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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased 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. 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. 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 autophagy assays, we hope to encourage technical innovation in the field

Microcin C is a ribosome-synthesized heptapeptide that contains a modified adenosine monophosphate covalently attached to the C-terminal aspartate. Microcin C is a potent inhibitor of bacterial cell growth. Based on the in vivo kinetics of inhibition of macromolecular synthesis, Microcin C targets translation, through a mechanism that remained undefined. Here, we show that Microcin C is a subject of specific degradation inside the sensitive cell. The product of degradation, a modified aspartyl-adenylate containing an N-acylphosphoramidate linkage, strongly inhibits translation by blocking the function of aspartyl-tRNA synthetase.Microcins are a class of small (Ͻ10 kDa) ribosomally synthesized peptide antibiotics produced by Enterobacteriaceae (1). Whereas some microcins are active as unmodified peptides (2), others are produced as polypeptide precursors that are heavily modified by dedicated maturation enzymes (3). Interest is attached to such post-translationally modified microcins due to their highly unusual structures and the fact that they target important cellular processes that are attractive targets for antibacterial drug development.Genes responsible for microcin production are usually plasmidborne. Plasmids encoding microcin structural and maturation genes also encode determinants of immunity specific to the microcin produced. Based on cross-immunity, post-translationally modified microcins can be subdivided into the B, C, and J types. Microcin B (MccB) 4 is a 43-residue peptide with 8 thiazole and oxazole rings that are synthesized by the McbBCD maturation enzyme complex from multiple serine and cysteine residues present in the MccB precursor (4). MccB is a potent inhibitor of DNA gyrase; it traps the enzyme at the stage of DNA strand passage (5). Microcin J, a 21-amino acid peptide, contains an unusual lactam bond between its N-terminal glycine and the ␦-carboxyl group of an internal glutamate; it assumes a highly unusual threaded-lasso structure (6 -8). MccJ inhibits bacterial RNA polymerase by occluding a narrow channel that is used to traffic transcription substrates, NTPs, to the catalytic center of the enzyme (9, 10).The structure of the subject of this study, Microcin C (McC) is shown in Fig. 1A. McC is a heptapeptide containing a modified adenosine monophosphate covalently attached to its C terminus through an N-acylphosphoramidate linkage (11, 12). The phosphoramidate group of the nucleotide part of McC is additionally modified by a propylamine group. Additionally, in mature McC, the peptide moiety, which is encoded by the mccA gene, is modified and the C-terminal asparagine residue specified by mccA is converted to an aspartate (18, 19), through an unknown mechanism. In vivo, McC appears to target translation (12). Guijarro et al. (12) also reported that large concentrations of McC, as well as of synthetic peptide of the same sequence but without the nucleotide modification, mildly inhibit translation in vitro. They therefore concluded that the peptide part of McC is responsible for transl...

