In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Activation of mammalian innate and acquired immune responses must be tightly regulated by elaborate mechanisms to control their onset and termination. MicroRNAs have been implicated as negative regulators controlling diverse biological processes at the level of posttranscriptional repression. Expression profiling of 200 microRNAs in human monocytes revealed that several of them (miR-146a/b, miR-132, and miR-155) are endotoxin-responsive genes. Analysis of miR-146a and miR-146b gene expression unveiled a pattern of induction in response to a variety of microbial components and proinflammatory cytokines. By means of promoter analysis, miR-146a was found to be a NF-κB-dependent gene. Importantly, miR-146a/b were predicted to base-pair with sequences in the 3′ UTRs of the TNF receptor-associated factor 6 and IL-1 receptor-associated kinase 1 genes, and we found that these UTRs inhibit expression of a linked reporter gene. These genes encode two key adapter molecules downstream of Toll-like and cytokine receptors. Thus, we propose a role for miR-146 in control of Toll-like receptor and cytokine signaling through a negative feedback regulation loop involving down-regulation of IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6 protein levels.

https://www.pnas.org/content/pnas/103/33/12481.full.pdf

NF-κB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses

Stimulation of Toll-like receptors (TLRs) triggers activation of a common MyD88-dependent signaling pathway as well as a MyD88-independent pathway that is unique to TLR3 and TLR4 signaling pathways leading to interferon (IFN)-beta production. Here we disrupted the gene encoding a Toll/IL-1 receptor (TIR) domain-containing adaptor, TRIF. TRIF-deficient mice were defective in both TLR3- and TLR4-mediated expression of IFN-beta and activation of IRF-3. Furthermore, inflammatory cytokine production in response to the TLR4 ligand, but not to other TLR ligands, was severely impaired in TRIF-deficient macrophages. Mice deficient in both MyD88 and TRIF showed complete loss of nuclear factor kappa B activation in response to TLR4 stimulation. These findings demonstrate that TRIF is essential for TLR3- and TLR4-mediated signaling pathways facilitating mammalian antiviral host defense.

https://science.sciencemag.org/content/sci/301/5633/640.full.pdf

Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway.

Type I interferons are central mediators for antiviral responses. Using high-throughput functional screening of interferon inducers, we have identified here a molecule we call interferon-beta promoter stimulator 1 (IPS-1). Overexpression of IPS-1 induced type I interferon and interferon-inducible genes through activation of IRF3, IRF7 and NF-kappaB transcription factors. TBK1 and IKKi protein kinases were required for the IPS-1-mediated interferon induction. IPS-1 contained an N-terminal CARD-like structure that mediated interaction with the CARD of RIG-I and Mda5, which are cytoplasmic RNA helicases that sense viral infection. 'Knockdown' of IPS-1 by small interfering RNA blocked interferon induction by virus infection. Thus, IPS-1 is an adaptor involved in RIG-I- and Mda5-mediated antiviral immune responses.

IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction

The transcription factors interferon regulatory factor 3 (IRF3) and NF-kappaB are required for the expression of many genes involved in the innate immune response. Viral infection, or the binding of double-stranded RNA to Toll-like receptor 3, results in the coordinate activation of IRF3 and NF-kappaB. Activation of IRF3 requires signal-dependent phosphorylation, but little is known about the signaling pathway or kinases involved. Here we report that the noncanonical IkappaB kinase homologs, IkappaB kinase-epsilon (IKKepsilon) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-kappaB activation, are also essential components of the IRF3 signaling pathway. Thus, IKKepsilon and TBK1 have a pivotal role in coordinating the activation of IRF3 and NF-kappaB in the innate immune response.

IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway.

The activation of NF‐κB by receptors in the tumor necrosis factor (TNF) receptor and Toll/interleukin‐1 (IL‐1) receptor families requires the TRAF family of adaptor proteins. Receptor oligomerization causes the recruitment of TRAFs to the receptor complex, followed by the activation of a kinase cascade that results in the phosphorylation of IκB. TANK is a TRAF‐binding protein that can inhibit the binding of TRAFs to receptor tails and can also inhibit NF‐κB activation by these receptors. However, TANK also displays the ability to stimulate TRAF‐mediated NF‐κB activation. In this report, we investigate the mechanism of the stimulatory activity of TANK. We find that TANK interacts with TBK1 (TANK‐binding kinase 1), a novel IKK‐related kinase that can activate NF‐κB in a kinase‐dependent manner. TBK1, TANK and TRAF2 can form a ternary complex, and complex formation appears to be required for TBK1 activity. Kinase‐inactive TBK1 inhibits TANK‐mediated NF‐κB activation but does not block the activation mediated by TNF‐α, IL‐1 or CD40. The TBK1–TANK–TRAF2 signaling complex functions upstream of NIK and the IKK complex and represents an alternative to the receptor signaling complex for TRAF‐mediated activation of NF‐κB.

/pdf/nf-kb-activation-by-a-signaling-complex-containing-traf2-7x6cffyej2.pdf

NF‐κB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK‐related kinase

Two Pathways to NF-κB

https://www.cell.com/immunity/pdf/S1074-7613(05)00310-9.pdf

Phosphorylation of the CARMA1 Linker Controls NF-κB Activation

Computer modeling suggested that transcription factors with novel sequence specificities could be designed by combining known DNA binding domains. This structure-based strategy was tested by construction of a fusion protein, ZFHD1, that contained zinc fingers 1 and 2 from Zif268, a short polypeptide linker, and the homeodomain from Oct-1. The fusion protein bound optimally to a sequence containing adjacent homeodomain (TAATTA) and zinc finger (NGGGNG) subsites. When fused to an activation domain, ZFHD1 regulated promoter activity in vivo in a sequence-specific manner. Analysis of known protein-DNA complexes suggests that many other DNA binding proteins could be designed in a similar fashion.

https://science.sciencemag.org/content/sci/267/5194/93.full.pdf

Structure-based design of transcription factors

NF‐κB is a critical target of signaling downstream of the T cell receptor (TCR) complex, but how TCR signaling activates NF‐κB is poorly understood. We have developed an expression cloning strategy that can identify catalytic and noncatalytic molecules that participate in different pathways of NF‐κB activation. Screening of a mouse thymus cDNA library yielded CARD11, a membrane‐associated guanylate kinase (MAGUK) family member containing CARD, PDZ, SH3 and GUK domains. Using a CARD‐deleted variant of CARD11 and RNA interference (RNAi), we demonstrate that CARD11 mediates NF‐κB activation by αCD3/αCD28 cross‐linking and PMA/ionomycin treatment, but not by TNFα or dsRNA. CARD11 is not required for TCR‐mediated induction of NFAT or AP‐1. CARD11 functions upstream of the IκB‐kinase (IKK) complex and cooperates with Bcl10 in a CARD domain‐dependent manner. RNAi‐rescue experiments suggest that the CARD, coiled‐coil, SH3 and GUK domains of CARD11 are critical for its signaling function. These results implicate CARD11 in factor‐ specific activation of NF‐κB by the TCR complex and establish a role for a MAGUK family member in antigen receptor signaling.

/pdf/card11-mediates-factor-specific-activation-of-nf-kb-by-the-t-3pj3di3doa.pdf

Joel L. Pomerantz

Papers

NF‐κB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK‐related kinase

Two Pathways to NF-κB

Phosphorylation of the CARMA1 Linker Controls NF-κB Activation

Structure-based design of transcription factors

CARD11 mediates factor-specific activation of NF-κB by the T cell receptor complex