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As a significant member of the immune checkpoint, programmed cell death 1 ligand 1 (PD-L1) plays a critical role in cancer immune escape and has become an important target for cancer immunotherapy. Clinically approved drugs mainly target the extracellular domain of PD-L1. Recently, the small cytoplasmic domain of PD-L1 has been reported to regulate PD-L1 stability and function through multiple pathways. Therefore, the intracellular domain of PD-L1 and its regulatory pathways could be promising targets for cancer therapy, expanding available strategies for combined immunotherapy. Here, we summarize the emerging roles of the PD-L1 cytoplasmic domain and its regulatory pathways. The conserved motifs, homodimerization, and posttranslational modifications of the PD-L1 cytoplasmic domain have been reported to regulate the membrane anchoring, degradation, nuclear translocation, and glycosylation of PD-L1. This summary provides a comprehensive understanding of the functions of the PD-L1 cytoplasmic domain and evaluates the broad prospects for targeted therapy. [ABSTRACT FROM AUTHOR]

L-type voltage-gated Ca(2+) channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The pore-forming α1-subunit comprises four repeated domains (I-IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca(2+) levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane. Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function.

Abstract Background Autophagy is an essential process in eukaryotic cells in which autophagosomes form to deliver cellular organelles and long-lived proteins to lysosomes for degradation. Many studies have recently identified the regulatory mechanisms involved in the interaction between viral infection and autophagy. Methods LC3 turnover and the proteins in the endoplasmic reticulum (ER) stress pathway were investigated using western blot analysis. The formation and degradation of autophagosomes were detected using immunofluorescence staining. Results Autophagy was activated by porcine reproductive and respiratory syndrome virus (PRRSV) NSP3, NSP5 and NSP9, which are two transmembrane proteins and an RNA-dependent RNA polymerase, respectively. The formation of autophagosomes was induced by NSP3 and NSP5 and developed from the ER; the fusion of these autophagosomes with lysosomes was limited. Although NSP3 and NSP5 are ER transmembrane proteins, these proteins did not activate the ER stress signaling pathways. In addition, the cytoplasmic domain of NSP3 plays a pivotal role in activating autophagy. Conclusions The data presented in this study reveal an important relationship between PRRSV NSPs and autophagy and provide new insights that improve our understanding of the involvement of PRRSV NSPs in the autophagy process.

As a significant member of the immune checkpoint, programmed cell death 1 ligand 1 (PD-L1) plays a critical role in cancer immune escape and has become an important target for cancer immunotherapy. Clinically approved drugs mainly target the extracellular domain of PD-L1. Recently, the small cytoplasmic domain of PD-L1 has been reported to regulate PD-L1 stability and function through multiple pathways. Therefore, the intracellular domain of PD-L1 and its regulatory pathways could be promising targets for cancer therapy, expanding available strategies for combined immunotherapy. Here, we summarize the emerging roles of the PD-L1 cytoplasmic domain and its regulatory pathways. The conserved motifs, homodimerization, and posttranslational modifications of the PD-L1 cytoplasmic domain have been reported to regulate the membrane anchoring, degradation, nuclear translocation, and glycosylation of PD-L1. This summary provides a comprehensive understanding of the functions of the PD-L1 cytoplasmic domain and evaluates the broad prospects for targeted therapy., (© The Author(s) (2023). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

A three-dimensional reconstruction of Sindbis virus at 7.0 Å resolution presented here provides a detailed view of the virion structure and includes structural evidence for key interactions that occur between the capsid protein (CP) and transmembrane (TM) glycoproteins E1 and E2. Based on crystal structures of component proteins and homology modeling, we constructed a nearly complete, pseudo-atomic model of the virus. Notably, this includes identification of the 33-residue cytoplasmic domain of E2 (cdE2), which follows a path from the E2 TM helix to the CP where it enters and exits the CP hydrophobic pocket and then folds back to contact the viral membrane. Modeling analysis identified three major contact regions between cdE2 and CP, and the roles of specific residues were probed by molecular genetics. This identified R393 and E395 of cdE2 and Y162 and K252 of CP as critical for virus assembly. The N-termini of the CPs form a contiguous network that interconnects 12 pentameric and 30 hexameric CP capsomers. A single glycoprotein spike cross-links three neighboring CP capsomers as might occur during initiation of virus budding.

