Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction

The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for two LDL receptor family members, the very low density lipoprotein receptor and the apoE receptor-2, in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor protein Disabled-1 (DAB1). Here, we identify an extended set of cytoplasmic proteins that might also participate in signal transmission by the LDL receptor gene family. Most of these novel proteins are adaptor or scaffold proteins that contain PID or PDZ domains and function in the regulation of mitogen-activated protein kinases, cell adhesion, vesicle trafficking, or neurotransmission. We show that binding of DAB1 interferes with receptor internalization suggesting a mechanism by which signaling through this class of receptors might be regulated. Taken together, these findings imply much broader physiological functions for the LDL receptor family than had previously been appreciated. They form the basis for the elucidation of the molecular pathways by which cells respond to the diversity of ligands that bind to these multifunctional receptors on the cell surface.     

Asef is a Cdc42-specific guanine nucleotide exchange factor

Asef is a member of the Dbl-family of guanine nucleotide exchange factors (GEFs) with a proposed specificity for the small GTPase Rac1. Here we investigated the specificity and regulation of Asef by measuring its GEF activity in vitro and observed hardly any activity towards Rac1, Rac2 and Rac3, or RhoA and TC10. In contrast, various purified Asef protein fragments catalyzed the nucleotide exchange reaction of Cdc42. The Cdc42GEF activity of the Dbl homology (DH) domain of Asef was significantly higher in the presence of the pleckstrin homology (PH) domain. Our data strongly suggest that Asef is a canonical Cdc42GEF, which employs its PH domain to efficiently stabilize its autoinhibited state, but also to facilitate nucleotide exchange activity of the DH domain after its activation by upstream signals.     

Cardiac sarcomere mechanics in health and disease

The sarcomere is the fundamental structural and functional unit of striated muscle and is directly responsible for most of its mechanical properties. The sarcomere generates active or contractile forces and determines the passive or elastic properties of striated muscle. In the heart, mutations in sarcomeric proteins are responsible for the majority of genetically inherited cardiomyopathies. Here, we review the major determinants of cardiac sarcomere mechanics including the key structural components that contribute to active and passive tension. We dissect the molecular and structural basis of active force generation, including sarcomere composition, structure, activation, and relaxation. We then explore the giant sarcomere-resident protein titin, the major contributor to cardiac passive tension. We discuss sarcomere dynamics exemplified by the regulation of titin-based stiffness and the titin life cycle. Finally, we provide an overview of therapeutic strategies that target the sarcomere to improve cardiac contraction and filling.     

Triple-negative breast cancer cells rely on kinase-independent functions of CDK8 to evade NK-cell-mediated tumor surveillance

Triple-negative breast cancer (TNBC) is an aggressive malignant disease that is responsible for approximately 15% of breast cancers. The standard of care relies on surgery and chemotherapy but the prognosis is poor and there is an urgent need for new therapeutic strategies. Recent in silico studies have revealed an inverse correlation between recurrence-free survival and the level of cyclin-dependent kinase 8 (CDK8) in breast cancer patients. CDK8 is known to have a role in natural killer (NK) cell cytotoxicity, but its function in TNBC progression and immune cell recognition or escape has not been investigated. We have used a murine model of orthotopic breast cancer to study the tumor-intrinsic role of CDK8 in TNBC. Knockdown of CDK8 in TNBC cells impairs tumor regrowth upon surgical removal and prevents metastasis. In the absence of CDK8, the epithelial-to-mesenchymal transition (EMT) is impaired and immune-mediated tumor-cell clearance is facilitated. CDK8 drives EMT in TNBC cells in a kinase-independent manner. In vivo experiments have confirmed that CDK8 is a crucial regulator of NK-cell-mediated immune evasion in TNBC. The studies also show that CDK8 is involved in regulating the checkpoint inhibitor programmed death-ligand 1 (PD-L1). The CDK8–PD-L1 axis is found in mouse and human TNBC cells, underlining the importance of CDK8-driven immune cell evasion in these highly aggressive breast cancer cells. Our data link CDK8 to PD-L1 expression and provide a rationale for investigating the possibility of CDK8-directed therapy for TNBC.Subject terms: Breast cancer, Immune evasion     

Role of Human Sec63 in Modulating the Steady-State Levels of Multi-Spanning Membrane Proteins

The Sec61 translocon of the endoplasmic reticulum (ER) membrane forms an aqueous pore, allowing polypeptides to be transferred across or integrated into membranes. Protein translocation into the ER can occur co- and posttranslationally. In yeast, posttranslational translocation involves the heptameric translocase complex including its Sec62p and Sec63p subunits. The mammalian ER membrane contains orthologs of yeast Sec62p and Sec63p, but their function is poorly understood. Here, we analyzed the effects of excess and deficit Sec63 on various ER cargoes using human cell culture systems. The overexpression of Sec63 reduces the steady-state levels of viral and cellular multi-spanning membrane proteins in a cotranslational mode, while soluble and single-spanning ER reporters are not affected. Consistent with this, the knock-down of Sec63 increases the steady-state pools of polytopic ER proteins, suggesting a substrate-specific and regulatory function of Sec63 in ER import. Overexpressed Sec63 exerts its down-regulating activity on polytopic protein levels independent of its Sec62-interacting motif, indicating that it may not act in conjunction with Sec62 in human cells. The specific action of Sec63 is further sustained by our observations that the up-regulation of either Sec62 or two other ER proteins with lumenal J domains, like ERdj1 and ERdj4, does not compromise the steady-state level of a multi-spanning membrane reporter. A J domain-specific mutation of Sec63, proposed to weaken its interaction with the ER resident BiP chaperone, reduces the down-regulating capacity of excess Sec63, suggesting an involvement of BiP in this process. Together, these results suggest that Sec63 may perform a substrate-selective quantity control function during cotranslational ER import.     

