Envien su pedido a partir de medio dia del viernes 29.
1. Emerging roles of lncRNAs in senescence (febs13679)
Abstract. Cellular senescence is a complex stress response that leads to an irreversible
state of cell growth arrest. Senescence may be induced by various stimuli such
as telomere shortening, DNA damage or oncogenic insult, among others.
Senescent cells are metabolically highly active, producing a wealth of cytokines
and chemokines that, depending on the context, may have a beneficial or deleterious
effect on the organism. Senescence is considered a tightly regulated
stress response that is largely governed by the p53/p21 and p16/Rb pathways.
Many molecules have been identified as regulators of these two networks, such
as transcription factors, chromatin modifiers and non-coding RNAs. The
expression level of several long non-coding RNAs is affected during different
types of senescence; however, which of these are important for the biological
function remains poorly understood. Here we review our current knowledge of
the mechanistic roles of lncRNAs affecting the main senescence pathways, and
discuss the importance of identifying new regulators.
2. The Role of Protein “Stability Patches” in Molecular Recognition: A Case Study of the Human Growth Hormone-Receptor Complex (jcc24276)
Dynamic characteristics of protein surfaces are among the factors
determining their functional properties, including their
potential participation in protein-protein interactions. The
presence of clusters of static residues—“stability patches”
(SPs)—is a characteristic of protein surfaces involved in intermolecular
recognition. The mechanism, by with SPs facilitate
molecular recognition, however, remains unclear. Analyzing
the surface dynamic properties of the growth hormone and of
its high-affinity variant we demonstrated that reshaping of the
SPs landscape may be among the factors accountable for the
improved affinity of this variant to the receptor. We hypothesized
that SPs facilitate molecular recognition by moderating
the conformational entropy of the unbound state, diminishing
enthalpy–entropy compensation upon binding, and by augmenting
the favorable entropy of desolvation. SPs mapping
emerges as a valuable tool for investigating the structural
basis of the stability of protein complexes and for rationalizing
experimental approaches, such as affinity maturation, aimed at
3. The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes (paper 3-5)
Abstract: Mass spectrometry (MS) is a powerful tool for determining the mass of biomolecules with
high accuracy and sensitivity. MS performed under so-called “native conditions” (native MS) can
be used to determine the mass of biomolecules that associate noncovalently. Here we review the
application of native MS to the study of protein2ligand interactions and its emerging role in elucidating
the structure of macromolecular assemblies, including soluble and membrane protein complexes.
Moreover, we discuss strategies aimed at determining the stoichiometry and topology of
subunits by inducing partial dissociation of the holo-complex. We also survey recent developments
in "native top-down MS", an approach based on Fourier Transform MS, whereby covalent bonds
are broken without disrupting non-covalent interactions. Given recent progress, native MS is anticipated
to play an increasingly important role for researchers interested in the structure of macromolecular
4. A Laboratory Exercise to Illustrate Protein–Membrane Interactions (paper 3-12)
The laboratory protocol presented here takes about 3 hours to perform and investigates protein and lipid interactions. Students first purify His6-tagged human apolipoprotein A-I (apoA-I) with Ni-NTA affinity resin in a simple batch protocol and prepare multilamellar vesicles (MLV) from predried phospholipid films. When apoA-I is added to the MLV, much smaller protein/lipid nanodisc complexes are formed in some instances. Nanodisc formation can be monitored by a decrease in light-scattering intensity at 340 nm using a simple spectrophotometer. Students will observe nanodisc formation with MLV formed from the anionic phospholipid dimyristoylphosphatidyl glycerol, which pack poorly into lipid bilayers, but not with MLV formed from the zwitterionic phospholipid dimyristoyl phosphatidylcholine, which form stable bilayers. This laboratory exercise is accompanied by questions and exercises that enable students a deeper of the dimensions of apoA-I and nanodiscs as well as the biological relevance of nanodisc formation in the process of reverse cholesterol transport.
