Reversible Oligonucleotide Chain Blocking Enables Bead Capture and Amplification of T‑Cell Receptor α and β Chain mRNAs

Next-generation sequencing (NGS) has proven to be an exceptionally powerful tool for studying genetic variation and differences in gene expression profiles between cell populations. However, these population-wide studies are limited by their inability to detect variation between individual cells within a population, inspiring the development of single-cell techniques such as Drop-seq, which add a unique barcode to the mRNA from each cell prior to sequencing. Current Drop-seq technology enables capture, amplification, and barcoding of the entire mRNA transcriptome of individual cells. NGS can then be used to sequence the 3′-end of each message to build a cell-specific transcriptional landscape. However, current technology does not allow high-throughput capture of information distant from the mRNA poly-A tail. Thus, gene profiling would have much greater utility if beads could be generated having multiple transcript-specific capture sequences. Here we report the use of a reversible chain blocking group to enable synthesis of DNA barcoded beads having capture sequences for the constant domains of the T-cell receptor α and β chain mRNAs. We demonstrate that these beads can be used to capture and pair TCRα and TCRβ sequences from total T-cell RNA, enabling reverse transcription and PCR amplification of these sequences. This is the first example of capture beads having more than one capture sequence, and we envision that this technology will be of high utility for applications such as pairing the antigen receptor chains that give rise to autoimmune diseases or measuring the ratios of mRNA splice variants in cancer stem cells

Subcellular fractionation and protease protection.

<p>7.5 ug of mitochondrial protein was processed and loaded in each lane. HB refers to treatment with hypo-osmotic buffer (20 mM KCl) for mitoplast isolation, PK to proteinase K (1 mg/mL) treatment and SDS to sodium dodecyl sulfate (0.5% w/v) treatment for mitoplast lysis. Markers for each compartment include hsp60—matrix, vdac1—outer mitochondrial membrane, tim23—inner mitochondria membrane and cytc—intermembrane space.</p

Proposed model of Mitochondrial Heme Metabolism Protein Complex.

<p>Proteins in the complex are shown in the inner mitochondrial membrane and labeled with their roles in porphyrin, iron and heme homeostasis. Additional proteins which may be involved in bridging between protein partners are not shown. IMM refers to the inner mitochondrial membrane, OMM to the outer mitochondrial membrane and EN to the endosome.</p

Graphical representation of affinity purification and MS analysis of FLAG-FECH (red), FLAG-FECH Variants (purple), FLAG-PPOX (orange) and FLAG-CPOX (blue).

<p>Each panel represents an identified mouse protein recovered with the bait human protein listed in the legend of panel A. Panels are as follows: (A)—fech, (B)—ppox, (C)—cpox and (D)—alas2, (E)—sucla2, (F)—abcb10 and (G)—abcb7. Number of unique peptides (x axis), % sequence coverage (y axis) and spectral counts (bubble size) for each was calculated using the maximal values obtained minus the maximal values observed in the control samples (empty vector). Size of bubbles represents the % of the total spectral counts identified for each mouse protein. The maximal spectral counts of each of the mouse proteins was fech = 971, ppox = 71, cpox = 256, alas-2 = 16, sucla2 = 16, abcb10 = 51 and abcb7 = 36. Note no mouse peptides for alas2, sucla2, or abcb10 were obtained using FLAG-CPOX as bait above that of empty vector.</p

Immunoblot from Affinity Purification of FLAG elutions.

<p>Each lane represents the FLAG elutions from the affinity purification using empty vector (lane 1), WT FECH (lane 2), M76H FECH variant (lane 3) and E343K FECH variant (lane 4). Blots were probed for abcb7, abcb10, sucla2, ppox and fech. The dashed line indicates non consecutive lanes on the same gel.</p

Native PAGE and Western blot analysis of mitochondrial protein complexes.

<p>(A) Solubilized proteins complexes from mitochondrial preparations of differentiated MEL cells were separated by Native PAGE. (B) Regions of the Native PAGE gel were excised and proteins further separated by SDS-PAGE. Western blot of the SDS-PAGE was carried out for ppox and fech.</p

Porphyrin and metallated porphyrin levels in undifferentiated MEL cells.

<p>(A) Hemin levels from undifferentiated control (n = 3) and FECH overexpressing (n = 4) MEL cells.. (B) Levels of protoporphyrin IX, Zn-protoporphyrin IX and porphyrin precursors (8–4 COOH porphyrins) for undifferentiated control (n = 3) and FECH overexpressing (n = 4) MEL cells. * indicates a <i>p</i> value of ≤ 0.006.</p

A Novel Role for Progesterone Receptor Membrane Component 1 (PGRMC1): A Partner and Regulator of Ferrochelatase

Heme is an iron-containing cofactor essential for multiple cellular processes and fundamental activities such as oxygen transport. To better understand the means by which heme synthesis is regulated during erythropoiesis, affinity purification coupled with mass spectrometry (MS) was performed to identify putative protein partners interacting with ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Both progesterone receptor membrane component 1 (PGRMC1) and progesterone receptor membrane component 2 (PGRMC2) were identified in these experiments. These interactions were validated by reciprocal affinity purification followed by MS analysis and immunoblotting. The interaction between PGRMC1 and FECH was confirmed in vitro and in HEK 293T cells, a non-erythroid cell line. When cells that are recognized models for erythroid differentiation were treated with a small molecule inhibitor of PGRMC1, AG-205, there was an observed decrease in the level of hemoglobinization relative to that of untreated cells. In vitro heme transfer experiments showed that purified PGRMC1 was able to donate heme to apo-cytochrome <i>b</i>₅. In the presence of PGRMC1, in vitro measured FECH activity decreased in a dose-dependent manner. Interactions between FECH and PGRMC1 were strongest for the conformation of FECH associated with product release, suggesting that PGRMC1 may regulate FECH activity by controlling heme release. Overall, the data illustrate a role for PGRMC1 in regulating heme synthesis via interactions with FECH and suggest that PGRMC1 may be a heme chaperone or sensor

Regulation of intracellular heme trafficking revealed by subcellular reporters

Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models

Patient Demographics and Risk Factors for PCT.

<p>Patient Demographics and Risk Factors for PCT.</p