The epidermal growth factor receptor (EGFR) is a membrane protein that regulates cell proliferation, differentiation and survival, and is a drug target for cancer therapy. Ligand-induced activation of the EGFR kinase is generally regarded to require ligand-bound-dimers, while phosphorylation and down-stream signalling is modulated by oligomers. Recent work has unveiled changes in EGFR dynamics from ligand-induced dimerization in membranes extracted from cells, however, less is known about the changes in EGFR dynamics that accompany the ligand-induced oligomerization in a live cell environment. Here, we determine the dynamics of a c-terminal GFP tag attached to EGFR in the unliganded dimer and in the liganded oligomers. By means of the single-frequency polarized phasor ellipse approach we extracted two correlation times on the sub-nanosecond and super-nanosecond timescales, respectively. EGF binding to the EGFR–GFP dimer lengthened the sub-nanosecond correlation time (from 0.1 to 1.3 ns) and shortened the super-nanosecond correlation time (from 210 to 56 ns) of the c-terminal GFP probe. The sub-nanosecond depolarization processes were assigned to electronic energy migration between proximal GFPs in the EGFR dimer or oligomer, while the super-nanosecond correlation times were assigned to nanosecond fluctuations of the GFP probe in the EGFR complex. Accordingly, these results show that ligand binding increased the average separation between the c-terminal tags and increased their rotational mobility. We propose that the dynamics are linked to an inhibitory function of the c-terminal tail in the un-liganded dimer and to the requirement of facile stochastic switching between kinase activation and cytoplasmic adaptor/effector binding in the active oligomers. [ABSTRACT FROM AUTHOR]

Integrins are cell surface adhesion receptors that are essential for the development and function of multicellular animals. Here we summarize recent findings on the regulation of integrin affinity for ligand (activation), one mechanism by which cells modulate integrin function. The focus is on the structural basis of integrin activation, the role of the cytoplasmic domain in integrin affinity regulation, and potential mechanisms by which activation signals are propagated from integrin cytoplasmic domains to the extracellular ligand-binding domain.

Background: Autophagy is an essential process in eukaryotic cells in which autophagosomes form to deliver cellular organelles and long-lived proteins to lysosomes for degradation. Many studies have recently identified the regulatory mechanisms involved in the interaction between viral infection and autophagy. Methods: LC3 turnover and the proteins in the endoplasmic reticulum (ER) stress pathway were investigated using western blot analysis. The formation and degradation of autophagosomes were detected using immunofluorescence staining. Results: Autophagy was activated by porcine reproductive and respiratory syndrome virus (PRRSV) NSP3, NSP5 and NSP9, which are two transmembrane proteins and an RNA-dependent RNA polymerase, respectively. The formation of autophagosomes was induced by NSP3 and NSP5 and developed from the ER; the fusion of these autophagosomes with lysosomes was limited. Although NSP3 and NSP5 are ER transmembrane proteins, these proteins did not activate the ER stress signaling pathways. In addition, the cytoplasmic domain of NSP3 plays a pivotal role in activating autophagy. Conclusions: The data presented in this study reveal an important relationship between PRRSV NSPs and autophagy and provide new insights that improve our understanding of the involvement of PRRSV NSPs in the autophagy process. [ABSTRACT FROM AUTHOR]

