receptors regulate cellular signaling through G proteins and arrestins. While G protein interactions are well understood, the molecular basis of arrestin recognition remains unclear due to the dynamic nature of GPCR*arrestin complexes. We show that arrestin recognition of the {beta}1-adrenergic receptor occurs independently of the receptors conformational state or ligand binding. Using NMR, cryo-EM, and biochemical assays, we find that arrestin engagement is driven by a conformational change within arrestin itself, triggered by a non-receptor-attached phosphopeptide, whereas electrostatic attraction towards receptor phosphosites may only play an auxiliary role. These findings provide new insights into arrestin activation, explain its ability to recognize diverse GPCRs, with significant implications for understanding biased signaling mechanisms and designing of selective therapeutic strategies.

biochemistry↗

bioRxiv2022

A key GPCR phosphorylation motif discovered in arrestin2-CCR5 phosphopeptide complexes

The two non-visual arrestin isoforms, arrestin2 and arrestin3 recognize and bind hundreds of G protein-coupled receptors (GPCRs) with different phosphorylation patterns leading to distinct functional outcomes. The impact of phosphorylation on arrestin interactions has been well studied only for very few GPCRs. Here we have characterized the interactions between the phosphorylated CC chemokine receptor 5 (CCR5) and arrestin2. We detected several new CCR5 phosphorylation sites, which are necessary for stable complex formation with arrestin2. Crystal structures of arrestin2 in apo form and in complexes with CCR5 C-terminal phosphopeptides together with NMR spectroscopy, biochemical and functional assays revealed three phosphoresidues in a pXpp motif that are essential for the arrestin2 interactions and activation. The same phosphoresidue cluster is present in other receptors, which form stable complexes with arrestin2. We propose that the identified pXpp motif is responsible for robust arrestin2 recruitment in many GPCRs. An analysis of available sequences, structural and functional information on other GPCR*arrestin interactions suggests that a particular arrangement of phosphoresidues within the GPCR intracellular loop 3 and C-terminal tail determines arrestin2 and 3 isoform specificity. Taken together, our findings demonstrate how multi-site phosphorylation controls GPCR*arrestin interactions and provide a framework to probe the intricate details of arrestin activation and signaling. One-sentence summaryA structural and functional analysis of arrestin2 in apo form and complexes with several CCR5 phosphopeptides reveals key phosphorylation sites responsible for stable GPCR*arrestin interactions and their contributions to CCR5-arrestin2 function.

biophysics↗

bioRxiv2021

Hsp40s display class-specific binding profiles, serving complementary roles in the prevention of tau amyloid formation

The microtubule-associated protein, tau, is the major subunit of neurofibrillary tangles, forming insoluble, amyloid-type aggregates associated with neurodegenerative conditions, such as Alzheimers disease. Tau aggregation, however, can be prevented in the cell by a class of proteins known as molecular chaperones, which play important roles in maintaining protein homeostasis. While numerous chaperones are known to interact with tau, though, little is known about the detailed mechanisms by which these prevent tau aggregation. Here, we describe the effects of the ATP-independent Hsp40 chaperones, DNAJA2 and DNAJB1, on tau amyloid fiber formation and compare these to the well-studied small heat shock protein HSPB1. We find that each chaperone prevents tau aggregation differently, by interacting with distinct sets of tau species along the aggregation pathway and thereby affecting their incorporation into fibers. Whereas HSPB1 only binds tau monomers, DNAJB1 and DNAJA2 recognize aggregation-prone tau conformers and even mature fibers, thus efficiently preventing formation of tau amyloids. In addition, we find that both Hsp40s bind tau seeds and fibers via their C-terminal domain II (CTDII), with DNAJA2 being further capable of recognizing tau monomers by a second, different site in CTDI. These results provide important insight into the molecular mechanism by which the different members of the Hsp40 chaperone family counteract the formation, propagation, and toxicity of tau aggregates. Furthermore, our findings highlight the fact that chaperones from different families and different classes play distinct, but complementary roles in preventing pathological protein aggregation.

biophysics↗

bioRxiv2020

SARS-CoV-2 infection induces mixed M1/M2 phenotype in circulating monocytes and alterations in both dendritic cell and monocyte subsets

Clinical manifestations of SARS-CoV-2 infection range from mild to critically severe. The aim of the study was to highlight the immunological events associated with the severity of SARS-CoV-2 infection, with an emphasis on cells of innate immunity. Thirty COVID-19 patients with mild/moderate symptoms and 27 patients with severe/critically severe symptoms were recruited from the Clinical Center of Kragujevac during April 2020. Flow cytometric analysis was performed to reveal phenotypic and functional alterations of peripheral blood cells and to correlate them with the severity of the disease. In severe cases, the number of T and B lymphocytes, dendritic cells, NK cells, and HLA-DR-expressing cells was drastically decreased. In the monocyte population proportion between certain subsets was disturbed and cells coexpressing markers of M1 and M2 monocytes were found in intermediate and non-classical subsets. In mild cases decline in lymphocyte number was less pronounced and innate immunity was preserved as indicated by an increased number of myeloid and activated dendritic cells, NK cells that expressed activation marker at the same level as in control and by low expression of M2 marker in monocyte population. In patients with severe disease, both innate and adoptive immunity are devastated, while in patients with mild symptoms decline in lymphocyte number is lesser, and the innate immunity is preserved.

immunology↗