S100A9 is a biliary protein marker of disease activity in primary sclerosing cholangitis

Background and Aims

Bile analysis has the potential to serve as a surrogate marker for inflammatory and neoplastic disorders of the biliary epithelium and may provide insight into biliary pathophysiology and possible diagnostic markers. We aimed to identify biliary protein markers of patients with primary sclerosing cholangitis (PSC) by a proteomic approach.

Methods

Bile duct-derived bile samples were collected from PSC patients (n = 45) or patients with choledocholithiasis (n = 24, the control group). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to analyse the proteins, 2-D-gel patterns were compared by densitometry, and brush cytology specimens were analysed by RT-PCR.

Results

A reference bile-duct bile proteome was established in the control group without signs of inflammation or maligancy comprising a total of 379 non-redundant biliary proteins; 21% were of unknown function and 24% had been previously described in serum. In PSC patients, the biliary S100A9 expression was elevated 95-fold (p<0.005), serum protein expression was decreased, and pancreatic enzyme expression was unchanged compared to controls. The S100A9 expression was 2-fold higher in PSC patients with high disease activity than in those with low activity (p<0.05). The brush cytology specimens from the PSC patients with high disease activity showed marked inflammatory activity and leukocyte infiltration compared to the patients with low activity, which correlated with S100A9 mRNA expression (p<0.05).

Conclusions

The bile-duct bile proteome is complex and its analysis might enhance the understanding of cholestatic liver disease. Biliary S100A9 levels may be a useful marker for PSC activity, and its implication in inflammation and carcinogenesis warrants further investigation.     

Mechanics on myocardium deficient in the N2B region of titin: the cardiac-unique spring element improves efficiency of the cardiac cycle

Titin (also known as connectin) is an intrasarcomeric muscle protein that functions as a molecular spring and generates passive tension upon muscle stretch. The N2B element is a cardiac-specific spring element within titin's extensible region. Our goal was to study the contribution of the N2B element to the mechanical properties of titin, particularly its hypothesized role in limiting energy loss during repeated stretch (diastole)-shortening (systole) cycles of the heart. We studied energy loss by measuring hysteresis from the area between the stretch and release passive force-sarcomere length curves and used both wild-type (WT) mice and N2B knockout (KO) mice in which the N2B element has been deleted. A range of protocols was used, including those that mimic physiological loading conditions. KO mice showed significant increases in hysteresis. Most prominently, in tissue that had been preconditioned with a physiological stretch-release protocol, hysteresis increased significantly from 320 ± 46 pJ/mm²/sarcomere in WT to 650 ± 94 pJ/mm²/sarcomere in N2B KO myocardium. These results are supported by experiments in which oxidative stress was used to mechanically inactivate portions of the N2B-Us of WT titin through cysteine cross-linking. Studies on muscle from which the thin filaments had been extracted (using the actin severing protein gelsolin) showed that the difference in hysteresis between WT and KO tissue cannot be explained by filament sliding-based viscosity. Instead the results suggest that hysteresis arises from within titin and most likely involves unfolding of immunoglobulin-like domains. These studies support that the mechanical function of the N2B element of titin includes reducing hysteresis and increasing the efficiency of the heart.     

A new technique for in vivo imaging of specific GLP-1 binding sites: first results in small rodents

EXPERIMENTAL OBJECTIVES: In vivo imaging of GLP-1 receptor-positive tissues may allow examination of physiologic and pathophysiologic processes. Based on the GLP-1 analog Exendin 4, we have developed a radiolabeled compound specifically targeting the GLP-1 receptor (DTPA-Lys40-Exendin 4). This work aims to detect GLP-1 receptor-positive tissues by biodistribution studies and in vivo small animal imaging studies. For in vivo imaging, a high-resolution multi-pinhole SPECT (single photon emission computed tomography) system was used in conjunction with an MRI (magnetic resonance imaging) system for image fusion. RESULTS: DTPA-Lys40-Exendin 4 can be labeled with 111In to high specific activity (40 GBq/micromol). The radiochemical purity reliably exceeded 95%. Using this compound for in vivo small animal imaging of rats and mice as well as for biodistribution studies, specific GLP-1 binding sites could be detected in stomach, pancreas, lung, adrenals, and pituitary. Receptor-positive tissues were visualized with a high-resolution SPECT system with a resolution of less than 1 mm. CONCLUSIONS: The new technique using DTPA-Lys40-Exendin 4 allows highly sensitive imaging of GLP-1 receptor-positive tissues in vivo. Therefore, intra-individual follow-up studies of GLP-1 receptor-positive tissue could be conducted in vivo.     

Localization of the Rh50-like protein to the contractile vacuole in Dictyostelium

The human Rhesus (Rh) family consists of three polytopic membrane proteins present at the cell surface of red blood cells. Although Rh proteins are essential for the expression of the blood group system their biological function remains unclear. In this study, the gene encoding a protein homologous to Rh50 in Dictyostelium discoideum was sequenced. The Rh50-like protein was localized to the contractile vacuole, the organelle responsible for maintenance of osmotic equilibrium within the cell. However, Rh50-like-deficient mutants in which the Rh50-like gene was disrupted did not appear to exhibit a phenotype related to osmoregulation. Nevertheless, these mutants may provide a valuable tool for studying the function of the rhesus protein.