5. Improving cold storage and processing traits in potato through targeted gene knockout
Cold storage of potato tubers is commonly used to reduce sprouting and extend postharvest
shelf life. However, cold temperature stimulates the accumulation of reducing sugars in
potato tubers. Upon high-temperature processing, these reducing sugars react with free
amino acids, resulting in brown, bitter-tasting products and elevated levels of acrylamide—a
potential carcinogen. To minimize the accumulation of reducing sugars, RNA interference
(RNAi) technology was used to silence the vacuolar invertase gene (VInv), which encodes a
protein that breaks down sucrose to glucose and fructose. Because RNAi often results in
incomplete gene silencing and requires the plant to be transgenic, here we used transcription
activator-like effector nucleases (TALENs) to knockout VInv within the commercial potato
variety, Ranger Russet. We isolated 18 plants containing mutations in at least one VInv allele,
and five of these plants had mutations in all VInv alleles. Tubers from full VInv-knockout
plants had undetectable levels of reducing sugars, and processed chips contained reduced
levels of acrylamide and were lightly coloured. Furthermore, seven of the 18 modified plant
lines appeared to contain no TALEN DNA insertions in the potato genome. These results
provide a framework for using TALENs to quickly improve traits in commercially relevant
autotetraploid potato lines.
6. Ubiquitin-Like Proteasome System Represents a Eukaryotic-Like Pathway for Targeted Proteolysis in Archaea (paper4-1)
ABSTRACT The molecular mechanisms of targeted proteolysis in archaea are poorly understood, yet they may have deep evolutionary roots shared with the ubiquitin-proteasome system of eukaryotic cells. Here, we demonstrate in archaea that TBP2, a TATA-binding protein (TBP) modified by ubiquitin-like isopeptide bonds, is phosphorylated and targeted for degradation by proteasomes. Rapid turnover of TBP2 required the functions of UbaA (the E1/MoeB/ThiF homolog of archaea), AAA ATPases (Cdc48/p97 and Rpt types), a type 2 JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN_) homolog (JAMM2), and 20S proteasomes. The ubiquitin-like protein modifier small archaeal modifier protein 2 (SAMP2) stimulated the degradation of TBP2, but SAMP2 itself was not degraded. Analysis of the TBP2 fractions that were not modified by ubiquitin-like linkages revealed that TBP2 had multiple N termini, including Met1-Ser2, Ser2, and Met1-Ser2(p) [where (p) represents phosphorylation]. The evidence suggested that the Met1-Ser2(p) form accumulated in cells that were unable to degrade TBP2. We propose a model in archaea in which the attachment of ubiquitin-like tags can target proteins for degradation by proteasomes and be controlled by N-terminal degrons. In support of a proteolytic mechanism that is energy dependent and recycles the ubiquitin-like protein tags, we find that a network of AAA ATPases and a JAMM/MPN_metalloprotease are required, in addition to 20S proteasomes, for controlled intracellular proteolysis.
7. HCF1 and OCT2 Cooperate with EBNA1 To Enhance OriP-Dependent Transcription and Episome Maintenance of Latent Epstein-Barr Virus (paper4-3)
Epstein-Barr virus (EBV) establishes latent infections as multicopy episomes with complex patterns of viral gene transcription and chromatin structure. The EBV origin of plasmid replication (OriP) has been implicated as a critical control element for viral transcription, as well as viral DNA replication and episome maintenance. Here, we examine cellular factors that bind OriP and regulate histone modification, transcription regulation, and episome maintenance. We found that OriP is enriched for histone H3 lysine 4 (H3K4) methylation in multiple cell types and latency types. Host cell factor 1 (HCF1), a component of the mixedlineage leukemia (MLL) histone methyltransferase complex, and transcription factor OCT2 (octamer-binding transcription factor 2) bound cooperatively with EBNA1 (Epstein-Barr virus nuclear antigen 1) at OriP. Depletion of OCT2 or HCF1 deregulated latency transcription and histone modifications at OriP, as well as the OriP-regulated latency type-dependentCpromoter (Cp) andQ promoter (Qp). HCF1 depletion led to a loss of histone H3K4me3 (trimethylation of histone H3 at lysine 4) and H3 acetylation at Cp in type III latency and Qp in type I latency, as well as an increase in heterochromatic H3K9me3 at these sites. HCF1 depletion resulted in the loss of EBV episomes from Burkitt’s lymphoma cells with type I latency and reactivation from lymphoblastoid cells (LCLs) with type III latency. These findings indicate that HCF1 and OCT2 function at OriP to regulate viral transcription, histone modifications, and episome maintenance. As HCF1 is best known for its function in herpes simplex virus 1 (HSV-1) immediate early gene transcription, our findings suggest that EBV latency transcription shares unexpected features with HSV gene regulation.