The exposure and release of TF is regulated by post-translational modifications of its cytoplasmic domain. Here, the potential of Pin1 to interact with the cytoplasmic domain of TF, and the outcome on TF function was examined. MDA-MB-231 and transfected-primary endothelial cells were incubated with either Pin1 deactivator Juglone, or its control Plumbagin, as well as transfected with Pin1-specific or control siRNA. TF release into microvesicles following activation, and also phosphorylation and ubiquitination states of cellular-TF were then assessed. Furthermore, the ability of Pin1 to bind wild-type and mutant forms of overexpressed TF-tGFP was investigated by co-immunoprecipitation. Additionally, the ability of recombinant or cellular Pin1 to bind to peptides of the C-terminus of TF, synthesised in different phosphorylation states was examined by binding assays and spectroscopically. Finally, the influence of recombinant Pin1 on the ubiquitination and dephosphorylation of the TF-peptides was examined. Pre-incubation of Pin1 with Juglone but not Plumbagin, reduced TF release as microvesicles and was also achievable following transfection with Pin1-siRNA. This was concurrent with early ubiquitination and dephosphorylation of cellular TF at Ser253. Pin1 co-immunoprecipitated with overexpressed wild-type TF-tGFP but not Ser258 → Ala or Pro259 → Ala substituted mutants. Pin1 did interact with Ser258-phosphorylated and double-phosphorylated TF-peptides, with the former having higher affinity. Finally, recombinant Pin1 was capable of interfering with the ubiquitination and dephosphorylation of TF-derived peptides. In conclusion, Pin1 is a fast-acting enzyme which may be utilised by cells to protect the phosphorylation state of TF in activated cells prolonging TF activity and release, and therefore ensuring adequate haemostasis. [ABSTRACT FROM AUTHOR]

Retroviruses are a family of viruses that cause a broad range of pathologies in animals and humans, from the apparently harmless, long-term genomic insertion of endogenous retroviruses, to tumors induced by the oncogenic retroviruses and acquired immunodeficiency syndrome (AIDS) resulting from human immunodeficiency virus infection. Disease can be the result of diverse mechanisms, including tumorigenesis induced by viral oncogenes or immune destruction, leading to the gradual loss of CD4 T-cells. Of the virally encoded proteins common to all retroviruses, the envelope (Env) displays perhaps the most diverse functionality. Env is primarily responsible for binding the cellular receptor and for effecting the fusion process, with these functions mediated by protein domains localized to the exterior of the virus. The remaining C-terminal domain may have the most variable functionality of all retroviral proteins. The C-terminal domains from three prototypical retroviruses are discussed, focusing on the different structures and functions, which include fusion activation, tumorigenesis and viral assembly and lifecycle influences. Despite these genetic and functional differences, however, the C-terminal domains of these viruses share a common feature in the modulation of Env ectodomain conformation. Despite their differences, perhaps each system still has information to share with the others.

Background: HIV-1 transmitted/founder viruses (TF) are selected during the acute phase of infection from a multitude of virions present during transmission. They possess the capacity to establish infection and viral dissemination in a new host. Deciphering the discrete genetic determinant of infectivity in their envelope may provide clues for vaccine design. Methods: One hundred twenty-six clade B HIV-1 consensus envelope sequences from untreated acute and early infected individuals were compared to 105 sequences obtained from chronically infected individuals using next generation sequencing and molecular analyses. Results: We identified an envelope amino acid signature associated with TF viruses. They are more likely to have an isoleucine (I) in position 841 instead of an arginine (R). This mutation of R to I (R841I) in the gp41 cytoplasmic tail (gp41CT), specifically in lentivirus lytic peptides segment 1 (LLP-1), is significantly enriched compared to chronic viruses (OR = 0.2, 95% CI (0.09, 0.44), p = 0.00001). Conversely, a mutation of lysine (K) to isoleucine (I) located in position six (K6I) of the envelope signal peptide was selected by chronic viruses and compared to TF (OR = 3.26, 95% CI (1.76−6.02), p = 0.0001). Conclusions: The highly conserved gp41 CT_ LLP-1 domain plays a major role in virus replication in mediating intracellular traffic and Env incorporation into virions in interacting with encoded matrix protein. The presence of an isoleucine in gp41 in the TF viruses’ envelope may sustain its role in the successful establishment of infection during the acute stage.