Xeroderma pigmentosum (XP) complementation group E gene product, damaged DNA-binding protein 2 (DDB2), is a subunit of the DDB heterodimeric protein complex with high specificity for binding to a variety of DNA helix-distorting lesions. DDB is believed to play a role in the initial step of damage recognition in mammalian nucleotide excision repair (NER) of ultraviolet light (UV)-induced photolesions. It has been shown that DDB2 is rapidly degraded after cellular UV irradiation. However, the relevance of DDB2 degradation to its functionality in NER is still unknown. Here, we have provided evidence that Cullin 4A (CUL-4A), a key component of CUL-4A-based ubiquitin ligase, mediates DDB2 degradation at the damage sites and regulates the recruitment of XPC and the repair of cyclobutane pyrimidine dimers. We have shown that CUL-4A can be identified in a UV-responsive protein complex containing both DDB subunits. CUL-4A was visualized in localized UV-irradiated sites together with DDB2 and XPC. Degradation of DDB2 could be blocked by silencing CUL-4A using small interference RNA or by treating cells with proteasome inhibitor MG132. This blockage resulted in prolonged retention of DDB2 at the subnuclear DNA damage foci within micropore irradiated cells. Knock down of CUL-4A also decreased recruitment of the damage recognition factor, XPC, to the damaged foci and concomitantly reduced the removal of cyclobutane pyrimidine dimers from the entire genome. These results suggest that CUL-4A mediates the proteolytic degradation of DDB2 and that this degradation event, initiated at the lesion sites, regulates damage recognition by XPC during the early steps of NER.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy with a median survival of 3 years despite chemoimmu-notherapy. Rituximab, a chimeric anti-CD20 monoclonal antibody (mAb), has shown only modest activity as single agent in MCL. The humanized mAb milatu-zumab targets CD74, an integral membrane protein linked with promotion of B-cell growth and survival, and has shown preclinical activity against B-cell malig-nancies. Because rituximab and milatu-zumab target distinct antigens and potentially signal through different pathways, we explored a preclinical combination strategy in MCL. Treatment of MCL cell lines and primary tumor cells with immobilized milatuzumab and rituximab resulted in rapid cell death, radical oxygen species generation, and loss of mitochon-drial membrane potential. Cytoskeletal distrupting agents significantly reduced formation of CD20/CD74 aggregates, cell adhesion, and cell death, highlighting the importance of actin microfilaments in rituximab/milatuzumab-mediated cell death. Cell death was independent of caspase activation, Bcl-2 family proteins or modulation of autophagy. Maximal inhibition of p65 nuclear translocation was observed with combination treatment, indicating disruption of the NF-B pathway. Significant in vivo therapeutic activity of combination rituximab and milatuzumab was demonstrated in a preclinical model of MCL. These data support clinical evaluation of combination milatuzumab and rituximab therapy in MCL. (Blood. 2011; 117(17):4530-4541) Introduction Mantle cell lymphoma (MCL) is a B-cell malignancy with a variable histology and clinical course, distinguished by the characteristic translocation t(11;14)(q13, q32) that results in overexpres-sion of cyclin D 1 and consequent dysregulation of cell-cycle control. 1 In addition, MCL exhibits alterations in cell survival pathways, including constitutive activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling 2 and nuclear factor-B (NF-B). 3 Despite the hallmark genetic translocation in MCL, the clinical course of MCL is variable with some patients experiencing indolent disease, 4 whereas others exhibit rapid progression. 5 MCL patients have a median overall survival (OS) of approximately 3 years, and no consensus exists for standard first-line therapy. 6-9 Although aggressive therapies including chemoimmunotherapy 10,11 or stem cell transplantation 12,13 have been shown to improve outcomes, no therapy offers the potential for cure. Given the absence of curative therapy and the limited number of options for patients with relapsed/refractory MCL, novel treatment approaches are essential. Rituximab (Genentech), a chimeric anti-human CD20 monoclo-nal antibody (mAb), has been used in multiple strategies to treat patients with MCL. 14 As a single agent, rituximab has been tested in patients with newly diagnosed and relapsed/refractory MCL with response rates (RR) of 27% to 38% and a median response duration of 6 to 12 months. 15,16 Interestingly, the RR obtained in untreated patients was not higher than in relapsed...

Functional defect in DNA damage binding (DDB) activity has a direct relationship to decreased nucleotide excision repair (NER) and increased susceptibility to cancer. DDB forms a complex with cullin 4A (Cul4A), which is now known to ubiquitylate DDB2, XPC, and histone H2A. However, the exact role of DDB1 in NER is unclear. In this study, we show that DDB1 knockdown in human cells impaired their ability to efficiently repair UV-induced cyclobutane pyrimidine dimers (CPD) but not 6-4 photoproducts (6-4PP). Extensive nuclear protein fractionation and chromatin association analysis revealed that upon irradiation, DDB1 protein is translocated from a loosely bound to a tightly bound in vivo chromatin fraction and the DDB1 translocation required the participation of functional DDB2 protein. DDB1 knockdown also affected the translocation of Cul4A component to the tightly bound form in UV-damaged chromatin in vivo as well as its recruitment to the locally damaged nuclear foci in situ. However, DDB1 knockdown had no effect on DNA damage binding capacity of DDB2. The data indicated that DDB2 can bind to damaged DNA in vivo as a monomer, whereas Cul4A recruitment to damage sites depends on the fully assembled complex. Our data also showed that DDB1 is required for the UV-induced DDB2 ubiquitylation and degradation. In summary, the results suggest that (a) DDB1 is critical for efficient NER of CPD; (b) DDB1 acts in bridging DDB2 and ubiquitin ligase Cul4A; and (c) DDB1 aids in recruiting the ubiquitin ligase activity to the damaged sites for successful commencement of lesion processing by NER. (Cancer Res 2006; 66(17): 8590-7)