A Novel, Fully Automated, and Reagent-Agnostic Transient Transfection Protocol.

Transfection is a potent technique to introduce foreign nucleic acids into eukaryotic cells. The capacity of the technique to alter the genetic content of host cells means it is useful for a wide range of applications, including the study of typical cellular processes, disease molecular mechanisms, and gene therapy effects. Here, we discuss a highly reliable and fully automated transient transfection protocol that utilizes an open-source liquid handler and accompanying HEPA Module.

Inositol Pyrophosphate Synthesis by Diphosphoinositol Pentakisphosphate Kinase-1 is Regulated by Phosphatidylinositol(4,5)bisphosphate.

5-diphosphoinositol tetrakisphosphate (5-InsP) and bisdiphosphoinositol tetrakisphosphate (InsP) are 'energetic' inositol pyrophosphate signaling molecules that regulate bioenergetic homeostasis. Inositol pyrophosphate levels are regulated by diphosphoinositol pentakisphosphate kinases (PPIP5Ks); these are large modular proteins that host a kinase domain (which phosphorylates 5-InsP to InsP), a phosphatase domain that catalyzes the reverse reaction, and a polyphosphoinositide-binding domain (PBD). Here, we describe new interactions between these three domains in the context of full-length human PPIP5K1.

The significance of the 1-kinase/1-phosphatase activities of the PPIP5K family.

The inositol pyrophosphates (diphosphoinositol polyphosphates), which include 1-InsP, 5-InsP, and InsP, are highly 'energetic' signaling molecules that play important roles in many cellular processes, particularly with regards to phosphate and bioenergetic homeostasis. Two classes of kinases synthesize the PP-InsPs: IP6Ks and PPIP5Ks. The significance of the IP6Ks - and their 5-InsP product - has been widely reported.

Asp1 from Schizosaccharomyces pombe binds a [2Fe-2S](2+) cluster which inhibits inositol pyrophosphate 1-phosphatase activity.

Iron-sulfur (Fe-S) clusters are widely distributed protein cofactors that are vital to cellular biochemistry and the maintenance of bioenergetic homeostasis, but to our knowledge, they have never been identified in any phosphatase. Here, we describe an iron-sulfur cluster in Asp1, a dual-function kinase/phosphatase that regulates cell morphogenesis in Schizosaccharomyces pombe. Full-length Asp1, and its phosphatase domain (Asp1(371-920)), were each heterologously expressed in Escherichia coli.

MORC2 signaling integrates phosphorylation-dependent, ATPase-coupled chromatin remodeling during the DNA damage response.

Chromatin dynamics play a central role in maintaining genome integrity, but how this is achieved remains largely unknown. Here, we report that microrchidia CW-type zinc finger 2 (MORC2), an uncharacterized protein with a derived PHD finger domain and a conserved GHKL-type ATPase module, is a physiological substrate of p21-activated kinase 1 (PAK1), an important integrator of extracellular signals and nuclear processes. Following DNA damage, MORC2 is phosphorylated on serine 739 in a PAK1-dependent manner, and phosphorylated MORC2 regulates its DNA-dependent ATPase activity to facilitate chromatin remodeling.

Signaling-dependent phosphorylation of mitotic centromere-associated kinesin regulates microtubule depolymerization and its centrosomal localization.

Background: Although PAK1 regulates cytoskeleton and microtubule dynamics, its role in controlling the functions of MCAK remains unknown.

Results: PAK1 phosphorylates MCAK and thereby regulates both its localization and function.

Conclusion: MCAK is a cognate substrate of PAK1.

Mechanism of MTA1 protein overexpression-linked invasion: MTA1 regulation of hyaluronan-mediated motility receptor (HMMR) expression and function.

Even though the hyaluronan-mediated motility receptor (HMMR), a cell surface oncogenic protein, is widely up-regulated in human cancers and correlates well with cell motility and invasion, the underlying molecular and nature of its putative upstream regulation remain unknown. Here, we found for the first time that MTA1 (metastatic tumor antigen 1), a master chromatin modifier, regulates the expression of HMMR and, consequently, its function in breast cancer cell motility and invasiveness. We recognized a positive correlation between the levels of MTA1 and HMMR in human cancer.

Metastasis-associated protein 1 drives tumor cell migration and invasion through transcriptional repression of RING finger protein 144A.

Metastasis-associated protein 1 (MTA1), a component of the nucleosome-remodeling and histone deacetylase complex, is widely up-regulated in human cancers and significantly correlated with tumor invasion and metastasis, but the mechanisms involved remain largely unknown. Here, we report that MTA1 transcriptionally represses the expression of RING finger protein 144A (RNF144A), an uncharacterized gene whose protein product possesses potential E3 ubiquitin ligase activity, by recruiting the histone deacetylase 2 (HDAC2) and CCAAT/enhancer-binding protein α (c/EBPα) co-repressor complex onto human RNF144A promoter. Furthermore, an inverse correlation between the expression levels of MTA1 and RNF144A was demonstrated in publicly available breast cancer microarray datasets and the MCF10 breast cancer progression model system.

Lactoferrin-endothelin-1 axis contributes to the development and invasiveness of triple-negative breast cancer phenotypes.

Triple-negative breast cancer (TNBC) is characterized by the lack of expression of estrogen receptor-α (ER-α), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2). However, pathways responsible for downregulation of therapeutic receptors, as well as subsequent aggressiveness, remain unknown. In this study, we discovered that lactoferrin (Lf) efficiently downregulates levels of ER-α, PR, and HER-2 in a proteasome-dependent manner in breast cancer cells, and it accounts for the loss of responsiveness to ER- or HER-2-targeted therapies.