Distinct roles in autophagy and importance in infectivity of the two ATG4 cysteine peptidases of leishmania major

Macroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite

Chaperoning junior faculty: Institutional support and guidance can relieve challenges for early-career group leaders and imporove academic performance

The initial excitement of finally leading an independent research group is quickly followed by the realization that it comes with novel challenges. The first day as a principal investigator sets the clock ticking on limited time and opportunities to publish and apply for grants and awards that all are required for tenure or the next job. Expectations are high: PIs must be outstanding scholars who establish their own research program, excel in teaching, and are helpful colleagues and mentors for their students and postdocs. Meeting such high expectations with little experience can cause anxiety and stress. Moreover, we are often our own worst critics; meeting high self‐expectations can be demanding even without external pressure. Based on our experiences as junior faculty, we herewith suggest a set of measures that could help early‐career group leaders to better handle this stress and allow them—and their host institutes—to flourish

Measurement of the Activity of the ATG4 Cysteine Proteases

While only one Atg4 is present in yeast, there are four Atg4 homologues in human and in mouse with different substrate specificities and catalytic efficiencies. The molecule Atg4 is a type of cysteine protease, and is known for its crucial roles in cleavage of the Atg8 family proteins before they can be conjugated to phospholipids, and also in cleavage of the conjugated Atg8 molecules from the membrane, a process known as deconjugation. Both processes are required for the maximal efficiency in autophagosome biogenesis. Atg4 could thus be a target for intervention of the autophagy process. It is thus important to measure Atg4 activity to determine and to modulate the autophagy function. Here we review the catalytic functions and regulatory mechanisms of human Atg4 proteases, and discuss the methodology for analyzing Atg4 activity in details

Endogenous HMGB1 regulates autophagy

HMGB1 displaces Bcl-2 from Beclin1 to induce and sustain autophagy in response to cell stress

Current concepts on the role of inflammation in COPD and lung cancer

Chronic obstructive pulmonary disease (COPD) and lung cancer are leading cause of death, and both are associated with cigarette smoke exposure. It has been shown that 50–70% of patients diagnosed with lung cancer suffer from COPD, and reduced lung function is an important event in lung cancer suggesting an association between COPD and lung cancer. However, a causal relationship between COPD and lung tumorigenesis is not yet fully understood. Recent studies have suggested a central role of chronic inflammation in pathogenesis of both the diseases. For example, immune dysfunction, abnormal activation of NF-κB, epithelial-to-mesenchymal transition, altered adhesion signaling pathways, and extracellular matrix degradation/altered signaling are the key underlying mechanisms in both COPD and lung cancer. These parameters along with other processes, such as chromatin modifications/epigenetic changes, angiogenesis, and autophagy/apoptosis are altered by cigarette smoke, are crucial in the development of COPD and lung cancer. Understanding the cellular and molecular mechanisms underlying these processes will provide novel avenues for halting the chronic inflammation in COPD and devising therapeutic strategies against lung cancer

The RNase Rny1p cleaves tRNAs and promotes cell death during oxidative stress in Saccharomyces cerevisiae

The cellular response to stress conditions involves a decision between survival or cell death when damage is severe. A conserved stress response in eukaryotes involves endonucleolytic cleavage of transfer RNAs (tRNAs). The mechanism and significance of such tRNA cleavage is unknown. We show that in yeast, tRNAs are cleaved by the RNase T2 family member Rny1p, which is released from the vacuole into the cytosol during oxidative stress. Rny1p modulates yeast cell survival during oxidative stress independently of its catalytic ability. This suggests that upon release to the cytosol, Rny1p promotes cell death by direct interactions with downstream components. Thus, detection of Rny1p, and possibly its orthologues, in the cytosol may be a conserved mechanism for assessing cellular damage and determining cell survival, analogous to the role of cytochrome c as a marker for mitochondrial damage

Fibroblasts from long‐lived mutant mice exhibit increased autophagy and lower TOR activity after nutrient deprivation or oxidative stress

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92378/1/j.1474-9726.2012.00833.x.pd

The Reprogramming of Tumor Stroma by HSF1 Is a Potent Enabler of Malignancy

Stromal cells within the tumor microenvironment are essential for tumor progression and metastasis. Surprisingly little is known about the factors that drive the transcriptional reprogramming of stromal cells within tumors. We report that the transcriptional regulator Heat-Shock Factor 1 (HSF1) is frequently activated in cancer-associated fibroblasts (CAFs), where it is a potent enabler of malignancy. HSF1 drives a transcriptional program in CAFs that complements, yet is completely different from, the program it drives in adjacent cancer cells. This CAF program is uniquely structured to support the malignant potential of cancer cells in a non-cell-autonomous way. Two central stromal signaling molecules—TGFβ and stromal-derived factor 1 (SDF1) – play a critical role. In early stage breast and lung cancer, high stromal HSF1 activation is strongly associated with poor patient outcome. Thus, tumors co-opt the ancient survival functions of HSF1 to orchestrate malignancy in both cell-autonomous and non-cell-autonomous ways, with far-reaching therapeutic implications

Heat Shock Factor 1-dependent extracellular matrix remodeling mediates the transition from chronic intestinal inflammation to colon cancer

In the colon, long-term exposure to chronic inflammation drives colitis-associated colon cancer (CAC) in patients with inflammatory bowel disease. While the causal and clinical links are well established, molecular understanding of how chronic inflammation leads to the development of colon cancer is lacking. Here we deconstruct the evolving microenvironment of CAC by measuring proteomic changes and extracellular matrix (ECM) organization over time in a mouse model of CAC. We detect early changes in ECM structure and composition, and report a crucial role for the transcriptional regulator heat shock factor 1 (HSF1) in orchestrating these events. Loss of HSF1 abrogates ECM assembly by colon fibroblasts in cell-culture, prevents inflammation-induced ECM remodeling in mice and inhibits progression to CAC. Establishing relevance to human disease, we find high activation of stromal HSF1 in CAC patients, and detect the HSF1-dependent proteomic ECM signature in human colorectal cancer. Thus, HSF1-dependent ECM remodeling plays a crucial role in mediating inflammation-driven colon cancer.R35 GM118173 - NIGMS NIH HHS; U01 TR002625 - NCATS NIH HHS; P30 CA008748 - NCI NIH HHS; FC010144 - Cancer Research UK; FC010144 - Medical Research Council; FC010144 - Wellcome TrustPublished versio